Hans Peter Zippel

Ultrastructure of the olfactory epithelium in intact, axotomized, and bulbectomized goldfish, Carassius auratus.

The ultrastructure of the olfactory epithelium in intact, axotomized, and bulbectomized goldfish was studied by scanning and transmission electron microscopy. A total of 58 adult goldfish of various survival times were examined to determine whether the different types of surgery—either olfactory nerve transection or bulbectomy—yielded differences in the extent or time course of cellular degeneration and renewal. Control animals were also examined in detail to elucidate previous controversial findings concerning the types of olfactory receptor neurons present in goldfish. We found that the intact olfactory epithelium of unoperated control goldfish contains the previously observed ciliated and microvillous receptor neurons, and the crypt cell, a cell type not yet seen in the goldfish but recently reported in other species of teleosts. Following either olfactory nerve transection or bulbectomy, the olfactory receptor neurons showed similar signs of degeneration and subsequent cell death, but, surprisingly, the thickness of the olfactory epithelium did not change significantly with either treatment. The time course of receptor cell renewal was different in axotomized and bulbectomized goldfish. In axotomized goldfish, the amount of receptor cells decreased continuously until 8–13 days after surgery, followed by rapid cell renewal. For bulbectomized goldfish, cell replacement began almost immediately after surgery, with degeneration and cell renewal occurring simultaneously. Six weeks after bulbectomy, cell death and cell proliferation reached a “steady state,” and the epithelia did not further improve. Microsc. Res. Tech. 45:325–338, 1999. 

High correlation between microvillous olfactory receptor cell abundance and sensitivity to pheromones in olfactory nerve-sectioned goldfish

Abstract 1. To determine whether microvillous olfactory receptor cells mediate responses to pheromonal cues, the olfactory nerves of mature male goldfish were axotomized and both the olfactory and behavioral sensitivity of these animals to olfactory stimuli investigated after which the histological condition of their olfactory epithelia was determined. 2. Behavioral responsiveness to food odor returned within 2 weeks but responsiveness to sexually-active females (pheromones) took 4–10 weeks to return. 3. Electro-olfactogram recordings from the olfactory epithelium of axotomized fish found that olfactory responsiveness to amino acids and pheromones changed little during the first week subsequent to axotomy. However, olfactory sensitivity decreased rapidly during the second week. During the course of the third week, electro-olfactogram sensitivity to amino acids remained while exposure to pheromones evoked no recordable electro-olfactogram. During week 4, sensitivity to amino acids increased further, and weak sensitivity to some pheromones became evident. Further recovery of electro-olfactogram sensitivity to all odorants was slow and erratic over the next 6 months, particularly to the pheromones. 4. Histological examination of the olfactory epithelia of axotomized fish demonstrated that while ciliated receptor cells were present within 2 weeks, microvillous receptor cells took approximately 4 weeks to regenerate. 5. Together these data suggest that microvillous receptor cells mediate responsiveness to pheromones in this species.

In goldfish the qualitative discriminative ability for odors rapidly returns after bilateral nerve axotomy and lateral olfactory tract transection

After amino acid discrimination training (Arg vs. Gln) in 5 groups of 2 fish the olfactory nerves and in 5 groups the lateral olfactory tracts were intracranially and bilaterally dissected. Immediately after this operation both groups of fish were unable to discriminate concentration differences and contaminated stimuli. Two weeks after the operation, following functional regeneration, both groups again were able to discriminate stimulus concentration differences and contaminations as they did before the operation. Therefore functional regeneration of the olfactory nerves (peripheral regeneration) and lateral olfactory subtracts (central regeneration) is highly specific.

Spontaneous behaviour, training and discrimination training in goldfish using chemosensory stimuli

The present behavioural experimental paradigm made use of the responsiveness of goldfish to natural and non-familiar chemosensory stimuli in the context of feeding. With the exception of Tubifex food extract, which was spontaneously preferred, goldfish exhibited no spontaneously recordable response to low concentrations of the stimuli tested. Training experiments using non-familiar stimuli (amyl acetate, α-ionone, β-phenylethanol, 10-6, 10-7M) required 2–3 months of daily training prior to the animals reaching a 70% positive response level for discrimination. This discrimination was dependent upon a functioning olfactory system as no responses were recorded after bilateral exclusion of olfaction, e.g. dissection of olfactory nerve or olfactory tracts. Amino acids (Ala, Arg, Gln, Gly, Lys), more natural stimuli than those listed above, were preferred when applied at concentrations < 10-5M. Goldfish were able to discriminate amino acid odours applied at 10-6 or 10-7M, but these stimuli elicited no spontaneous response below 10-5M. Ten to twenty reinforcements were sufficient to achieve discrimination between amino acids, which again was eliminated after bilateral exclusion of olfactory pathways. In contrast to the 4-week period for long-term memory to non-familiar odours, long-term memory for amino acids lasted at least 3 months.

