Functional and morphological regeneration of olfactory tracts and subtracts in goldfish

Goldfish are ideal vertebrates for the study of regeneration within the central nervous system. The present behavioural and neuroanatomical investigations after bilateral transection of the entire olfactory tracts of either lateral or medial subtracts have been designed (1) to examine the relationship between morphological changes and changes in the perception of spontaneously preferred chemosensory stimuli, (2) to investigate the animals' ability to qualitatively discriminate amino acids in olfactory concentrations (below taste threshold, 10^-6–10^-8 M), one of which had been rewarded preoperatively (specific regeneration), and (3) to examine the discriminative ability for amino acids at concentrations above taste threshold (> 10^-5 M) in intact sham-operated, and in operated specimens at various time intervals before functional regeneration. Within 10–14 days after bilateral transection of the lateral olfactory tracts, specific regeneration was observed. After bilateral transection of the medial olfactory tracts, no immediate behavioural change was recorded for 1 week. Thereafter, goldfish behaviour became unstable and dropped to the chance level for 3–4 weeks. Subsequent to this time the goldfish returned to the preoperative level. Following bilateral crushing of the olfactory tracts and after total tractotomy, a specific regeneration was observed after 4 weeks and 6–8 weeks, respectively, post op. HRP studies showed that after bilateral lesioning a qualitative reinnervation of the respective nuclei within the forebrain by the medial and lateral olfactory subtracts was evident.Abbreviations FB funnel biting - FO funnel orientation - HRP horseradish peroxidase - LOT lateral olfactory tract - MOT medial olfactory tract     

Topographical relation between the olfactory bulb and the olfactory tract in tench (Tinca tinca L)

Electrical activity was recorded from single cells in the olfactorybulb when electrically stimulating the medial and lateral olfactorytract and when stimulating the olfactory epithelium with aminoacids. Bulbar units excited by stimulation of the medial olfactorytract were found in the medial and middle parts of the bulb.Neurones in the dorso-lateral part of the bulb were excitedby stimulation of lateral tract. Units inhibited by stimulationof the lateral or medial olfactory tracts had a reversed distributionwith the majority found in the medial or lateral parts of thebulb respectively. The chemicals tested induced changes in thedischarge of units mainly situated in the lateral part of thebulb.     

Patterns of olfactory projections in the desert iguana,Dipsosaurus dorsalis

The neural organization of the olfactory system in the desert iguana, Dipsosaurus dorsalis, has been investigated by using the Fink-Heimer technique to trace the efferents of the main and accessory olfactory bulbs, and Golgi preparations to determine the spatial relations between olfactory afferents and neurons in the primary olfactory centers. The accessory olfactory bulb projects to the ipsilateral nucleus sphericus via the accessory olfactory tract. The main olfactory bulb projects to the ipsilateral telen-cephalon via four tracts. The medial olfactory tract projects to the rostral continuation of medial cortex and to the septum. The intermediate olfactory tract projects to the olfactory tubercle and retrobulbar formation. The lateral olfactory tract projects to the rostral part of lateral cortex. The intermediate and lateral olfactory tracts also merge caudally to form the stria medullaris, which crosses the midline in the habenular commissure and distributes fibers to the contralateral hemisphere via two tracts. The lateral corticohabenular tract terminates in the contralateral lateral cortex. The anterior olfactohabenular tract terminates in the contralateral olfactory tubercle, retrobulbar formation and septum. The relation of olfactory afferents to neurons in the medial cortex, lateral cortex, nucleus sphericus, and septum corresponds to a pattern of organization that is typical of many olfactorecipient structures. Such structures are trilaminar, with neurons whose somata are situated in the intermediate layer (layer 2) sending spine-laden dendrites into an outer, molecular layer (layer 1). Olfactory afferents intersect the distal segments of these dendrites. By contrast, other olfactorecipient structures in Dipsoaurus deviate from the familiar pattern. Olfactory afferents intersect somata lying in layer 2 of the retrobulbar formation. Olfactory afferents include some fibers which course perpendicularly to the surface of the olfactory tubercle and extend deep to layer 2.     

