Bidirectional movements of mitochondria along axons of an isolated nerve segment

Summary The sciatic nerve of the rat was constricted at two sites about 10 mm apart. Three and seventeen hours later, a part of the nerve including the intermediate segment between the two crushed regions was dissected out and studied electron microscopically. Mitochondria and other organelles accumulated at the ends of interrupted fibres both proximally and distally of either crushed region. In the nerve segment between the two constrictions mitochondria collected at the fibre ends, whereas their number became significantly reduced in axons of the middle part of the segment. It is inferred that this translocation of mitochondria is brought about by a bidirectional shift of particles towards the fibre ends at which they become deposited.Skillful technical assistance of Mrs. M. Sobotková and Mr. M. Doubek is gratefully acknowledged.     

Fluorescence studies on neural structures and endocrine cells in the pancreas of the cat

Summary With the use of the Falck-Hillarp histochemical technique for the detection of monoamines, nerve fibre fluorescence is observed throughout the tail of the pancreas of the cat and the arrangement and distribution of the nerve fibres can be studied in both the exocrine and endocrine tissue. In the exocrine pancreas, adrenergic nerve fibres innervate arterioles, larger veins and major pancreatic ducts. Adrenergic nerve fibres also appear to terminate on the non-adrenergic nerve cell bodies of the intrapancreatic ganglia. In the islets of Langerhans, adrenergic nerve fibres innervate both the endocrine cells and blood vessels. Some of the islet cells exhibit fluorescence with the Falck-Hillarp technique and these cells have been identified as alpha cells. In animals treated with reserpine, the fluorescence in nerve fibres and in alpha cells is absent.The author wishes to thank ProfessorG. C. Schofield and Dr.G. C. Smith for their encouragement and valuable criticism during the course of this study. The assistance of MissJ. Bennett and MissW. Kemp and the photographic help of Mr.J. S. Simmons, F.R.P.S., are gratefully acknowledged. The diagram was drawn by MissS. Flett.     

Ultrastructural transformation of fast chicken muscle fibres induced by nerve cross-union

Summary The fast posterior latissimus dorsi (PLD) muscle of newly hatched chickens was transposed and cross-innervated by the slow-type nerve originally innervating the anterior latissimus dorsi (ALD) muscle. The innervation and the ultrastructure of the cross-innervated posterior latissimus dorsi (PLD-X) muscle was investigated from one week up to 18 months after the operation and compared with that of the control fast (PLD-C) and control slow (ALD-C) muscles. All nerve terminals in the PLD-X muscle were of the slow type. Yet the degree of ultrastructural transformation differed from fibre to fibre. Only about 30% of PLD-X fibres had transformed ultrastructure closely resembling the control slow fibres. In this group of maximally altered fibres, the myofibrils had large diameters, wide Z lines and indistinct M lines as the control slow fibres. The amount of mitochondria was increased to levels found in control slow fibres. The mean percentage of triads was also comparable to that of control slow fibres, being approximately by two thirds lower than in control fast fibres.The differences in the degree of ultrastructural transformation are presumably due to different plasticity of muscle cells at the time of cross-innervation. In the transposed PLD-X muscles large areas undergo degeneration and regeneration. It is suggested that an almost complete changeover of the fibre type is only brought about after cross-innervation of newly differentiating muscle cells, whereas partial alteration occurs after reinnervation of young myofibres.The skillful technical assistance of Dr. Z. Liková, Mrs. M. Sobotková, Ing. M. Doubek and Mr. H. Kunz is gratefully acknowledged.     

The sensory papilla X-organ in Boreomysis arctica (Krøyer) (Crustacea Malacostraca Mysidacea)

Summary The SPX-organ in Boreomysis arctica (Krøyer) (Crustacea Malacostraca Mysidacea) was investigated light and electron microscopically. The organ consists of a group of cells (the SPX-cells) and a vesicle surrounded by a connective tissue sheath. It is situated near the base of the sensory papilla of the eye-stalk. Neurosecretory material is produced in the SPX-cells and transported in axon-like projections from these cells into the vesicle. These processes contain no neurotubuli. Numerous fibres from an afferent nerve emanating from the medulla terminalis also enter the vesicle, where they form a dense irregular meshwork. This nerve transports no neurosecretory material. There are numerous synaptic contacts between the afferent nerve fibres and the neurosecretory processes from the SPX-cells. The neurosecretory material released from them accumulates in haemocoelic spaces in the vesicle. Release is most probably effected by the afferent nerve. Acknowledgements. We are indebted to Prof. H. Brattström and the staff of Biologisk Stasjon, Espegrend for working facilities and material. Mrs B. Morawetz and Mrs L. Eriksson gave us skilled technical assistance. The investigation was made possible by grants from the Swedish Natural Science Research Council.     