Die Oberflächenstruktur der olfaktorischen Drüsen des Goldfisches (Carassius auratus)

SummaryThe appearance and fine structure of the surface of endoepithelial glands in the regio olfactoria of Carassius auratus are described. The phases of accumulation of secretory droplets, their excretion, and their significance for the production of the terminal mucous film are demonstrated and discussed.Two different kinds of endoepithelial glands formation are described. It is possible that these glands are only modifications of a single cell type. The first modification is referred to as goblet cells; the second as endoepithelial glands with balloon-like projections over the surface of the olfactory epithelium.On the surface of the goblet cells, two classes of microvilli, which differ in structure and pattern of distribution, can be seen. In addition, membrane protrusions, which are interpreted as bundles of fused cilia, can sometimes be found.The goblet cells always appear with ciliated cells in well defined areas. The cilia of ciliated cells are characterized by the existence of “microspines”.The terminal mucous film, which until now was described only in land-living vertebrates is demonstrated in fish for the first time.ZusammenfassungIn der Regio olfactoria des Goldfisches (Carassius auratus) werden intraepitheliale Drüsen und deren Oberflächenstruktur beschrieben. Die Stadien der Sekretanhäufung, der Sekretausscheidung und der Bedeutung für die Bildung des Schleimfilmes über der olfaktorischen Rosette werden demonstriert und diskutiert. Zwei unterschiedliche Erscheinungsbilder von Drüsen werden beschrieben, bei denen es sich jedoch möglicherweise nur um zwei Modifikationen ein und desselben Zelltyps handelt: 1. Drüsen, die als Becherzellen bezeichnet werden und 2. Drüsen, deren ballonartige Vorwölbungen ihrer distalen Zellpole weit über die Oberfläche des olfaktorischen Epithels hinausragen. Diese beiden Drüsen unterscheiden sich in ihrer Häufigkeit, ihrer Verteilung auf den Lamellen der olfaktorischen Rosette, ihrem Sekretionsmechanismus und ihrer Oberflächenbeschaffenheit, nicht jedoch im lichtmikroskopischen Erscheinungsbild ihrer Sekrettropfen.Auf der Oberfläche der Becherzellen lassen sich zwei verschiedene Typen von Mikrovilli mit jeweils unterschiedlichem Verteilungsmuster erkennen: 1) ca. 0,15–0,5 μ lange Mikrovilli die in wechselnder Dichte über die gesamte Oberfläche der Drüsenzellen verteilt sein können und 2) bis ca. 1 μ lange Mikrovilli, die wie ein Stäbchensaum den Verlauf benachbarter Zellmembranen markieren. Außer diesen beiden Oberflächenprofilen kommen noch Strukturen vor, die in Ein- oder Mehrzahl auf der Oberfläche der Becherzellen erscheinen können und als Bündel verwachsener Zilien interpretiert werden. Sie sind bis zu 5 μ lang, ihr Basisdurchmesser beträgt ca. 1 μ und sie verjüngen sich im allgemeinen zur Spitze hin.Die Existenz von „microspines“ an den Zilien der den Becherzellen stets benachbarten Flimmerzellen wird beschrieben.Die Existenz eines terminalen Filmes auch über dem olfaktorischen Saum von Teleostiern konnte erstmalig nachgewiesen werden.

Histologische, funktionelle und spezifische Regeneration nach Durchtrennung der Fila olfactoria beim Goldfisch (Carassius auratus)

Summary1.Alter dissection of the olfactory libres (fila olfactoria, Fig. 1) in the goldfisch, histological (Fig. 5) as well as functional regeneration takes place.2.When regeneration of the olfactory fibres was complete, no differences in training and learning behaviour between operated and untreated control animals could be observed (Fig. 2).3.Specific regeneration could also be proved after dissection of the olfactory fibres: Preoperatively trained discriminative behaviour returned (Figs. 3, 4) when histological regeneration had reached an advanced state (Figs. 6, 7).4.Higher concentrations of all substances used for odour training were capable of exciting the taste receptors or other struktures outside the olfactory mucosa. The concentrations used for olfactory training, however, were below the thresholds for non-olfactory perception.5.Neither the surgical procedure nor the anesthesia influenced the memory function.Zusammenfassung1.Goldfische können die durchschnittenen Bahnen der Fila olfactoria anatomisch und funktionell regenerieren.2.Nach Regeneration der Fila olfactoria unterscheiden sich Lernverlauf und Dressurleistungen nicht von intakten Tieren.3.Nach Durchschneidung der Fila olfactoria konnte eine spezifische Regeneration nachgewiesen werden: Zum Zeitpunkt der fortgeschrittenen histologischen Regeneration stellen sich auch die präoperativ andressierten Verhaltensweisen wieder ein.4.Alle in der Dressur verwendeten Geruchsstoffe wirken in genügend hoher Konzentration auch als Geschmacksreize (oder Reize auf Rezeptoren außerhalb der Mucosa olfactoria). Die im Geruchstest verwendeten Geruchsstoff-Konzentrationen lagen jedoch unter diesen nicht-olfaktorischen Schwellen.5.Die Operation und die Narkose selbst beeinflußten die Gedächnisleistungen nicht.