Decline and recovery of olfactory receptor expression following unilateral bulbectomy

The effects of unilateral olfactory bulb ablation upon the odorant receptor expression were studied during the degeneration/regeneration process in the olfactory epithelium of adult rats. Using the in situ hybridization approach, we compared the time course of decay and recovery of expression for three different receptor subtypes (OR14, OR5, OR124). The number of neurons expressing receptor subtypes dramatically decreased in the olfactory epithelium on the lesioned side and reached a minimum at day 5 postsurgery. A progressive recovery was then observed from day 5 to day 15 postlesion, when a plateau was reached. Noticeable differences in the recovery level of receptor expression were observed according to the zonal patterning: the recovery level for neurons located in the lateral zone reached 70% of the control side value while the recovery levels in the dorsal and medial zones represented 35% and 53% of this value, respectively. Axotomy experiments suggest that zone-specific differences in receptor reexpression reported after bulbectomy might be related to the trophic influence of the olfactory bulb.     

Cell proliferation and growth-associated protein 43 expression in the olfactory epithelium in Poecilia reticulata after copper solution exposure

This study investigated the regeneration in the olfactory mucosa of the teleostean fish Poecilia reticulata when returned to dechlorinated tap water after 4-day exposure to 30 microg/L of Cu(2+). The regeneration process in the olfactory tissue was examined in fishes at 0, 3, 6 and 10 days of recovery in well water. Jade B staining permitted to evaluate the rate of the damage which was especially extended to olfactory neurons. Immediately after the end of exposure, a massive mitotic activity in the basal region of the mucosa was detected by immunostaining with PCNA. After 3 days of recovery the nuclei of the newly formed cells had already finished their migration to the upper portion of the epithelium, and cellular division was much less intense. Simultaneously, immunoreactivity for the neural growth-associated phosphoprotein GAP-43 increased respect to control levels, revealing that the new differentiating PCNA-positive elements belonged to immature neurons. After 6 days in well water no mitotic activity was detected, while the GAP-43 labelling appeared particularly concentrated in the apical surface of the olfactory epithelium. After 10 days the aspect of the olfactory epithelium was almost identical to the control. The present results suggest that after 10 days regeneration seems to be complete and integrity of the tissue restored. Furthermore, the epithelium reconstitution does not show apparent divergence from other fishes or mammals.     

Volumetric and horseradish peroxidase tracing analysis of rat olfactory bulb following reversible olfactory nerve lesions

Olfactory receptor neurons can regenerate from basal stem cells. Receptor neuron lesion causes degenerative changes in the olfactory bulb followed by regeneration as new olfactory receptor axons innervate the olfactory bulb. To our knowledge, parametric analyses of morphometric changes in the olfactory bulb during degeneration and regeneration do not exist except in abstract form. To better characterize olfactory bulb response, we performed morphometric analysis in rats following reversible olfactory nerve lesion with diethyldithiocarbamate. We also performed anterograde tracing of the olfactory nerve with wheatgerm agglutinin linked to horseradish peroxidase. Results of morphometry and tracing were complementary. The glomerular layer and external plexiform layer showed shrinkage of 45 and 26%, respectively, at 9 days. No significant shrinkage occurred in any other layer. Individual glomeruli shrank by 40-50% at 3 and 9 days following lesion. These data show that degenerative changes occur both in the glomeruli and transneuronally in the external plexiform layer. Olfactory nerve regeneration (identified by WGA-HRP transport) paralleled volumetric recovery. Recovery occurred first in ventral and lateral glomeruli between 9 and 16 days followed by recovery in medial and dorsal glomeruli. These data indicate substantial transynaptic degeneration in the olfactory bulb and a heretofore unrecognized gradient in olfactory nerve regeneration that can be used to systematically study recovery of a cortical structure.     

