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Subcellular and polypeptide distributions of slowly transported proteins in the garfish olfactory nerve.

In the garfish olfactory nerve proteins labeled with [3H]leucine are transported by slow axonal flow as a well-defined crest of radioactivity. At 21 degrees C slow flow moves along the axon with a velocity of 0.92 +/- 0.02 mm/day. It has been possible to analyze 4 subcellular fractions (soluble, mitochondrial and 2 membranous) as well as their polypeptide composition, in areas of the nerve containing (1) the slow moving crest, (2) the material remaining in the nerve behind the crest, and (3) the labeling present in front of the slow crest. Analyses were done 70 and 110 days after isotope deposition. The crest of slow moving radioactivity is characterized by a close parallelism between labeling and protein concentration in the subcellular fractions as well as among the polypeptides constituting these fractions. The radioactivity is mainly associated with mol. wt. of 14,000, 30-45,000, 58,000 and 68,000. This last peak corresponds to a protein not labeled by fast transport, present only in the light membranous fraction. The composition of the moving crest remains essentially constant during the 40-day period investigated. Most of the slow-moving molecules remain in the axon behind the moving crest. This deposited material appears to be redistributed and/or to be turning over more rapidly than the molecules still moving in the crest. A large amount of radioactivity was recovered in front of the moving crest. This might be produced by molecules deposited by fast transport and by material released from the cell body at rates intermediate between the fast and slow phases of transport. The subcellular and polypeptide compositions of this area of the nerve remain constant and are intermediate between the compositions of fast and slow flow. The slowly transported labeled polypeptides in the mitochondrial fraction are of low molecular weight, and were found to be similar in the various areas of the nerve and at the two time points studied, and were even similar to the polypeptide distribution determined for fast transport.

Animals↗

Glial fibrillary acidic protein (GFAP)-like immunoreactivity in normal and transected rat olfactory nerve.

Normal and transected rat olfactory nerves were stained immunohistochemically using a monoclonal antibody previously shown to selectively detect GFAP-like immunoreactivity in central astrocytes but not in peripheral Schwann cells. Low levels of "central" type GFAP were found in the olfactory nerves, presumably in ensheathing cells. The levels of GFAP increased dramatically after nerve transection. A population of strongly GFAP-positive cells was detected at the junction between the olfactory epithelium and initial part of the nerves, of possible relevance to the regenerative abilities of this pathway.

Animals↗

Neural regeneration and functional reconnection following olfactory nerve transection in hamster.

The olfactory sensory neurons in the vertebrate nervous system are unique in that they undergo continuous neurogenesis and replacement. Anatomical studies have shown that transection of the olfactory nerves leads to a degeneration of sensory neurons followed by a neurogenesis and replacement with newly formed cells. Replacement neurons grow axonal processes that are capable of reestablishing morphological connections with cells in the olfactory bulb. To determine the functional capacity of these anatomical reconnections, single unit responses to odor stimuli were recorded from cells in the olfactory bulb following recovery from unilateral olfactory nerve transection. A total of 56 cells were studied, taken from hamsters with recovery times of 4,35,60,90,120,180 and 270 days. At day 4, although there was spontaneous activity recorded from cells on the experimental side (n = 10), they did not respond to stimulation of the olfactory epithelium with odors. Control cells (n = 9) from the unoperated side of the same animals showed normal odor responses. By day 35, some of the cells tested on the experimental side responded to odor stimulation, indicating that connections had been reestablished with sensory neurons. With longer recovery times, an increasing percentage of cells responded to odor stimuli. In addition, concentration response functions showed that cells were capable of signaling differences in stimulus intensity. The response of cells to four odors (amyl acetate, 1-butanol, ethyl acetate and ethyl butyrate) showed differences in odor selectivity, suggesting their ability to discriminate among odors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Intercellular interactions in the mammalian olfactory nerve.

