Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts

Summary Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with ³H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later.In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The present experiments provide a direct proof of utilization of donor satellite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.     

A proposed neural pathway for vocalization in South African clawed frogs,Xenopus laevis

Vocalizations of South African clawed frogs are produced by contractions of laryngeal muscles innervated by motor neurons of the caudal medulla (within cranial nerve nucleus IX-X). We have traced afferents to laryngeal motor neurons in male and female frogs using retrograde axonal transport of horseradish peroxidase conjugated to wheat germ agglutinin (HRP-WGA). After iontophoretic injection of HRP-WGA into n. IX-X, retrogradely labelled neurons were seen in the contralateral n. IX-X, in rhombencephalic reticular nuclei, and in the pre-trigeminal nucleus of the dorsal tegmental area (DTAM) of both males and females.     

The organization of corticocortical projections from area 17 to area 18 of the cat's visual cortex

Retrogradely transported tracers were injected into area 18 of the visual cortex of the adult cat to study the organization of corticocortical projections from area 17 to area 18. All injections, whether very small or relatively large, and irrespective of their exact location in area 18, produced a discontinuous, clustered distribution of labelled cells, mainly in layers II, III and upper IV, in a topographically related region of area 17. The mean centre-centre distance between neighbouring patches was about 750 microns. We conclude that the overall population of cells projecting to area 18 is genuinely distributed in a patchy fashion and that they provide an efficient spatial sample of information from area 17. Comparison of the dimensions of each injection site and of the retrogradely labelled territory suggested that each region in area 18 receives a convergent input from a zone in area 17 whose visual field representation is about 0.8 M-1 deg larger in all directions (where M is the magnification factor in millimetres per degree at the termination site in area 18). Pairs of injection were made in area 18 by placing small volumes of two fluorescent tracers, fast blue and diamidino yellow, side-by-side in either a rostrocaudal or a mediolateral plane, with different distances between them. When the boundaries of the dense central cores of two injection sites were separated, at their closest points, by about 1.6 mm, the two corresponding distributions of labelled cells in area 17 were just non-overlapping, suggesting that each group of cells in area 17 sends a divergent projection in innervate a zone about 0.8 mm larger in all directions in area 18. More closely spaced injections led to overlap of the distributions of labelling by the two dyes, with shared clusters containing a mixture of labelled cells. The proportion of double-labelled cells in these shared clusters never exceeded 4.4% (but was 70% after sequential injection of the two dyes at a single point). We conclude that, although each cluster of cells sends a divergent projection to area 18, the majority of individual axons terminate more discretely, perhaps providing specific inter-connections between functionally corresponding 'columns' in the two areas.     

The sizes of motoneurons supplying hindlimb muscles in the mouse

Motoneurons supplying identified muscle groups in the mouse spinal cord were labelled by retrograde transport of horseradish peroxidase. The size of motoneurons was estimated by measuring perimeter and cross-sectional area at the level of the nucleolus for the following seven major muscle groups: quadriceps femoris, adductors and gracilis, gluteal musculature, hamstring muscles, posterior crural musculature, anterolateral crural musculature and intrinsic musculature of the foot. The qualitative observation of two size ranges of motoneuron was supported by the measurements. Frequency distribution histograms of motoneuronal cross sectional area were bimodal for all motoneuronal groups except for the foot musculature. The population parameters and proportions for the six bimodal histograms were estimated by the method of maximum likelihood. It was found that the mean area of the small neuron component, which were presumed to be gamma motoneurons, was similar for the six bimodal systems. In contrast to this the mean area of the large neuron component, presumed to be alpha motoneurons, was found to be different for the six bimodal systems; motoneurons supplying more proximal muscles showed a larger mean area than those supplying distal muscles. The mean area of both components was unaffected by survival time and this was interpreted as indicating that changes in survival time did not label greater numbers of small or large motoneurons. The proportion of motoneurons in the small neuron component was found to vary from 9 to 27%.     