Effects of methyl bromide on the rat olfactory system

The nature and extent of damage produced by methyl bromide (MeBr)exposure, and recovery of function after exposure, were studiedusing a multifacct approach which included behavioral, morphologicaland neurochemical endpoints. Thirty adult male Long–Evansrats were exposed to 200 p.p.m. MeBr for 4 h/day, 4 days a weekfor 2 weeks. Fifteen control rats were exposed to filtered aironly. On the first day following the onset exposure to MeBr,extensive damage to the olfactory epithelium as well as greatlyimpaired olfactory function were observed. However, even withcontinuous MeBr exposure, olfactory function was essentiallynormal after 4 days of exposure. Repair of the epithelium wasin progress by day 4 although morphology was atypical. The levelsof carnosine in both the olfactory epithelium and bulbs wereseverely depleted by day 4. Recovery, both in terms of structuralrepair and return of normal carnosine concentrations, laggedfar behind recovery of the ability to detect an odor stimulus.Even with repeated exposure, olfactory function recovered rapidly,even faster than anatomical repair. Measurement of overall carnosinelevels correlated well with the results obtained from representativeareas of tissues selected for histopathology. Morphometric analysisprovided quantitative detail on the nature of insult resultingfrom MeBr exposure. These data indicate that the olfactory systemis a most resilient system and that normal function is possibleeven after repeated insult by a toxic agent.     

Renewing olfactory receptor neurons in goldfish do not require contact with the olfactory bulb to develop normal chemical responsiveness

This study investigated whether contact with the olfactory bulb was necessary for developing and renewing olfactory receptor neurons (ORNs) to attain normal odorant responsiveness, and whether the anatomical and functional recoveries of the olfactory epithelium were similar in both bulbectomized (BE) and bilaterally axotomized (AX) preparations. In vivo electrophysiological recordings were obtained in response to amino acids, a bile acid [taurolithocholic acid sulfate(TLCS)] and a pheromonal odorant [17α, 20β,-dihydroxy-4-pregnen-3-one (17,20P)] from sexually immature goldfish. Both transmission and scanning electron microscopy indicated that the olfactory epithelium degenerated in BE and AX goldfish. Within 1–2 weeks subsequent to the respective surgeries, responses to high concentrations (>0.1 mmol · l⁻¹) of the more stimulatory amino acids remained, whereas responses were no longer obtainable to TLCS and 17,20P. At 4 weeks, responses to amino acid stimuli recovered to control levels, while responses to TLCS and 17,20P were minimal. By 7 weeks post bilateral axotomy, the olfactory epithelium recovered to a condition similar to control sensory epithelium; however, the rate of degeneration and proliferation of receptor neurons in BE preparations appeared to remain in balance, thus blocking further recovery of the olfactory epithelium. At 7 weeks post surgery, odorant responses of AX and BE goldfish to TLCS and 17,20P were still recovering. Accepted: 14 June 1997     

Immunohistochemical distribution of galectin-1, galectin-3, and olfactory marker protein in human olfactory epithelium

The expression pattern of galectin-1 and galectin-3 in the human olfactory epithelium was investigated in relation to olfactory marker protein (OMP) using confocal laser immunofluorescence in human specimens and postmortem biopsies. OMP expression was found in olfactory receptor neurons (ORNs) in the olfactory mucosa and in fibers of the olfactory nerve crossing the submucous connective tissue. Galectin-1 was expressed in both the connective tissue of the nasal cavity and in the basal layer of the olfactory epithelium. In contrast, galectin-3 expression was limited to cells of the upper one-third of the olfactory epithelium. Expression of galectin-3 occurred in a subset of OMP-positive cells. However, between areas of galectin-1 and galectin-3 expression in the lower and upper portion of the epithelium, OMP-positive ORNs did not stain for both galectins. Considering the potential role of galectin-1 and galectin-3 in cell differentiation and maturation, the differential localization of galectins in the olfactory epithelium appears to be consistent with a significant role of these molecules in the physiological turnover of ORNs. Accepted: 20 December 1999     

Functional correlates of degeneration and renewal of cilia and knobs of olfactory receptor neurons in the frog

Changes in surface structures of the olfactory epithelium, olfactorynerve and olfactory nerve layer in the olfactory bulb followingolfactory nerve section were studied, by scanning electron microscopy,in the frog. Correlative neurophysiological responses were recordedfrom the olfactory epithelium in response to odor stimulation.Examination of the epithelial surface showed degeneration andloss of the dense ciliary matrix and olfactory knobs by day10, which exposed the microvillar surface of the sustentacularcells. The amplitude of slow voltage transients recorded fromthe epithelial surface systematically decreased through day10. By day 40, the olfactory epithelium became responsive toodor stimulation. At this time partial renewal of the ciliarymatrix on the epithelial surface and bundles of receptor cellaxons in the olfactory nerve layer of the olfactory bulb wereobserved. There was substantial replacement of the ciliary matrixby day 100; in contrast, considerably less recovery of the slowvoltage transient was evident. Recovery of odor-evoked responsivity lagged behind recovery of the ciliary matrix. Therefore,these data imply that the reappearance of olfactory knobs andcilia is causally related to the recovery of the slow voltagetransients.     