Some aspects of the fine structure of the statocysts of the molluscsPecten andPterotrachea

Summary The statocyst ofPecten is composed of hair cells and supporting cells. The hair cells bear kinocilia and microvilli at their distal ends and the supporting cells bear microvilli. The cilia have a 9+2 internal filament content, and arise from basal bodies that have roots, basal feet and microtubular connections. Two different ciliary arrangements are described, one with a small number of cilia arranged in a ring, and another with many more cilia arranged in rows. Below the hair cells are probable synapses. A ciliated duct connects to the lumen of the static sac and passes through the centre of the static nerve. The hair cells in the statocyst ofPterotrachea bear kinocilia and microvilli. The possible importance of cilia and microvilli in the transduction process is discussed.We would like to thank ProfessorJ. Z. Young for bringing specimens ofPterotrachea from Naples and also the staff of the Stazione Zoologica for the provision of specimens, Dr.M. Land for providing specimens ofPecten, the Science Research Council (U.K.) for providing the electron microscope used in much of the study and also for a grant to one of us (V.C.B.), and Mrs.J. Parkers and Mr.R. Moss and Mrs.J. Hamilton for much photographic and technical assistance.     

Early postmortem changes in the Organ of Corti (guinea pig)

Summary Early post mortem changes in the Organ of Corti are described. 15 minutes after death, when kept at room temperature, 20° C (63° F), an oedematous swelling is observed within the cytoplasm of hair cells and nerve endings, the latter being more severe affected. After 30 minutes post mortem the mitochondria of the hair cells have also become significantly swollen. Three hours post mortem the general character of the hair cells is still recognizable, but most of the nerve endings have been completely destroyed. Acknowledgement. We wish to express our appreciation of the skilful technical assistance of Mrs. B. Flock, Miss A.-M. Lundberg, Miss Sonja Löfvenius, Mr. G. Bornholm and Mr. Rune Ragnefjell.This work was supported by grants from the Swedish Medical Research Council, the National Institute of Health grant (no NB 03956-02), the Therese and Johan Anderssons minne, the Gustav and Tyra Svenssons fund and the King Gustav V Research Fund.     

Fluoreszenzmikroskopische und elektronenmikroskopische Untersuchungen an regenerierenden adrenergischen Nervenfasern

Zusammenfassung Die Regeneration peripherer adrenergischer Nervenfasern wurde fluoreszenzmikroskopisch nach einer von Falck u. Mitarb. angegebenen Methode, ferner elektronenmikroskopisch untersucht. Schon 24 Std nach einer Durchschneidung des N. ischiadicus (Kaninchen) schwellen die Axone unmittelbar am Schnittrand des zentralen Stumpfes an. Diese Anschwellungen weisen einen großen Gehalt an Catecholaminen auf. Die primären Axonanschwellungen fallen der initialen Degeneration anheim. Die nachfolgende Axonaussprossung beginnt mit der Bildung von Wachstumsendkolben, die sich von der Trümmerzone des zentralen Stumpfes aus in die Nervennarbe vorschieben. Die Wachstumsendkolben adrenergischer Fasern sind elektronenmikroskopisch durch das vesicular erweiterte neuroendoplasmatische Reticulum und durch eine große Zahl von Catecholaminkörpern gekennzeichnet. Es wird angenommen, daß die Catecholaminkörper der adrenergischen Regenerate überwiegend Noradrenalin enthalten, das im Bereich der Wachstumsendkolben vermehrt an das Grundplasma und das Interstitium abgegeben wird. Die Feinstruktur der adrenergischen Wachstumsendkolben hat große Ähnlichkeit mit normalen adrenergischen Nervenfaserendigungen.