Peripheral nerve lesion-induced uptake and transport of choleragenoid by capsaicin-sensitive c-fibre spinal ganglion neurons

In the present experiments the effect of systemic capsaicin treatment on the retrograde labelling of sensory ganglion cells was studied following the injection of choleratoxin B subunit-horseradish peroxidase conjugate (CTX-HRP) into intact and chronically transected peripheral nerves. In the control rats CTX-HRP injected into intact sciatic nerves labelled medium and large neurons with a mean cross-sectional area of 1,041 +/- 39 gm2. However, after injection of the conjugate into chronically transected sciatic nerves of the control rats, many small cells were also labelled, shifting the mean cross-sectional area of the labelled cells to 632 +/- 118 microm2. Capsaicin pretreatment per se induced a moderate but significant decrease in the mean cross-sectional area of the labelled neurons (879 +/- 79 microm2). More importantly, systemic pretreatment with capsaicin prevented the peripheral nerve lesion-induced labelling of small cells. Thus, the mean cross-sectional areas of labelled neurons relating to the intact and transected sciatic nerves, respectively, did not differ significantly. These findings provide direct evidence for a phenotypic switch of capsaicin-sensitive nociceptive neurons after peripheral nerve injury, and suggest that lesion-induced morphological changes in the spinal cord may be related to specific alterations in the chemistry of C-fibre afferent neurons rather than to a sprouting response of A-fibre afferents.     

Localization and morphometric analysis of masticatory muscle afferent neurons in the nucleus of the mesencephalic root of the trigeminal nerve in the cat

Injections of horseradish peroxidase (HRP) were made into the ipsilateral temporal muscle and contralateral masseter muscle of 10 cats in order to identify and characterize neurons in the nucleus of the mesencephalic root of the trigeminal nerve that innervate muscle receptors in the orofacial periphery. Neurons labelled by HRP injections and unlabelled cells from 5 control cats were measured with a computer-based image analyzer, and their position was mapped on a stereotaxic graph. Cells that innervate the masseter and temporal muscles were identified throughout the rostrocaudal extent of the nucleus. There was no indication of a somatotopic pattern nor of a specific segregation within the nucleus for cells innervating muscle receptors. The nucleus contained small, rounded unipolar neurons located primarily in the dorsal border of the periaqueductal gray (PAG) matter in the rostral part of the nucleus and larger oval unipolar neurons which were scattered throughout the nucleus, but were predominant in the pontine portion of the nucleus. HRP injections labelled both large and small cells, as well as occasional multipolar cells. The last-mentioned tended to be located in the lateral margins of the PAG. The mean geometric values obtained for the control group were: area 552.7 microns2 perimeter 110.3 microns; maximum diameter 36.0 microns. and diameter of an equivalent circle 26.1 microns. The mean values of the labelled neurons were: area 606.6 microns2; perimeter 100.1 microns; maximum diameter 36.0 microns, and diameter of an equivalent circle 27.2 microns.     

Features of the angioarchitectonics of the papillary muscles and the chordae tendineae of the human heart

In persons of mature, elderly and old age vascularization of the papillary muscles and tendinous chordae in the right and left cardiac ventricles has been studied depending on peculiarities of their structure and sources of their blood supply using injection, macro-microscopical, roentgenoangiographic methods, and silver nitrate impregnation. Certain data concerning the distribution zones in the papillary muscles of the coronary artery branches at various types of the cardiac blood supply are presented. Angioarchitectonics of the papillary muscles are described along their whole extention--in the basal area, in the middle part and at the apex. Architectonic peculiarities of the blood vessels in the muscular-tendinous part of the papillary muscles and in some tendinous chordae are described.     

An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats

Summary Satellite cells were traced autoradiographically during the regeneration of skeletal muscle in young Sprague-Dawley rats. Approximately 31% of the satellite cells in uninjured muscles appeared labelled after three injections of tritiated thymidine; none of the myonuclei were labelled in the same muscles. Four to six days after transplanting the radioactive muscles to non-radioactive littermates, regenerating myotube nuclei in the host appeared labelled. Thus, this study confirms that satellite cells in young rats can differentiate into multinucleated myotubes following muscle injury.Supported by NIH grant No. 5 S01-RR05356-13I wish to acknowledge the excellent technical assistance of Ms. Amy Erisman     

Organization of the cortico-ponto-cerebellar pathway to the dorsal paraflocculus. An experimental study with anterograde and retrograde transport of WGA-HRP in the cat.