The small, unmyelinated axons of olfactory sensory neurons project to the olfactory bulb in densely packed fascicles, an arrangement conducive to axo-axonal interactions. We recently demonstrated ephaptic interactions between these axons in the olfactory nerve layer, the layer of the olfactory bulb in which the axon fascicles interweave and rearrange extensively. In the present study, we hypothesized that the axons, which express connexins, may have another mode of communication: gap junctions. Previous transmission electron microscopy (TEM) studies have failed to demonstrate such junctions. However, the definitive method for detecting gap junctions, freeze fracture, has not been used to examine the interaxonal connections of the olfactory nerve layer. Here, we apply a combined approach of TEM and freeze fracture to determine if gap junctions are present between the olfactory axons. Gap junctions involving olfactory axons were not found. However, by freeze fracture, P faces of both the axons and ensheathing cells (glia that surround the axon fascicles) contained distinctive linear arrays of particles, aligned along the small columns of extracellular space. In axons, few intramembranous particles were present outside of these arrays. Multi-helix proteins, including ion channels and connexin hemichannels, have been shown to be visible as particles by freeze fracture. This suggests that the proteins important for signal transmission are confined to the linear arrays. Such an arrangement would facilitate ephaptic transmission, calcium waves, current oscillations, and paracrine communication and may be important for olfactory neural code processing.

Animals↗

Expression of galectin-1 in the olfactory nerve pathway of rat.

The olfactory neuroepithelium is characterised by the mosaic distribution of primary olfactory neurons that express different odorant receptors and cell surface glycoconjugates. Carbohydrates are believed to form a glycocode that mediates sorting out and fasciculation of primary olfactory axons through interactions with carbohydrate-binding proteins such as galectin-1. In the present study, we describe in detail the expression pattern of galectin-1 in the developing and adult rat olfactory system. We demonstrate that galectin-1 is expressed by olfactory ensheathing cells both in olfactory nerve and within the nerve fibre layer of the olfactory bulb of the embryonic and adult rat. In the adult rat, galectin-1 was preferentially expressed by olfactory ensheathing cells in the nerve fibre layer of the ventromedial and lateral surfaces of the olfactory bulb. Galectin-1 was also expressed by subsets of periglomerular cells and granule cells, particularly in the ventromedial region of the olfactory bulb. In adult rat, the galectin-1 ligand, N-acetyl-lactosamine, was expressed by primary olfactory axons that terminated in glomeruli present in the ventromedial and lateral olfactory bulb. These results suggest that expression of galectin-1 may provide a mechanism for the sorting of subpopulations of axons in the nerve fibre layer of the olfactory bulb during development as well as play a role in the postnatal maintenance of specific glomerular connections.

Aging↗

Numbers of olfactory receptor cells and fine structure of olfactory nerves in various birds.

The numbers of olfactory receptor cells from electron micrographs in various species of birds were counted and the fine structure of their olfactory nerves was observed using electron micrographs. The birds were domestic ducks, a slay-backed gull, quails, budgerigars and bengalees. Data from pigeons obtained from a previous study were also included for comparison. The approximate numbers of olfactory cells on each side were 5,800,000 in the duck, 2,700,000 in the gull, 570,000 in the quail, 130,000 in the budgerigar, and 110,000 in the bengalee. From a cross section of an olfactory nerve, the nerve was observed to be divided roughly into several fascicles by perineurium. Each fascicle was composed of many small bundles which were surrounded by endoneurium. Each small bundle was separated into several divisions by mesaxon which originated from Schwann cells. In the anterior part of the nerve, the number of axons surrounded by mesaxon ranged between one and several dozen. The number in the middle part was much larger than in the anterior part. In the posterior part the number decreased again. The diameter of an axon was 0.21-0.26 microns on average. The axon contained neurotubules, neurofilaments, mitochondria and axonal smooth ER. The fine structure of the olfactory nerve and the numbers of olfactory cells in these birds are discussed and compared with those of other vertebrates.

Animals↗

[Degeneration and regeneration of olfactory epithelial neurons after olfactory nerve sectioning].