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Summary The regeneration of peripheral adrenergic nerve fibres is investigated with the method described by Falck et al. and by means of electron microscopy. Within 24 hours after the severance of the N. ischiadicus (rabbit) a swelling of the axons, which are situated in the immediate vicinity of the sectioned area in the central stump, is observed. These swellings have a high content of catechol amines. These primary axon swellings undergo degeneration. The subsequent proliferation of axons starts with a formation of end-bulbs, which grow from the debris-zone of the central stump into the scar of the nerve. The end bulbs of adrenergic fibres are electron microscopically distinguishable through their neuroendoplasmic reticulum expanded to form vesicles and a large number of catecholamine-bodies. It is suggested that the cate-cholamine-bodies in the adrenergic regeneration tissue contain predominantly noradrenaline, which in the region of the end-bulbs is secreted predominantly into the interstitial ground substance and the interstitium. The fine structure of the adrenergic end-bulbs has a similar appearance to that of normal adrenergic nerve fibre endings.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft     

Localization of melanin synthesis within the pigment cell: Determination by a combination of electron microscopic autoradiography and topographic planimetry

Summary Localization of melanin synthesis within the pigment cells of the Cloudman S-91 mouse melanoma was determined by means of a combination of high resolution autoradiography and topographic planimetry. Initial melanin biosynthesis occurred predominantly in the endoplasmic reticulum and associated ribonucleoprotein particles of the melanocytes. By measuring a number of cell organelles and employing the index of relative specific localization it could be shown that the nucleus and mitochondria are of little or no importance in the process of melanogenesis.This investigation has been supported by the following research grants: CA 06548 CB, NIH, PHS and an Institutional Grant from the American Cancer Society (to H. M. H.); CA-05887, NIH, PHS (to A. S. Z.); M-00388 and NB-00782, NIH, PHS (to J. F. H.). One of us (H. M. H.) holds a Research Cancer Development Award (5-K 3-GM-2634) of the National Institute of General Medical Sciences, Public Health Service.We are grateful to Mr. Ronald Abler for his help with the topographic measurements; to Dr. Erhard Haus for help and advice; to Mr. J. Thornby and Mr. A. P. Basu for assistance with the statistical aspects of this study; and to Mrs. Lenore Mottaz, Miss Bernice Uittenbogaard, and Mrs. Judith Strong for careful technical assistance.     

Remarkable adrenergic nerves in the exocrine pancreas

Summary The adrenergic nerves in the pancreas of mice, rats, guinea-pigs, rabbits, and cats were investigated with the fluorescence method of Falck and Hillarp. The relations between the adrenergic fibres and the vessels were studied by the injection of india ink into the vessels.Besides the normal manifestation of adrenergic fibres at the large vessels, some vessels of capillary size were also accompanied by adrenergic fibres. These fibres had a very weak fluorescence, and showed up regularly only when the animal had been treated with Nialamide and L-DOPA or dopamine to increase the catecholamine content of the adrenergic fibres. The weakness of the fluorescence is perhaps due to low transmitter concentration or to small size of the nerve fibres, or to both. A rough estimate indicated that either the transmitter concentration of the nerve fibre is at least approximately 100 times below that seen in adrenergic nerves in other tissues, or that the radius of the varicosities of the nerve fibres is less than 0.2 . Neither alternative has previously been recognized.The secretory acini of the pancreas seem to lack a direct adrenergic supply. In the intrapancreatic ganglia, non-fluorescent nerve cells were reached by adrenergic terminals. No adrenergic nerve cells were detected in the pancreas of rats and cats. Small intensely fluorescent catecholamine-containing cells were observed in connexion with the intrapancreatic ganglia of rats.The research reported in this document has been sponsored by the Air Force Office of Scientific Research under grant AF EOAR 67-15 through the European Office of Aerospace Research (OAR), United States Air Force, by the United States Public Health Service (grant NB 06701-01) by the Swedish Medical Research Council (project B 67-12X-712-02A), and by the Faculty of Medicine, University of Lund, Sweden.     