Alarm reaction in the crucian carp is mediated by the medial bundle of the medial olfactory tract

Experiments were performed to determine which bundles of the olfactory tracts were essential for mediating alarm reaction in crucian carp (Carassius carassius L.). The fish were maintained in physiological saline after surgery to preserve the remaining tracts and postoperative inspections revealed the functionality of the intact tracts. Operations on the tracts were performed symmetrically on both sides. Sham-operated and non-operated fish showed the typical alarm behaviour of fast swimming to the bottom, dashing movements and aggregation when exposed to skin extract which contain alarm substance. Fish with only the medial bundle of the medial olfactory tract intact also displayed the alarm behaviour upon exposure; however, these fish did not react to the amino acid, L-alanine with either feeding response or alarm reaction. Crucian carp which had the medial bundle of the medial olfactory tract cut, leaving both the lateral bundle of the medial olfactory tract and the lateral olfactory tract intact, did not display any alarm reaction to skin extract; however, these fish reacted to exposure to L-alanine with feeding behaviour. There were statistically significant differences between the behaviour scores for the fish subject to different treatments. The present study demonstrates that the medial bundle of the medial olfactory tract appears to be both necessary and sufficient for mediation of the alarm reaction. The results also show that the sensory neurons which respond to alarm substance terminate and make synaptic connections with the secondary neurons that make up the medial bundle of the medial olfactory tract; thereby demonstrating the specificity of the spatial aspect of olfactory processing. The results are discussed with respect to the spatial aspect of organization within the olfactory system, the pattern of generalization across orders of fish, and the functional implications of the spatial arrangement of information transmission between the peripheral olfactory organ and the brain.     

Age-Related Changes of Elements in Human Olfactory Bulbs and Tracts and Relationships Among Their Contents

To elucidate compositional changes of the olfactory bulb and tract with aging, the authors investigated age-related changes of elements in the olfactory bulbs and tracts of Japanese and the relationships among the elements. After ordinary dissection at Nara Medical University was finished, the olfactory bulbs were resected with the olfactory tracts from 40 subjects. The subjects consisted of 15 men and 25 women, ranging in age from 65 to 102 years (average age = 84.6 +/- 7.5 years). After ashing with nitric acid and perchloric acid, element contents in the olfactory bulbs and tracts were analyzed by inductively coupled plasma-atomic emission spectrometry. Seven elements of Ca, P, S, Mg, Zn, Fe, and Na did not change significantly in the olfactory bulbs and tracts with aging. The Ca, P, and S contents of major elements were less than 10 mg/g in all of the olfactory bulbs and tracts. Regarding the relationships among the elements, extremely or very significant direct correlations were found among the contents of Ca, P, Mg, Zn, and Na in the olfactory bulbs and tracts, with one exception. In addition, an extremely significant direct correlation was found between S and Mg contents and a very significant direct correlation was found between P and S contents. As P increased in the olfactory bulb and tract, Ca, Mg, Zn, Na, and S also increased in the olfactory bulb and tract.     

Neural and behavioral changes in rats following continuous exposure to an odor

Summary Continuous exposure of young rats to the almond-like odor of acetophenone or cyclohexanone for up to 4 months, resulted in distinct but similar patterns of degenerating mitral cells in their olfactory bulbs. Rats favored their exposure odor in olfactory preference tests (Fig. 2) and their acuity for it was not altered (Fig. 3). However, they appeared to exhibit a deficit in detecting a similar but novel odor. The results suggest that the remaining normal mitral cells in the bulbs of these animals are those stimulated by the exposure odor. Cells which show signs of degeneration (Fig. 4) may receive little or no input from the periphery. Controls exposed to a similar but non-odorous environment showed evidence of non-selective mitral cell degeneration. In addition they had a lower acuity for acetophenone and cyclohexanone than animals reared in a normal rat colony (Fig. 3). Anatomical and behavioral data from odor exposed and control groups, suggest that partial regeneration of altered mitral cells may have occurred during a 5 month period following exposure. Overall the results provide further evidence for a topographical projection of the olfactory receptor epithelium onto the olfactory bulb and spatial coding of different odors in the bulb.     