The morphological changes of olfactory mucous membrane have been investigated with light microscope and T.E.M. within a period of 3 days to 4 weeks after unilateral olfactory nerve sectioning. No obvious morphological changes of supporting cells and Bowman's gland cells were seen in any group postoperatively. A degeneration in some neurons began to appear on the third day following the operation and reached its peak about one week after operation. No changes of the basal cells were seen on the third day. After one week, however, they changed into globose type and some of which showed mitotic activity. Within 2-4 weeks, the globose cells differentiated gradually into mature neurons. Four weeks after the operation, the regenerated olfactory epithelium appeared similar to that of the control group. The main feature of neuron degeneration was the increase in plasma electron density. In serious cases, the organelle systems disappeared and the nuclei were pyknotic. The results obtained from this experiment shows that after the sectioning of olfactory nerve, degeneration and regeneration processes of olfactory neuron will occur; and the regenerated neuron may possibly be developed from basal cells.

Animals↗

A parametric study of the stimulation variables affecting the magnitude of the olfactory nerve response.

The magnitude of olfactory responses can be related to three primary variables [number of odorant molecules (N), sniff volume (V), and sniff duration (T)] and three derived variables [concentration (C = N/V), flow rate (F = V/T), and delivery rate (D = N/T)]. To evaluate the effects of these interdependent variables upon the olfactory response, the summated multiunit discharges were recorded from the olfactory nerves of nine frogs in response to octane presented at two levels (in 2:1 ratio) of each primary variable. This presentation defined eight "sniff" combinations representing three levels of each derived variable. In an ANOVA of the logs of the responses, the effect of each primary variable was highly significant, with no significant interactions. A multiplicative regression model incorporating the effects of the three primary variables represented responses exceedingly well, with positive effects of N and T and a negative effect of V. When, with this model, the effect of each of the derived variables was isolated from the effects of all other variables, the analysis showed a positive effect for C, a near-zero positive effect for D, and a negative effect for F. Placing certain constraints upon the model parameters generates 13 distinct one- and two-variable models (e.g., the [C, T] model requires N and V to have equal but opposite effects). In ranking these reduced models in terms of their ability to predict the neural response, the predictive ability of [F, N] and [C, T] was at least as good as that of the three-variable model.

Analysis of Variance↗

Synaptic actions on mitral and tufted cells elicited by olfactory nerve volleys in the rabbit.

1. A unitary study has been carried out of mitral and tufted cell responses to olfactory nerve volleys in the olfactory bulb of rabbits lightly anaesthetized with urethane-chloralose. 2. With volleys of different strengths, some mitral cells responded with a spike whose latency decreased considerably as the strength increased (elastic response); other cells responded at an invariant latency (inelastic response). The former may reflect diffuse olfactory nerve inputs to the dendritic tufts in the olfactory glomeruli, while tha latter may reflect input from discrete bundles of fibres. 3. The shortest spike latencies are consistent with monosynaptic excitation by the olfactory nerves; longer latencies may be due to longer pathways through the nerves, or polysynaptic pathways within the glomerular layer. 4. Facilitation, in terms of lower threshold and shorter spike latency, was found when testing with paired volleys of weak intensity at relatively short intervals (less than 40 msec). Suppression, in terms of raised threshold, longer latency and briefer repetitive discharges, was found at intervals up to several hundred msec. The facilitation and suppression are consistent with the hypothesis of synaptic excitation and inhibition, respectively, mediated through interneurones in the olfactory bulb. 5. Presumed tufted cells were similar in response properties to identified mitral cells. 6. Intracellular recordings revealed long-lasting hyperpolarization and in some cases, an initial depolarization leading to spike initiation, in response to an olfactory nerve volley.

Action Potentials↗

The alteration of odor-induced c-Fos immunoreactivity in the rat olfactory bulb after olfactory nerve transection.

We used the rats in which one olfactory nerve had been transected and observed the odor (Propionic acid) -induced c-Fos immunoreactivity in the bulb at different times (2, 4, 8 weeks) after nerve transection. The exposure to odor produced a strong cluster of c-Fos positive cells in the mediodorsal region of the intact bulb. On the other hand, the transected bulb showed much less reactivity 2 weeks after neurectomy; however, a large number of positive cells were observed in the whole of the bulb from 4 weeks after neurectomy. Furthermore, we measured the levels of mRNA for tyrosine hydroxylase (TH), which was the marker of odor-induced olfactory nerve activity in the bulb, by using real-time PCR. The level of TH mRNA decreased on the transected side at 2 weeks but recovered to the level of the contralateral side at 4 weeks after neurectomy. We firstly demonstrated that projection mapping of odor receptors was altered after olfactory nerve transection by using an immunohistochemical method.