A method for the demonstration of adrenergic nerve fibres in peripheral nerves

Summary The adrenergic nerve fibres running from the ganglia to the innervated tissues usually have too low a content of noradrenaline to be clearly visualized with the histochemical fluorescence method of Falck and Hillarp. They can easily be demonstrated, however, as early as 24 hours after axotomy (crushing or constriction of the nerves) due to the rapid accumulation of what is probably noradrenaline taking place proximally to the lesion. The fibres can be visualized even more clearly if axotomy is combined with the administration of l-dopa and with monoamine oxidase inhibition. In this way the presence, distribution and direction of adrenergic fibres can be directly studied in peripheral nerves.For generous supplies of drugs we are indepted to Swedish Ciba, Stockholm (reserpine) and Swedish Pfizer, Stockholm (nialamide). The investigation has been supported by research grants from the United States Public Health Service (NB 02854-04), the Swedish Medical Research Council, and Knut and Alice Wallenbergs Foundation.     

The nerve fibres in the basement membrane and related structures in Saccoglossus horsti (Enteropneusta)

Summary The structure of the basement membrane of Saccoglossus horsti has been examined with the electron microscope. The membrane consists of two lamellae each of two layers. An outer amorphous layer 150 nm across and an inner fibrillar layer 1–3 m across. The fibrils of the fibrillar layer are two sizes, the majority are 5–9 nm in diameter and at least 2 m long. The thicker 30 nm fibrils occur in small patches and have striations with a 30 nm period.Within the lamellae of the basement membrane are blood spaces. The only regularly found structures in these spaces are blood particles some 12–16 nm in diameter.Nerve fibres of varying diameters traverse all the layers of basement membrane. These fibres run longitudinally and obliquely through the basement membrane, and emerge amongst the muscle cells inserted into the coelomic side of the membrane. No motor end plates have been seen. Preliminary observations suggest that many of the nerve fibres have no sheath other than the cell membrane of the fibre itself.The muscle cells are attached to the basement membrane by structures that resemble hemidesmosomes. The blood vessels of Saccoglossus have a basement membrane on the lumenal side of the endothelial cell cytoplasm.I wish to thank Professor J. Z. Young, F. R. S. for continuous encouragement and advice. To Dr. R. Newell I am indebted for the collection and identification of the specimens. I am pleased to acknowledge my debt to Dr. R. Bellairs for the use of electron microscope facilities, and to Mrs. J. Hamilton and Mr. R. Moss for skillful technical assistance.     

Autoradiographic study of RNA synthesis in isolated cells in culture from chick embryo spinal ganglia

Summary Dissociated cells from 9, 12 and 15 day-old chick embryo spinal ganglia were cultivated in presence of total embryo-extract, brain embryo extract, or total embryo extract supplemented with purified nerve growth factor (NGF). The cells were maintained during 4 days in Maximow assembly and during 1 month in Rose chamber. Neurons showed growth of nerve fibres. The non-neural cells evolved to spindle cells, Schwann cells, or fibroblasts.Ribonucleic acid (RNA) synthesis was followed with tritiated uridine by autoradiography. Some nerve cells showed tritiated uridine incorporation. The highest incorporations for short-term cultures were at 15 hours in presence of NGF, at 48 hours in presence of total or brain extract, and for long-term cultures at 8 days. These periods corresponded to the highest growing activity of the nerve fibres. After 4 days all the non-neural cells incorporated tritiated uridine.The tritiated uridine was first incorporated into the RNA of the nucleus and, afterwards was found also in the cytoplasm. The presence of brain extract or of NGF stimulates the incorporation of labelled uridine into RNA. No labelling was found in the nerve fibres, even after 4 hours incubation.Chargée de Recherche au C.N.R.S.This communication is a part of the Doctorat és-Sciences thesis, presented by Mrs. J. Treska-Ciesielski.With the technical assistance of Mrs. M. F. Knoetgen and A. Bieth.     

Regeneration of motor axons in crayfish limbs: Distal stump activation followed by synaptic reformation

