Nebulised morphine: a new delivery method for pain relief.
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Biomedical subjects
M R Bennett
Publications and source records attributed to M R Bennett.
Motor neuron survival and neuritic extension from spinal cord explants induced by factors released from denervated muscle.
Extracts prepared from denervated adult skeletal muscle contain increased amounts of neurotrophic activity which promotes both survival of dissociated motor neurons and the outgrowth of neurites from explants of spinal cord maintained in serum-free defined media. The trophic activity is specific for motor neurons and reaches a peak within the first week post-denervation. In these most potent extracts the neurite outgrowth enhancement is a linearly increasing function of protein concentration at low concentrations; at higher concentrations the neurite activity-concentration relationship saturates and in the milligram range the relationship becomes inhibitory. When media containing active denervated muscle extract was preincubated over polycationic substrata, it lost the ability to promote neuritic growth; this could be restored if fresh extract was added to the cultures. Thus it was demonstrated that within the denervated muscle extract there are physically separable agents responsible for neuron survival and neurite expression. It is possible that the release of neurotrophic factors may be in part responsible for the in vivo phenomenon of nerve sprouting.
Animals↗
Effects of cultured astroglia on the survival of neonatal rat retinal ganglion cells in vitro.
Retinal ganglion cells (RGC), as identified by retrograde horseradish peroxidase (HRP) labeling technique, were cultured in a minimal medium; only 20% of them survived after 16 hr in vitro. However, superior colliculus-conditioned medium was capable of supporting 100% of RGC over this assay time; enhanced neurite expression also was evident. It was decided to investigate whether glial cells within the superior colliculus may provide a soluble factor capable of supporting RGC. Glial-conditioned medium prepared over monolayers of either predominantly flat astrocytes (relatively immature) or predominantly process-bearing (mature) astrocytes failed to maintain RGC. The possibility that astrocytes may provide support for RGC via membrane contact was then investigated. Dissociated retinae were grown on monolayers consisting primarily of either flat or process-bearing astrocytes. Cultures rich in flat astrocytes maintained over 70% of RGC originally present, and many of them exhibited extensive neurite outgrowth and elongation. Process-bearing astrocytes were unable to support RGC survival. Immature astroglial cells may therefore support RGC via glial-neuronal interaction.
Animals↗
Differentiation of fiber types in wing muscles during embryonic development: effect of neural tube removal.
The embryonic precursors of the avian slow (type I and III) and fast (type II) fibers can be distinguished from each other early in muscle formation (stage 28, V. Hamburger and H. L. Hamilton, J. Morphol, 88, 49-92, 1951) on the basis of the differential sensitivity of their myosin ATPases. To test the neural dependence of fiber type differentiation, the source of motor innervation was eliminated by excision of the brachial neural tube at stages 16-18 before muscles are innervated. Removal of the brachial neural tube did not affect the number of primary myotubes in a sample muscle of the forelimb (ulnimetacarpalis dorsalis, UMD) up until stage 36. Myosin ATPase staining at a variety of pHs revealed the typical patterns of fiber types in muscles of neural-tube free embryos in stages 35-37. These muscles included the anterior latissimus dorsi, brachialis, and UMD which showed presumptive type III staining (type IIIEMB), the pronator superficialis and flexor carpi ulnaris which showed embryonic type II staining (type IIEMB), and the triceps brachii muscles which showed characteristic arrangements of both type IEMB and type IIEMB fibers. The normal patterns of type IEMB and type IIEMB myotubes were also seen in muscles containing a heterogeneous mixture of fiber types such as the biceps brachii, extensor metacarpi radialis, and adductor indicis muscles, although the intensity of acid-stable ATPase staining of the type IEMB myotubes in these muscles was lower than in innervated muscles. It is concluded that the earliest differentiation of muscle fiber types is independent of the nervous system.
Adenosine Triphosphatases↗
Segmental motor projections to rat muscles during the loss of polyneuronal innervation.
The pattern of innervation of three muscles from rat (lateral gastrocnemius, biceps brachii, pectoralis minor) has been ascertained in the neonatal period (birth to 4 days) and from two to three weeks postnatal. Contraction, electrophysiological and histological methods were used to determine the pattern of innervation. There is a change in the segmental innervation during development which establishes the topographical projection of different segmental nerves found in mature muscles. Development of such topographical projections by different segmental nerves onto the muscles studied emerges as a consequence of elimination of terminals arising from the inappropriate segmental nerve.
Animals↗
Development of the topographical projection of motor neurons to a rat muscle accompanies loss of polyneuronal innervation.