Animals↗

Differences in the composition of the polypeptides deposited in the axon and the nerve terminals by fast axonal transport in the garfish olfactory nerve.

Proteins transported by the fast wave of axonal transport have been shown to be deposited both in the axon and in the nerve terminals. Differences in the nature of the molecules deposited in these two areas were studied in the garfish olfactory system. In order to avoid analysis of transported molecules in two different types of tissue like the olfactory nerve and the olfactory bulb, the study was conducted (1) by comparing the composition of the moving crest of radioactivity at two different points along the nerve: when the crest enters the axon and when it reaches a distance of approximatively 5 cm from the nerve endings, (2) by determining the composition of the molecules remaining in the axon behind the moving crest. Three subcellular fractions (two membranous fractions and a mitochondrial pellet) were investigated. In both membranous fractions the majority of the polypeptides deposited in the axon ranged from 50 to 150,000 daltons. No outstanding peak of radioactivity was found in either fraction. Radioactivity was relatively evenly distributed among the various polypeptides. In the lightest membranous fraction, however, a peak (mol. wt., 54-58,000) was more particularly deposited in the axon. The opposite situation was found for the molecules moving toward the synapses: transported radioactivity was concentrated in a few distinct polypeptides, while the others were significantly less labeled. Three peaks were found in the lightest membranous fraction (mol. wt., 35,000, 54-58,000 and 126,000). Only two peaks were determined in the heaviest fraction (mol. wt., 58,000 and 126,000). The 126,000 mol. wt. peak increases with distance in both membranous fractions from 9 to 12% of the total radioactivity and moves mainly toward the synapses. The 35,000 mol. wt. polypeptide presented some interesting properties: it was found in larger quantities in the lightest membranous fraction; labeling was very poor in the heaviest membranous fraction, and finally this polypeptide appeared to be largely transported to the synapses. Results concerning the polypeptide composition and the composition of the transported molecules indicated that the lightest fraction may contain more synaptosomal material. From this study it appears that most transported polypeptides are distributed in both the axon and the nerve terminals, but that the percentage delivered to each area varies. A few distinct polypeptides on the contrary are more selectively transported to the synapses and are even differently localized in subcellular fractions.

Animals↗

Study of regeneration in the garfish olfactory nerve.

Previous studies of the olfactory nerve, mainly in higher vertebrates, have indicated that axonal injury causes total degeneration of the mature neurons, followed by replacement of new neuronal cells arising from undifferentiated mucosal cells. A similar regeneration process was confirmed in the garfish olfactory system. Regeneration of the nerve, crushed 1.5 cm from the cell bodies, is found to produce three distinct populations of regenerating fibers. The first traverses the crush site 1 wk postoperative and progresses along the nerve at a rate of 5.8 +/- 0.3 mm/d for the leading fibers of the group. The second group of fibers traverses the crush site after 2 wk postcrush and advances at a rate of 2.1 +/- 0.1 mm/d for the leading fibers. The rate of growth of this group of fibers remains constant for 60 d but subsequently falls to 1.6 +/- 0.2 for the leading population of fibers. The leading fibers in the third group of regenerating axons traverse the crush site after 4 wk and advance at a constant rate of 0.8 +/- 0.2 mm/d. The multiple populations of regenerating fibers with differing rates of growth are discussed in the context of precursor cell maturity at the time of nerve injury and possible conditioning effects of the lesion upon these cells. Electron microscopy indicates that the number of axons decreases extensively after crush. The first two phases of regenerating axons represent a total of between 6 and 10% of the original axonal population and are typically characterized by small fascicles of axons surrounded by Schwann cells and large amounts of collagenous material. The third phase of fibers represents between 50 and 70% of the original axonal population.

Animals↗

The immunopathology of olfactory nerve injury and hemorrhage in gerbils.