Ciliated olfactory receptor neurons in goldfish (Carassius auratus) partially survive nerve axotomy, rapidly regenerate and respond to amino acids

Electro-olfactogram (EOG) recordings in response to amino acid stimulation were made from both control and experimental olfactory mucosae following unilateral axotomy. The recorded EOG amplitudes, amino acid stimulus relative effectiveness and dose-response relations for control and experimental mucosae were comparable in all pre- and postoperative recordings. Semi-thin investigations of olfactory mucosae showed degeneration of olfactory receptors but indicated that intact receptors were also present. SEM of olfactory mucosae revealed that ciliated receptor cells were present in both axotomized and control sides on postoperative days, whereas microvillous receptors completely degenerated and did not regenerate until 7 weeks post axotomy. The present findings along with previous behavioral observations suggest at least three possible sources of the EOGs recorded from the experimental olfactory mucosae following olfactory nerve transection: (1) young olfactory receptor neurons whose axons had not yet reached the region of the transected olfactory nerve; (2) newly-emerged olfactory receptor neurons; and (3) olfactory receptor neurons that had not degenerated.

The influence of repeated natural stimulation upon discharge patterns of mitral cells of the goldfish olfactory bulb

Summary1.The responses of goldfish mitral cells were analysed by extracellular recording. The stimulus was a rectangular pulse of odour of at least 30 s duration. Every cell was stimulated at least 40 times with the same stimulus in order to investigate changes of the responses in the course of the stimulus repetitions.2.The most common response to a single stimulus pulse was an initial phasic activity increase or decrease (about 3–5 s) followed by a fairly constant mean activity maintained during the pulse.3.The activity during the stimulus-free period (at least 30 s) between two stimulus periods usually began with a phasic reaction (about 3–5 s) followed by an interval of fairly constant activity.4.In 37 of 51 cases the responses to single stimuli were reproducible over all runs of the experiment.5.In 14 recordings the single run responses were not reproducible. They rather showed (a) abrupt changes from the first to the second run or (b) slow changes that extended over up to 30 runs. Every pattern of this kind became reproducible after a certain number of runs. The activity changes during the first runs can therefore be attributed to a process that leads to a more pronounced response to a repeated stimulus.

Geruchsdifferenzierungsvermögen der Karausche (Carassius carassius) nach funktioneller und histologischer Regeneration des Tractus olfactorius und der Commissura anterior

Summary1.After dissection of olfactory pathways (Fig. 1) (tractus olf., commissura ant.) in the teleost fish Carassius carassius, functional regeneration takes place within seven weeks.2.Following functional regeneration successful training of discrimination between a conditioning and a competing odour stimulus can he achieved (Figs. 3–7).3.Histological controls have proved that at the time of functional regeneration new olfactory tract fibres have grown connecting the olfactory bulb to the forebrain (Figs. 8, 9).Zusammenfassung1.Karauschen können die durchschnittenen Bahnen der Tractus olfactorii und der Commissura anterior innerhalb etwa 7 Wochen funktioneill regenerieren (Wiederkehr der Riechfähigkeit).2.Nach Abschluß der funktionellen Regeneration lassen sich die Tiere auf einen bedingten geruchlichen Reiz gegen einen konkurrierenden Geruchsstoff dressieren.3.Zum Zeitpunkt der Wiederherstellung der Riechfähigkeit (funktionelle Regeneration) lassen sich bei Kontrollfischen mit durchtrenntem Tractus olf. Fasern vom Bulbus olf. bis zum Vorderhirn verfolgen.

Specific Regeneration of Peripheral and Central Olfactory Pathways in Goldfish

In a first series, ten groups of two goldfish were trained to discriminate Arg 10−6 M (rewarded stimulus) from Gln 10−6M (not rewarded). After successful discrimination training, significant positive responses were recorded when the rewarded stimulus was applied in 80-fold lower concentration (Arg 5 × 10−8 M vs Gln 4 × 10−6 M) and during contamination of the rewarded stimulus up to 50% with the unrewarded stimulus (50% Arg 10−6 M + 50% Gln 10−6 M vs Gln 10−6 M). Thereafter, in five groups of fish, the olfactory nerves and in five groups, the lateral olfactory tracts were intracranially and bilaterally dissected. Immediately after this operation, both collectives were unable to discriminate concentration differences and contaminated stimuli. Two weeks after receptor axotomy and lateral tractotomy, following functional regeneration, both collectives again were able to discriminate concentration differences and contaminations, as they did before operation. The regeneration of the olfactory nerves (peripheral regeneration) and the lateral olfactory subtracts (central regeneration) therefore is highly specific, even if the discriminative task is at the threshold of the discriminative ability in intact goldfish [1]. Following functional regeneration, intrabulbar and telencephalic information processing results in a complete return of the preoperative discriminative ability.