The rat lateral gastrocnemius muscle receives a topographical projection from lumbar segmental nerves L4 and L5. A study has been made of the development of this projection during the period when polyneuronal innervation is eliminated. The tetanic contraction due to stimulation of each nerve was compared with that due to stimulation of both nerves simultaneously. This percentage of contraction declined from about 90% to 70% for L4 from birth to 2 postnatal weeks; it declined from about 90% to 30% for L5 over the same period. The innervation of about 60% of the cells by both L4 and L5 is therefore eliminated during 2 postnatal weeks. Tetanic and twitch contraction due to stimulation of ventral rootlets of segmental level L4 and L5 was compared to stimulation of the whole muscle directly. There was a general reduction in the size of the motor units from both L4 and L5. A selective reduction in the number of L5 motor units was observed during the first 5 postnatal days. The distribution of L4 and L5 terminals on the dorsal surface of the muscle was determined by intracellular impalement of muscle cells and was determined by recording endplate potentials (EPPs) due to stimulation of L4 and L5. Polyneuronal innervation of the lateral gastrocnemius is eliminated by about 2 postnatal weeks. The percentage of muscle cells innervated by L4 or L5 in each of six equal-size muscle sectors was ascertained during this period. This percentage of innervation of muscle cells by L5 declined in all sectors between 3 days and 2 weeks postnatal.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The growth of neurites from explants of brachial spinal cord exposed to different components of wing bud mesenchyme.
The extension of peripheral axons from the brachial spinal cord into the embryonic chick wing bud suggests that the target premuscle cell masses may act as the source of an adhesion gradient which establishes selective nerve pathways. Wing premuscle cell masses were explanted from different stage embryos and tested against age-matched brachial spinal cord for their ability to promote directional neurite outgrowth. It was found that target premuscle which does not contain myotubes could still elicit directional outgrowth in vitro. In contrast, skin and precartilage were unable to promote neuritic outgrowth significantly. Serum-free conditioned media were prepared from stages 20-38 premuscle and tested against age-matched spinal cord explants. There was an increase in the effects of conditioned media on neuritic outgrowth up to stage 35; conditioned media from older-stage premuscles had less effect than that of stage 35 premuscles. These results were shown to be dependent on the maturation of the premuscles and not on that of the spinal cord. When the premuscle conditioned media were preincubated over polylysine substrata, the ability to induce neuritic outgrowth was abolished from media derived from premuscle at stage 27 and older. Conditioned media derived from premuscle at stage 27 or older contain a polylysine-binding neurite-promoting factor which is present in greater amounts in more differentiated muscle. The time of first detection of neurite-promoting factors in stage 27 premuscle conditioned media correlates with the in vivo stages at which muscle-specific nerves branch from the main nerve trunks.
Animals↗
Growth of segmental nerves to the developing rat diaphragm: absence of pioneer axons.
A study has been made of the growth of cervical nerves C3-C6 to the rat diaphragm. At 11 days of embryonic age these cervical nerves first project out of the spinal cord toward the cardinal veins and later form the left and right phrenic nerve trunks. During the next 2 days, the phrenic nerves grow caudally in close association with the cardinal veins toward the diaphragm. At the growing tips of these nerve trunks the growth cones of axons were observed every 1-2 micrometers. The last axon did not project more than 2 micrometers ahead of any neighbouring axons. At 14 days the phrenic nerves reach the level of the developing diaphragm and converge into pools of premuscle cells. Previous studies have suggested that the phrenic nerve enters the premuscle masses of the diaphragm at an early developmental stage when the premuscle masses are at approximately the segmental levels C3-C6. This study shows that the phrenic nerves must grow to more caudal levels in order to reach the premuscle cells of the diaphragm. Furthermore, the leading axons of the phrenic nerve trunk do not project in a pioneering fashion, far in advance of the trailing axons.
Animals↗
Death of motorneurons during the postnatal loss of polyneuronal innervation of rat muscles.
A study was made of the decline in the number of motor neurons and axons of the brachial spinal cord of the rat during postnatal development. The injection of horseradish peroxidase (HRP) into the biceps muscle showed that it was innervated by motor neurons located in the dorsolateral position of the lateral motor column in segments C5 and C6; HRP injections into the triceps muscle showed that it was innervated by motor neurons located in the ventrolateral position of the lateral motor column in segments C7 and C8. There was no change in the position of these motor neuron pools between birth and maturity. However, there was a decline in the number of neurons in each pool during the postnatal period; over 35% of the neurons present at birth had disappeared by maturity. This loss of neurons was uniform throughout the rostrocaudal extent of each pool. It was accompanied by a similar percentage loss in the number of axons in a ventral root at the branchial level (C8). Electrophysiological measurements showed that the disappearance of motor neurons was accompanied by a loss in the polyneuronal innervation of synaptic sites in the biceps muscle. The possibility that a decrease in the number of neurons contributes to the loss of polyneuronal innervation is discussed.
Animals↗
Neonatal retinal ganglion cell cultures of high purity: effect of superior colliculus on their survival.