To reveal the organization and relative magnitude of connections from various parts of the cerebral cortex to the dorsal paraflocculus via the pontine nuclei, WGA-HRP was injected in the dorsal paraflocculus in conjunction with injection of the same tracer in various parts of the cerebral cortex in 17 cats. Termination areas of cortical fibres (anterogradely labelled) and pontine neurons projecting to the dorsal paraflocculus (retrogradely labelled) were carefully plotted in serial transverse sections. As an average of countings in ten cats, 90% of the labelled cells were found in the pontine nuclei contralateral to the injection, and the majority (70%) were located in the rostral half of the nuclei. The highest degree of overlap between anterograde and retrograde labelling was found after injections of the parietal association cortex (areas 5 and 7). In an experiment with double anterograde tracing, it was shown that both area 5 and 7 contribute substantially to the cerebral inputs to the dorsal paraflocculus. High degree of overlap also occurred after injections of several visual cortical areas (areas 17, 18, 19, 20 and the posteromedial lateral suprasylvian visual area, PMLS). Cases with injections restricted to individual visual areas indicate that they all contribute to the parafloccular input. Considerably less overlap occurred after injections of the primary sensorimotor region (SI, MI) and second somatosensory area (SII), while the supplementary motor area, the auditory cortex and gyrus cinguli probably have no or very restricted access to the dorsal paraflocculus. It is concluded that the dorsal paraflocculus has its major cortical input from the parietal association cortex and the visual cortical areas. Since all the various cortical regions studied project to largely different parts of the pontine nuclei, and overlap with neurons projecting to the dorsal paraflocculus takes place at numerous places, it follows that the pontine neurons projecting to the dorsal paraflocculus must consist of many subgroups differing with regard to their cortical input.     

Retinotopic organization of the lateral suprasylvian sulcus' posterior-medial area as revealed by analysis of the pattern of cortico-cortical connection with the cat 17th area

Using cortico-cortical connection analysis technique, the cat visual area PMLS (the area located on posterior medial wall of the lateral suprasylvian sulcus) retinotopic organization was investigated. A retrograde axonal tracer: horseradish peroxidase (HRP), was injected in the PMLS, and initial neurons were investigated in area 17. It was shown that after HRP injection in PMLS locus, where a central vision field is located, a labelled cell pattern in area 17 corresponded to the L. Palmer et al., 1978, retinotopic map. On the contrary, after HRP injection in PMLS locus, where an upper vision field must be located, as L. Palmer et al., 1978 predicted, initial neurons are visualized in area 17 loci where low visual periphery is displayed: -10 degrees to -60 degrees in vertical meridian and 40 degrees to 80 degrees in horizontal meridian. Such discrepancy in upper and lower visual field representation was also obtained in electrophysiological and topographic investigations by Grant, Shipp, 1991. The data suggest necessity of S. Grant and S. Shipp's retinotopic map use in the cat area PMLS morphofunctional investigation.     

Distribution of motoneurones innervating extraocular muscles in the brain of the marmoset (Callithrix jacchus)

Extraocular muscle motoneurones were localised in the oculomotor nucleus (ON), trochlear nucleus (TN) and abducens nucleus (AN) in the marmoset brain using the horseradish peroxidase (HRP) retrograde labelling technique. HRP pellets injected into individual extraocular muscles revealed one or more groups of labelled neurones occupying discrete loci within these nuclei. Relatively little overlap of motoneurone pools was observed, except in the case of the inferior oblique and superior rectus muscles. Injections of HRP into the medial rectus muscle revealed three separate populations of labelled cells in the ipsilateral ON. Motoneurones innervating the inferior rectus muscle were mainly localised in the lateral somatic cell column of the ipsilateral ON. A second smaller grouping was observed in the medial longitudinal fasciculus. The inferior oblique muscle motoneurones were localised in the ipsilateral medial somatic cell column intermingled with motoneurones supplying the superior rectus muscle of the opposite eye. The superior oblique muscle motoneurones occupied the entire TN and the lateral rectus muscle motoneurones the AN. It was concluded that the organisation of nuclei and subnuclei responsible for controlling the extraocular muscles in the marmoset is broadly similar to that of other primates.     

Localization of retinal dehydrogenase type 1 in the stomach and intestine

Rats were injected with liposomes containing iodixanol (CTP10 Injection; 100 mg iodine per kg body weight) followed by a second injection of 125I-tyramine-cellobiose-albumin microspheres. The amounts of phagocytosed and degraded labelled albumin in liver were measured. A reduced uptake and degradation of albumin microspheres was observed when the labelled microspheres were injected 2 h or 24 h after the liposomes compared with that obtained in control animals receiving saline. No effect on the uptake and degradation of labelled microspheres was observed when the time lag between the injection of liposomes and labelled microspheres was 1 week. The data show that the uptake and degradation of 125I-tyramine-cellobiose-albumin microspheres can be used as indicators of Kupffer cell phagocytotic function following drug uptake by these cells.     