Summary Servered distal stumps of limb motor axons in the crayfish Procambarus clarkii remain ultrastructurally intact for at least 2–3 ms after being severed from their cell body. Initial regeneration of a motor axon is associated with the appearance of up to 200 small profiles (satellite axons) having no glial sheath adjacent to the large surviving stump for about 1 cm distal to the lesion at 4–5 wks postoperatively. These satellite axons are seen 2–4 cm distally at the target muscles 3–4 ms postoperatively. By 14–15 ms postoperative, the motor sheaths from the lesion site to the target muscles contain small axonal processes having thick glial sheaths. Behavioral tests show that some axons that are reconnected to the CNS at 4–5 wks may not be connected at 14–15 ms, whereas other axons not connected by 3–4 ms may be connected at 14–15 ms when the original distal stumps have degenerated.We suggest that all these data can best be explained by the view that motor axons in crayfish limbs initially regenerate via activation of the surviving distal stump by satellite axons which grow out from proximal stump. In most cases, these satellite axons continue to activate the surviving distal stump as they slowly grow to the target muscle. Eventually the satellite axons reform synapses on the target muscle and the original distal stump degenerates.This work was supported by NSF grants BNS 77-27678 and 80-22248 and an NIH RCDA 00070 to GDB. The authors would like to thank Mr. Martis Ballinger, Mr. Robert Reiss, and Mrs. Mary Raymond for their excellent technical assistance. We would also like to thank Dr. Wesley Thompson and Mr. Douglas Baxter for helpful discussions.     

Differentiation of the membrane system in the cells of imaginai discs

Summary Imaginal discs from developing larvae of the fly Calliphora erythrocephala fixed in permanganate or osmium tetroxide and embedded in Epon 812 were observed by electron microscopy. When the larval growth ceases, the differentiation manifests itself through an enlargement of the endoplasmic reticulum, by which continuous membrane contact is established between all cell organelles. During the same time mitochondria swell up and transform into lipid granules and the intercellular contacts weaken.I am indebted to Mrs Mariann Carleson and Miss Brita Nilsson for technical aid.     

Further studies on the neurohypophysis of the hedgehog (Erinaceus europaeus)

Summary Further studies of the neurohypophysis of the hedgehog have confirmed the presence of two distinct types of neurosecretory fibre in the infundibular process. A third type of fibre, possibly containing catecholamines, is found in the upper infundibular stem.Lesions of the paraventricular nuclei result in vacuolation of type I nerve fibres, which contain crystal-like and aggregate bodies. Type II fibres, containing membranous inclusions, with or without osmiophilic material, apparently do not degenerate. The evidence suggests that type I fibres arise in both the paraventricular and supraoptic nuclei.Dedicated to Professor Berta Scharrer on the occasion of her 60th birthday.     

The structure of the nerve fibre layer and neurocord in the enteropneusts

Summary The nerve fibre layer and the neurocord of the Enteropneusts Saccoglossus horsti, Harrimania kupfferi and Ptychodera flava have been examined with the electron microscope. The nerve fibres vary in diameter between 0.15 to 10 m. The majority of the fibres are of the smaller diameters. The nerve fibre layer is intraepidermal, and is divided by processes running radially from the epithelial cells to the basement membrane that separates the nerve fibre layer from the muscle cells.The cells of origin of these nerve fibres are situated mainly in the innermost layers of the epidermal cells. The nerve fibre profiles contain numerous vesicles of very varied diameter and contents, together with larger granular inclusions that are also found in the nerve cell bodies.Morphologically recognisable synapses are rare, but the majority of fibres are in intimate contact with one another. Sometimes the mass of fibres is divided into bundles by the epithelial cell processes. The majority of giant fibres are situated near to the basement membrane of the neurocord. The giant fibres also have a varied content of vesicles as well as neurofilaments and neurotubules.The central canal in Ptychodera flava and the remnants of the central canal in Saccoglossus horsti are both lined by columnar cells that bear microvilli as well as cilia with the typical 9 + 2 pattern of tubules. Scattered amongst these cells are mucus secreting cells which open into the cavity of the canal.I (P.N.D.) should like to thank Professor J. Z. Young, F. B. S. for much advice and encouragement. Dr. R. Bellairs generously provided the electron microscope facilities, and Dr. R. Newell kindly collected and identified the Saccoglossus specimens. Mr. R. Moss, Mrs. J. Hamilton and Mr. A. Aldrich gave excellent technical and photographic assistance.     