A method is described for obtaining retinal ganglion cell (RGC) cultures of high purity. RGC were retrogradely labelled in vivo with either the fluorescent dye True Blue and horseradish peroxidase (HRP), or with FITC-conjugated HRP. Following dissociation, RGC were separated from the intrinsic cells of the retina using fluorescent activated cell sorters, on the basis of their greater size and fluorescent intensity. When the sorted cells were cultured, RGC could be subsequently identified by their HRP labelling. Using such criteria, cultures in which 75% of the cell population consisted of RGC could be regularly obtained. This represents a 150-fold increase in RGC concentration over whole retinal cultures. The survival of the sorted RGC could be greatly enhanced when they were cocultured with their target tissue, the superior colliculus.
Animals↗
Survival of purified motor neurones in vitro: effects of skeletal muscle-conditioned medium.
Spinal motor neurones in the adult mouse were labelled retrogradely with both True Blue and horseradish peroxidase (HRP). Using the cell sorter, motor neurones were separated on the basis of cell size and the intensity of True Blue fluorescence. Cultures of the sorted cells were then prepared and the motor neurones were identified by their HRP labelling. There was found to be a 4.8-fold increase in motor neurones over the unsorted population such that 40% of the cells present in culture was labelled. Medium conditioned over skeletal muscle was shown to enhance the survival of motor neurones in these cultures.
Animals↗
Evidence that the early postnatal reduction in the number of rat retinal ganglion cells is due to a wave of ganglion cell death.
Horseradish peroxidase (HRP) was injected into the retino-recipient nuclei of each hemisphere in newborn rats. The animals were perfused 3--9 days after the injections; the number of retinal ganglion cells in retinal wholemounts was estimated by counting cells containing granules of HRP reaction product. The mean number (150,500) of labelled cells in 3-day-old rats was significantly higher than those in older animals (117,000, 121,000, 113,000 respectively on 6th, 8th and 9th postnatal days). However, in animals of any of the ages studied, the estimated numbers of ganglion cells were virtually the same as those in the animals of the same age but injected with HRP only 15--20 h before the perfusion. Thus, the reduction in the number of retinal cells projecting to the central visual nuclei observed during the first few postnatal days is due to a wave of retinal ganglion cell death; ganglion cell death induced by the neonatal removal of the contralateral superior colliculus has a similar time course.
Animals↗
The growth of nerves in relation to the formation of premuscle cell masses in the developing chick forelimb.
A description is given of the growth of segmental nerves 13 to 16 (SN13 to SN16) from the brachial myotomes into the ventral and dorsal premuscle cell masses of the chick forelimb. At stage 25 (Hamburger and Hamilton, '51), segmental nerves have converged to form two nerve trunks which enter the ventral and dorsal premuscle cell masses. These nerves grow into the limb, parallel to its proximodistal axis. The nerve trunk in the ventral compartment, the brachialis longus inferior (bli n), grows on the brachial artery as far as the metacarpals. The nerve trunk in the dorsal compartment, the brachialis longus superior (bls n), grows on the precartilage adjacent to a dorsal premuscle density as far as the metacarpals. The brachialis longus inferior and superior nerve trunks give rise to a number of nerves during their growth to the metacarpals. Each of these nerves appears at the time of formation of a new premuscle cell density. At late stage 25, a posterior and ventral premuscle density forms in the middle of the stylopodium; posterior fascicles of the bli n diverge at this level and grow toward this new density to form the ulnar nerve. By stage 26 a posterior and ventral premuscle density is prominent over the bli n, at the junction of the stylopodium and the zeugopodium; ventral fascicles of the bli n grow into this density to form the flexor digitorum profundus nerve. At this stage a posterior and dorsal premuscle density forms just beyond the junction of the stylopodium and zeugopodium. Posterior fascicles of the bls n diverge at this level and grow toward this density to form a nerve from which the extensor metacarpi ulnaris, extensor digitorum communis, and radialis laterialis nerves are destined to form. At stage 29 the premuscle cell masses of the autopodium have formed. The remaining nerve fascicles comprising the bls n (the radialis profundus nerve) grow into the autopodium and branch to innervate each of the dorsal premuscles. The remaining nerve fascicles comprising the bli n (the middle nerve) grow into the autopodium and branch to innervate each of the ventral premuscles.
Animals↗
Cholinergic growth factor from skeletal muscle elevated following denervation.
The effect of skeletal muscle extracts upon cholinergic neuron in vitro survival was investigated. All muscle soluble protein extracts elicited survival of neurons above that of neurons alone in culture and were found to be active below a protein concentration of 1.0 mg/ml. However, skeletal muscle which had been previously denervated, was found to contain higher amounts of survival activity than innervated muscle extracts. This elevation of survival activity was greatest within the first 7 days post-denervation, but subsequently declined towards the innervated level.