大鼠延髓尾端加压区对外周血管紧张的影响

本文以血管的灌流压变化反映血管紧张性,观察Ｌ－谷氨酸及肾上腺素能受体阻断剂微量注射于大鼠延髓腹面心血管活动调控区对骨骼肌血管和肾血管紧张的影响。实验观察到,Ｌ－－ｇｌｕ兴奋延髓尾端加压区（ＣＰＡ）在动脉直升的同时,双后肢骨骼肌灌流压上升３７．５９％,左肾灌流压上升１７．０４％,如预先在延髓腹面加我注入普萘少尔或酚妥拉明均能明显减小Ｌ－ｇｌｕ兴奋ＣＰＡ时的灌流压和肾灌流压升高效应。提示ＣＰＡ对外周血     

Descending course of primary afferent collaterals in the cat's spinal cord.

After injecting horseradish-peroxidase into the lower thoracic, lumbar and sacral spinal cord segments of cats, labelled perikarya were found in several spinal ganglia localized cranially to the site of injection. The segmental distance between the injection site and the rostralmost localized ganglion with labelled cells varied depending on the medio-lateral localization of the injection. The longest distance (10 segments) was achieved by medial injections. Primary sensory neurones with long descending collaterals belong to large ganglion cells.     

Unstable nuclear DNA in hypoglossal neurons of adult mice

To demonstrate the existence of unstable or metabolic DNA in normal mammalian neurons and to study the effect of peripheral nerve injury on this metabolic DNA, adult mice were given repeated injections of high doses of 3H-thymidine (3H-T) on the day before injury to the left hypoglossal nerve. The animals were killed at intervals up to 33 days after the injections of 3H-T. Analyses of grain counts showed a low but significant elevation in the number of radioautographic grains per unit area of hypoglossal neuronal nuclei above background levels for up to 5 days after 3H-T injection. Digestion of the tissue with DNase lowered the nuclear grain counts to background levels, confirming that the DNA was indeed labelled. Although there was a loss of labelled material from the neuronal nuclei with time, there was no difference between injured and uninjured neurons at any of the intervals tested after injection of 3H-T.     

The presence and in vivo biosynthesis of fragments of CPP (the C-terminal glycopeptide of the rat vasopressin precursor) in the hypothalamo-neurohypophyseal system

The existence and rate of formation of fragments of the 39-residue C-terminal glycopeptide of propressophysin (CPP1-39) was investigated in the hypothalamo-neurohypophyseal system. Newly-prepared antisera to CPP were used to confirm the existence of smaller C-terminal fragments derived from CPP1-39. Radiolabelled fucose was injected into rats in vivo into the area of the supraoptic nucleus, and the labelled peptides formed in the neurohypophysis were examined at various time intervals up to five weeks after the injection. The products derived from the neurohypophyseal hormone precursors were separated by high-performance liquid chromatography. The level of the major immunoreactive C-terminal fragment (CPP22-39) was constant and represented about 5% of the intact CPP1-39 in the neurohypophysis. The appearance of newly-synthesized N-terminal fragment of CPP1-39 occurred only after 3 or 4 days. This fucose labelled fragment increased slowly thereafter until it reached the same level as the CPP C-terminal fragment immunoreactivity between 21 and 28 days after injection. The results suggest that CPP1-39 is extremely stable in the hypothalamo-neurohypophyseal neurons, and that the cleavage at Arg21-Leu22 is a delayed proteolytic event in the magnocellular neurons of the SON.     