Patients with head injury may experience olfactory dysfunction. Unfortunately, little is known about changes in the olfactory neuroepithelium caused by intracranial olfactory nerve injury and hemorrhage. We performed olfactory nerve section or bulbectomy in 53 Mongolian gerbils and subjected the olfactory neuroepithelium to immunohistochemical analysis. A decrease or disappearance of the antiserum to an olfactory marker protein containing olfactory receptor neurons was evident in the sectioned group. Degeneration of the olfactory neuroepithelium contralateral to olfactory nerve injury was also noted in animals with perineural hemorrhage. These findings suggest that olfactory dysfunction may occur not only with nerve injury but also secondary to intracranial hemorrhage.

Animals↗

Ensheathment of the olfactory nerves in the adult rat.

The ensheathment of the olfactory nerve fibres is achieved by cooperation of two cell types. The olfactory ensheathing cells have a rounded outer surface enclosed in a continuous single basal lamina, and enclose an inner compartment from which overlapping processes of the same and adjacent cells enwrap interweaving territories of tightly apposed aligned axons. The olfactory nerve fibroblasts are highly flattened, dense cells generating multiple layers of very thin processes encircling individual or groups of olfactory ensheathing cells. This paper illustrates the unique ultrastructural features of this ensheathment.

Animals↗

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.

Animals↗

Garfish olfactory nerve: easily accessible source of numerous long, homogeneous, nonmyelinated axons.

The olfactory nerve of the garfish, Lepisosteus, is about 1 millimeter in diameter and about 20 centimeters long, depending on the size of the fish; it is easily prepared by breaking off successive scored segments of the rostrum. It consists of a relatively homogeneous population of about 10(7) nonmyelineated nerve fibers, each about 0.24 micrometer in diameter. In most other nerves each fiber is separated from all others by an enfolding Schwann cell, but in the olfactory nerve the fibers are directly in contact with one another in groups of several hundred fibers. The Schwann cell, not directly concerned with propagation of the nerve impulse, forms a thin layer at the periphery of the group and makes up a small proportion of the total cellular material. The volume of axon cytoplasm is about five times greater than that of Schwann cell cytoplasm, and the axon surface is about 30 times the Schwann cell surface. The ratio of surface to volume for axons of a typical olfactory nerve is about 5400 times that for the squid axon of the same diameter. The large proportion of axonal membrane recommends this nerve for use in chemical and physical studies of properties of axon membranes.

Action Potentials↗

Transection of the rat olfactory nerve increases glial fibrillary acidic protein immunoreactivity from the olfactory bulb to the piriform cortex.

Astrocytic glial fibrillary acidic protein (GFAP) immunoreactivity in response to retrograde changes of motoneurons after axotomy has been the subject of a number of reports. In contrast, this study examined the astrocytic GFAP immunoreactivity in response to axotomy in a sensory system, the adult rat olfactory system. The purpose of this study was to determine, by immunolabeling GFAP, the extent and transience of astrocytic reactivity in the olfactory system. Unilateral transection of the olfactory nerve fascicles was performed intracranially at the level of the cribriform plate. Rats were allowed to survive from 24 hours to 1 month after axotomy. GFAP immunolabeling was examined throughout the rat olfactory system using the peroxidase-anti-peroxidase method. After axotomy, a transient increase occurred in the astrocytic GFAP immunoreactivity in the ipsilateral olfactory system. The greatest enhancement of GFAP immunoreactivity in the olfactory system occurred at 48 hours post-axotomy. Increased GFAP immunoreactivity occurred not only along the axons and synaptic endings of the injured primary olfactory neurons, but also along the dendrites, cell bodies, axons, and synaptic endings of the secondary sensory neurons. The increased GFAP immunoreactivity was specifically associated with the anatomical distribution pathways of the primary and secondary olfactory neurons. Increased GFAP immunoreactivity was not altered until 14 days post-axotomy. At 1 month post-axotomy, GFAP immunoreactivity returned to control levels. The time course and transience of increased GFAP immunoreactivity closely correlates with the time course of rat primary olfactory neuronal degeneration and regeneration after axotomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