Electron microscopic study of the termination of the centrifugal fibers in the goldfish olfactory bulb

Summary The terminals of centrifugal fibers to the olfactory bulbs of goldfish were studied by electron microscopy after transection of the medial, lateral or entire olfactory tract. The centrifugal fibers originate in the telencephalic hemisphere, pass through both the medial and the lateral olfactory tract, and form synaptic contacts with dendrites in the granule cell layer.     

Intranasal inoculation with the olfactory bulb line variant of mouse hepatitis virus causes extensive destruction of the olfactory bulb and accelerated turnover of neurons in the olfactory epithelium of mice

Viral upper respiratory infections are the most common cause of clinical olfactory dysfunction, but the pathogenesis of dysosmia after viral infection is poorly understood. Biopsies of the olfactory mucosa in patients that complain of dysosmia after viral infection fall into two categories: one in which no olfactory epithelium is seen and another in which the epithelium is disordered and populated mainly by immature neurons. We have used intranasal inoculation with an olfactory bulb line variant of MHV to study the consequences of viral infection on peripheral olfactory structures. MHV OBLV has little direct effect on the olfactory epithelium, but causes extensive spongiotic degeneration and destruction of mitral cells and interneurons in the olfactory bulb such that the axonal projection from the bulb via the lateral olfactory tract is markedly reduced. Moreover, surviving mitral cells apparently remain disconnected from the sensory neuron input to the glomerular layer, judging from retrograde labeling studies using Dil. The damage to the bulb indirectly causes a persistent, long-term increase in the turnover of sensory neurons in the epithelium, i.e. the relative proportion of immature to mature sensory neurons and the rate of basal cell proliferation both increase. The changes that develop after inoculation with MHV OBLV closely resemble the disordering of the olfactory epithelium in some patient biopsies. Thus, damage to the olfactory nerve or bulb may contribute to a form of post-viral olfactory dysfunction and MHV OBLV is a useful model for studying the pathogenesis of this form of dysosmia.     

Once and again: retinoic acid signaling in the developing and regenerating olfactory pathway

Retinoic acid (RA), a member of the steroid/thyroid superfamily of signaling molecules, is an essential regulator of morphogenesis, differentiation, and regeneration in the mammalian olfactory pathway. RA-mediated teratogenesis dramatically alters olfactory pathway development, presumably by disrupting retinoid-mediated inductive signaling that influences initial olfactory epithelium (OE) and bulb (OB) morphogenesis. Subsequently, RA modulates the genesis, growth, or stability of subsets of OE cells and OB interneurons. RA receptors, cofactors, and synthetic enzymes are expressed in the OE, OB, and anterior subventricular zone (SVZ), the site of neural precursors that generate new OB interneurons throughout adulthood. Their expression apparently accommodates RA signaling in OE cells, OB interneurons, and slowly dividing SVZ neural precursors. Deficiency of vitamin A, the dietary metabolic RA precursor, leads to cytological changes in the OE, as well as olfactory sensory deficits. Vitamin A therapy in animals with olfactory system damage can accelerate functional recovery. RA-related pathology as well as its potential therapeutic activity may reflect endogenous retinoid regulation of neuronal differentiation, stability, or regeneration in the olfactory pathway from embryogenesis through adulthood. These influences may be in register with retinoid effects on immune responses, metabolism, and modulation of food intake.     

Pheromone detection and olfactory pathways in the brain of the female African catfish,Clarias gariepinus