Location and connectivity of abdominal motoneurons in the embryo and larva of Drosophila melanogaster

The soma location and peripheral connectivity of motoneurons in abdominal segments of the embryo and larva of the fruitfly, Drosophila melanogaster are described as an initial step in determining the mechanisms by which motoneurons make connections with their target muscles in a genetically accessible organism. Embryonic motoneuron somata were retrogradely labelled by application of the fluorescent dye, DiI, to the whole peripheral nerve or to its separate anterior or posterior fascicles in segments A5-A7 of late stage 15/early stage 16 embryos. This technique reveals a stereotyped, segmentally repeated population of 34 motoneurons per hemisegment, several of which can be individually identified from their soma position. The same set of motoneurons was revealed in third instar larvae of D. melanogaster by cobalt backfilling of abdominal peripheral nerves, although the positions of some of these neurons change during larval development. The peripheral connectivity and axon morphology of several of the abdominal motoneurons was determined by intracellular injection with Lucifer Yellow in stage 16 embryos. For the motoneurons with axons in the anterior fascicle there is no clear relationship between somata groupings and the muscle targets innervated: contrary to earlier claims, these motoneurons arborize over both ventral and dorsal muscles. Individual motoneurons possess a stereotyped pattern of terminal arborization.     

Growth Fraction and Cycle Duration of Hepatocytes In the Three-Week-Old Rat

The proliferation of hepatocytes in the liver of 3-week-old rats has been investigated by autoradiographic methods. This investigation is a continuation of earlier work on the same topic (Schultze & Maurer, 1972; 1973). 21 days after birth, 102 rats received a single injection of ³H-TdR. the percentage of labelled mitoses was then determined 1 hr later and at various times throughout the interval up to 12 days after application of ³H-TdR. In agreement with earlier work, a first peak of labelled mitoses was found 7 hr after ³H-TdR injection. the area under the peak indicates an S phase duration of 8 hr. In addition a second very broad peak of labelled mitoses was found between 2 and 12 days after pulse labelling. the analysis of the results leads to the conclusion that the hepatocytes of the 3-week-old rat have a growth fraction close to 1 and a doubling time of 6–7 days. This is at variance with earlier results of Post, Huang & Hoffman (1963) and Grisham (1969) who had derived a value of 21.5 hr for the duration of the cell cycle and a value of only 0. 1–0.2 for the growth fraction of the hepatocytes.     

Effect of 4-aminopyrazolo[3,4-d]pyrimidine on the catabolism of high-density lipoprotein apolipoprotein A-I in the rat

The in vivo turnover and sites of catabolism of O-(4-diazo-3-[125I]iodobenzoyl)sucrose-labelled rat high-density lipoprotein (HDL) apolipoprotein A-I were studied in rats treated for 3 days with 4-aminopyrazolo-[3,4-d]pyrimidine (4APP). It was found that 4APP treatment decreases the serum cholesterol concentration to 6 mg/dl and stimulates the serum decay of labelled HDL. The latter effect could be attributed to an increased catabolism of radioactive HDL apolipoprotein A-I by the liver. When the serum cholesterol concentration was raised to physiological levels by a bolus injection of unlabelled rat HDL, at the time of administration of the labelled HDL, the serum decays and the tissue uptakes of apolipoprotein A-I labelled HDL were identical in 4APP-treated rats and control animals. When a bolus injection of unlabelled human low-density lipoprotein (LDL) was administered to 4APP-treated rats, the serum decay and tissue uptake of apolipoprotein A-I labelled HDL remained rapid. The recovery of radioactivity in the adrenal glands was increased almost 10 fold by 4APP treatment, a phenomenon which was reversed by a bolus injection of unlabelled HDL, but not by injection of unlabelled LDL. It is concluded that treatment of rats with 4APP does not affect the rate of catabolism of rat HDL apolipoprotein A-I, when the serum HDL concentration in the treated animals is restored to physiological levels.     

In vivo incorporation of labeled palmitic and oleic acids into skeletal muscle lipids of normal and thyroidectomized rats during swimming]

The purpose of this work was to study the incorporation of [1-14C] palmitic and and [9,10(-3)H] oleic acids, after intravenous administration in the lipids of rat hind leg muscles. The animals were fasting or fed, at rest or swimming during 10 min before test, euthyroid or thyroidectomised. All these animals have been taking the same daily swimming training, during 15 days before the injection of labelled molecules. They were killed 10 min (+/-1)later. The lipidic muscle composition, the incorporation rate of labelled fatty acids in these lipids and the radioactivity distribution among the different lipids in the various cases have been determined. Moreover the plasmatic non-esterified acid radioactivity has been measured. These various values are affected by nutritional, hormonal state, and by physical activity of the animals. Particularly, it seems that supplementary energy spent during swimming test will be covered by the oxidation of different nutriments, according to the nutritional and hormonal state of animals.