Animals↗
Development of neuromuscular synapses.
Quantal secretion at nerve terminals in mature muscles depends on the number of terminal branches and the size of release sites (sect. VB4). The physical length of SBL determines the length of terminal branch that can be laid down in a reinnervation experiment (sect. IVA4). A limit is set on the total length of terminal branches formed by a motoneuron; this limit is determined by the amount of TF (sect. IVB) made available from the neuron soma to the peripheral branches of the neuron (sect. VC). As a result of this limit, not all SBL needs to be occupied at a site by terminal branches. The SBL eventually disappears if it is not occupied by terminal branches (sect. IVA2). If a muscle is relatively inactive, it synthesizes and releases at synaptic sites additional amounts of NGF, which stimulates the growth of additional terminal branches. These may secrete sufficient amounts of AF to induce the formation of new SRs with associated SBL. In these circumstances a new synaptic site is formed or an extension of an existing site is created. If the size of a motor unit is decreased, the enhanced release of TF at the remaining terminals ensures that each occupies all the SBL at the synaptic site. Furthermore the enhanced release of AF per terminal induces more SBL, allowing additional terminal branches on the muscle cells to be established. Neither of these changes occurs unless the threshold amount of NGF is available from the muscle to stabilize the terminals. If this condition is met, an increase in quantal release per terminal occurs after reducing the size of a motor unit (sect. VC). An increase in quantal release per terminal also occurs after inactivation of a muscle. Such inactivation leads to an enhanced release of NGF per synaptic site (sect. VA4). Extra terminals may then form if sufficient TF is available; these may innervate existing but empty synaptic sites. In rare circumstances the extra terminal may induce SBL and innervate these new sites if sufficient AF is available. In both cases the quantal release per terminal increases. During development the secretory capacity of the axon terminal depends on the muscle cells with which it synapses. This secretory capacity can be enhanced either by increasing the number of terminal branch pairs or by increasing the secretory capacity of individual release sites. If two terminals innervate a synaptic site, their individual secretory capacity is reduced--in these circumstances the terminal's secretory capacity depends on the amount of NGF available to the terminal; two terminals must share their NGF.
Acetylcholinesterase↗
Evidence for neuron-survival and neurite-promoting factors from skeletal muscle: their effects on embryonic spinal cord.
Explants of 8- and 9-day embryonic chick spinal cord were cultured for 4 days in medium which had been conditioned by skeletal muscle cells. When the conditioned medium was preincubated over polylysine substrata, it lost the ability to induce extensive neuritic outgrowth from the spinal cord. This ability was restored when the spinal cord was exposed to preincubated conditioned medium on preincubated polylysine substrata. Skeletal muscle thus appears to contain two separable components; one is a soluble factor which supports neuronal survival, and the other is an adsorbable factor which binds to appropriate substrata and promotes neuritic outgrowth.
Animals↗
The survival of neonatal rat retinal ganglion cells in vitro is enhanced in the presence of appropriate parts of the brain.
The enzyme horseradish peroxidase (HRP) was injected into the visual centres of the brains of neonatal rats. Following dissociation of retinae into tissue culture, the ganglion cells could be identified by appropriate histochemical staining for HRP reaction product. Cultures were prepared of dissociated retinae from rats aged 2-6 days postnatal. After 3 h the cultures were fixed, and HRP-labelled cells visualized and counted. Estimates were made of the number of ganglion cells per retina at each age. Results indicated a loss of ganglion cells during the first few postnatal days. This loss paralleled that observed in vivo. It was further found the retinal ganglion cells died rapidly in vitro when cultured in a minimal medium. Only 50% of ganglion cells originally plated remained viable after 24 h. However, the survival rate could be increased to 100% by co-culturing the cells with diencephalon and mesencephalon; these contain the retinorecipient nuclei. Co-culturing with cerebellum did not result in such an enhanced survival rate. Ganglion cells could be maintained over longer periods of time by reinoculating the cultures with additional tissue containing diencephalon and mesencephalon. These results support the hypothesis that developing neurons require trophic factors from their target tissues in order to survive.
Animals↗
The loss of ganglion cells in the developing retina of the rat.
Horseradish peroxidase (HRP) was injected into both sides of the brain of newborn and adult rats. The number of retinal ganglion cells was estimated by counting cells containing granules of HRP reaction product. The mean number of labelled cells in 2-day-old animals was 169,500 (S.D. +/- 16,000, n = 6). By the tenth postnatal day the mean number of labelled cells had fallen to 113,500 (+/- 2900, n = 3). This value is similar to the mean number of labelled cells in the adult animal (113,000 +/- 2700; n = 4). Thus, during the first few postnatal days the number of retinal cells projecting to the central visual nuclei is reduced by at least 35%.
Animals↗