Summary The olfactory tract of the African catfish, Clarias gariepinus, consists of two tracts, the medial and lateral olfactory tract. Ovulated female catfish are attracted by male steroidal pheromones. Attraction tests with catfish in which the medial and lateral olfactory tract have been selectively lesioned show that the effects of these pheromones are mediated by the medial olfactory tract. The central connections of the medial and lateral olfactory tract have been studied by retro- and anterograde transport techniques using horseradish peroxidase as a tracer. Upon entering the forebrain, the medial olfactory tract innervates the posterior pars ventralis and pars supracommissuralis of the area ventralis telencephali and the nucleus preopticus periventricularis, the nucleus preopticus and the nucleus recessus posterioris. Application of horseradish peroxidase to the olfactory epithelium shows that part of the innervation of the area ventralis telencephali and the nucleus preopticus periventricularis can be attributed to the nervus terminalis, which appears to be embedded in the medial olfactory tract. The lateral olfactory tract sends projections to the same brain areas but also innervates the nucleus habenularis and a large terminal field in the area dorsalis telencephali pars lateralis ventralis. Furthermore, the medial olfactory tract carries numerous axons from groups of perikarya localized in the area dorsalis telencephali. Contralateral connections have been observed in the olfactory bulb, telencephalon, diencephalon and mesencephalon. It is suggested that processes of the medial olfactory tract innervating the preoptic region may influence the gonadotropin-releasing hormone system and in doing so may lead to behavioral and physiological changes related to spawning.     

Quantitative observations on the nasal epithelia and olfactory innervation in bats. Suggested design mechanisms for the olfactory bulb.

The nasal epithelia of two species of bats were quantified with respect to relative surface areas and olfactory epithelial volumes. In the macrosmatic Aribeus jamaicensis 55.9% of the nasal cavity surface was covered by olfactory epithelium (232.4 mm2), in contrast to only 28.9% in the microsmatic Myotis lucifugus (36.4 mm2). The roles of the various nasal epithelia have been discussed as they may relate to olfaction, respiration and echolocation. In the olfactory bulbs of both species, the estimated concentration of mitral cells approximated at 2,500/mm2 compared to an olfactory nerve concentration of 5/mm2. In Artibeus, calculated total volume of olfactory epithelium was on the order of 16 times greater than in Myotis, and Artibeus' olfactory bulb diameter was twice as great. These findings, together with previously published surface, volume and physiological relationships, suggest a developmental design mechanism for an olfactory bulb in which the number of olfactory receptors increases some 450-fold above an initially established ratio of 2:1 between receptors and mitral cells. Key governing factors could be requisite mechanical rigidity of the cribriform plate of the ethmoid bone and response thresholds of higher brain centers.     

Rhinotopy is disrupted during the re-innervation of the olfactory bulb that follows transection of the olfactory nerve

Re-innervation of the olfactory bulb was investigated after transection of the olfactory nerve using monoclonal antibody RB-8 to assess whether rhinotopy of the primary olfactory projection is restored. In normal animals RB-8 heavily stains the axons, and their terminals, that project from the ventrolateral olfactory epithelium onto glomeruli of the ventrolateral bulb (termed RB-8(+)). In contrast, axons from dorsomedial epithelium are unlabeled (RB-8(-)) and normally terminate in the dorsomedial bulb. Sprague-Dawley rats underwent unilateral olfactory nerve transection and survived for 6 weeks prior to perfusion, sectioning and immunostaining with RB-8. Nerve lesion does not shift the position of the boundary between RB-8(+) and RB-8(-) regions of the epithelium. However, following transection and bulb re-innervation, the distribution of RB-8(+) and RB-8(-) axons is markedly abnormal. First, in all 10 experimental animals RB-8(-) axons displace RB-8(+) axons from anterior glomeruli. Furthermore, the usual target of the RB-8(-) fibers, i.e. the dorsomedial bulb at more posterior levels of the bulb, remains denervated, judging by the lack of staining with antibodies that label axons derived from all epithelial zones. Finally, RB-8(+) fibers invade foreign territory in the dorsolateral bulb on the lesioned side in some cases. The shifts in terminal territory in the bulb after transection contrast with the restoration of the normal zonal patterning of the projection after recovery from methyl bromide lesion, but is consistent with reports of mistargeting by a receptor-defined subset of neurons after transection.     

Large olfactory responses of the carp after complete removal of olfactory cilia

To study the role of olfactory cilia on olfactory reception, the carp olfactory cilia were removed by modified "ethanol-calcium shock" and the bulbar responses were recorded before and after deciliation. Large olfactory responses to various amino acids were observed after complete deciliation. The relation between magnitude of olfactory response and alanine concentration before and after deciliation was essentially unchanged. The present results suggests that the olfactory cilia may not be necessary for receptor neuron function in the carp.