Adrenergic and cholinesterase-containing neurons of the heart

Summary The adrenergic and acetylcholinesterase-containing nerves of the hearts of mice, rats, guinea-pigs, rabbits, and cats were studied. The fluorescence technique of Falck and Hillarp was used for the demonstration of adrenergic nerves, whereas a modified Koelle cholinesterase technique was used for the cholinesterase-containing nerves. The inhibitors used were Mipafox, iso-OMPA and Nu 683. Microspectrofluorometry was used to identify the structures containing dopamine.Adrenergic as well as acetylcholinesterase-containing fibres were found in all parts of the heart, most abundantly in the atria. Dense nerve plexa supplied the sinoarial and atrioventricular nodes. There was a plexus of both fibre types in the endocardium and on the atrial side of the valves. In the valves, it could be shown that adrenergic and cholinesterase-containing fibres ran closely parallel to each other. Indirect evidence suggested that this applies also to the myocardium.No nerve fibres containing dopamine were revealed in the microspectrofluorometer. The dopamine previously found in the atria seems, instead, to be situated in so-called small intensely fluorescent cells.No adrenergic ganglion cells were found in the heart despite extensive search. The vagus of rabbits was found to contain only few adrenergic preterminals.     

Observations on the fate of muscle fibres temporarily isolated by transection of a muscle belly

Summary Transection of the sternohyoid muscle of the rat has been used as an experimental situation in which the fate of the portions of fibre isolated from their nerve supply by the lesion can be studied. Sections from the muscle were stained to demonstrate oxidative and glycolytic enzymes and cholinesterase. Biopsies performed after periods of up to ten weeks after operation revealed a series of changes that suggested that after passing through the early stages of denervation atrophy recovery of the fibres took place. There was no indication that new motor end-plates were formed among the isolated fibres and it was concluded that communication had been reestablished with innervated fibres and that this reunion had been followed by a redetermination of the metabolic activity of the isolated fibres.I would like to thank Professor J. Z. Young for his advice and encouragement and Mrs. C. A. Joseph for her histochemical preparations.     

The fine structure of the eye of the mollusc Pecten maximus

Summary The retina of Pecten maximus is divided into two light sensitive layers forming the distal and proximal retinae. The cells from these layers have different electrophysiological responses, the distal cells giving primary off responses, and the proximal cells giving on responses. The receptor surfaces of the distal retinal cells are formed from lamellae produced by the outer membranes of flattened cilia. These cilia have a basal body, basal foot, no root system and a 9 + 0 internal filament content. Each cell gives rise to an axon from its distal side, and this process goes up to the basement membrane, which is present below the cellular lens, passes along beneath it, and joins the distal optic nerve. The receptor part of the proximal retinal cells is formed from a vast array of microvilli. Each of these cells also bears one or two cilia with a probable 9 + 0 internal filament complement and no roots. The proximal cells give rise to axons, forming the proximal optic nerve. Below the proximal retina is a reflecting layer, the argentea, and below this is a pigment cell layer.We would like to acknowledge the advice and encouragement of Professor A. F. Huxley, Professor J. Z. Young and Dr. E. G. Gray. — We would like to thank Mrs. J. I. Astafiev for drawing Fig. 1, Mr. S. Waterman for photographic help and Miss C. Martin for clerical assistance.     

Stretch receptors in urodele limb muscles

Summary Non-encapsulated, fine beaded nerve endings were found histologically on some muscle fibres in a number of limb muscles in newts and axolotls. They were present in newt muscles that had been chronically de-efferented, and in which no efferent activity survived, and were therefore likely to be sensory. They were located only on muscle fibres on or near the outside surface of the muscle. These small-diameter muscle fibres were characterised histochemically by low lipid, SDH and phosphorylase content; ultrastructurally by low glycogen content, and relatively large myofilaments poorly delimited by a sparse SR. There were many of this type (Type 1) that did not support sensory endings. A few endings occurred on another larger-diameter type of fibre (Type 2) whose properties were opposite to those listed above for Type 1. There was virtually no specialization of muscle fibre structure beneath the sensory endings.Physiological experiments involving ramp-and-hold and sinusoidal stretch applied to the muscle whilst recording single-unit afferent responses in m.ext. dig. III of axolotls showed unit responses very similar to those known from muscle spindles, particularly those of the frog.We are grateful to the M.R.C. for an equipment grant (to R.M.A.P.R.), and to Mrs. Janis Taberner for her technical help. Part of this work was done during the tenure of a Nuffield-NRC Lectureship (Q.B.) which is gratefully acknowledged, as is financial support by Prof. J.R. Nussall during a visit to the University of Alberta at Edmonton.