Increased quantal size in transmission at slow but not fast neuromuscular synapses of apolipoprotein E deficient mice.

Uncertainties from the literature concerning the role of apolipoprotein E (apoE) in central cholinergic function prompted us to investigate what effect apoE may have on transmission at the neuromuscular junction. Both spontaneous and evoked release were measured in isolated extensor digitorum longus (edl) and soleus muscles from both wild-type and apoE-deficient mice. Miniature endplate and nerve-evoked endplate potentials (MEPPs and EPPs, respectively) were indistinguishable in edl muscles in both groups of mice; however, MEPP amplitudes in soleus muscles were significantly larger (by an average of 23%) in apoE-deficient mice compared with 5- to 7-week-old age-matched wild-type mice. The EPP amplitudes were also larger in soleus muscles in the mutant mice, but this was a reflection of the larger quantal size in this muscle because quantal content, determined from the ratio of the average EPP amplitude to average MEPP amplitude, was unchanged from normal in the mutant mice. The MEPP frequency and the percent of nerve stimulations failing to produce an EPP were unchanged from normal in both muscle types in the mutant mice. The difference in quantal size in soleus muscle transmission between mutant and wild-type mice was abolished in the presence of neostigmine, an acetylcholinesterase inhibitor. The results suggest that apoE normally associates with acetylcholinesterase in the synaptic cleft of slow muscles, modulating the activity of the enzyme and therefore quantal size.

Purinergic receptors in auditory neurotransmission.

The effects of ATP (adenosine 5' triphosphate) analogs on gross cochlear potentials and single primary afferent discharge properties were studied by intracochlear perfusion in anesthetized guinea pigs. ATP-gamma-S was most potent, with betagammamethylene-ATP and Bz-ATP being significantly less effective. These data are consistent with the notion that purinergic receptors activated by scala tympani perfusion contain subunits of the P2X(2) variant. The relative ineffectiveness of Bz-ATP (a P2X(7) agonist) suggests that while this variant has been reported to be expressed in the cochlea, it may not play a major functional role under normal conditions. Changes in the threshold of the gross DC receptor potential (summating potential, SP) and the compound action potential (CAP) were consistent with a combination of effects on both early and final stages of the transduction process, as reported by previous workers. Effects of ATP-gamma-S on single-neuron spontaneous firing rates varied according to the initial spontaneous rate of each primary afferent. Effects on single-neuron tuning curves were consistent with an action mainly on the outer hair cell transduction with betagammamethylene-ATP (elevation of tuning curve tips), but with ATP-gamma-S changes in sensitivity across the full extent of the tuning curve indicated an additional action on inner hair cell-afferent neurotransmission. In agreement with previous reports on ATP-gamma-S, it was found that all ATP analogs produced significant increases in the DC potential in scala media (endocochlear potential, EP). However, the relationship between changes in EP (a major component of the driving force on ions through hair cells) and the alterations in gross and single unit measures of cochlear activity was not clear.

Structural basis for the spotted appearance of amphibian neuromuscular junctions stained for synaptic vesicles.

We have compared the distribution of vesicles in amphibian motor nerve terminals determined by electron microscopy and by functional labeling with the styryl dye, FM2-10. Our aim was to resolve apparent discrepancies in the literature on the distribution of vesicles determined by the two procedures. Electron photomicrographs of non-serial cross sections of terminal branches were analyzed by stereological procedures to obtain indices of the terminal and vesicle areas. Terminal cross sectional area varied 3-fold on average along terminal branches and was largest particularly when active zone was present in the section. The vesicle area index (a measure of vesicle abundance) was highly correlated with the terminal area index, suggesting that the average density of vesicles is constant throughout the branches. When the data were separated according to whether active zone was present or not in a section, we found a small (26%) but significant increase in the average density of vesicles in active zone compared with non-active zone regions in the terminal. The distribution of spots along terminal branches following vesicle staining with FM2-10, as well as with antibodies to vesicle proteins, suggested that vesicles were distributed in highly concentrated clusters. However, the degree of variation between spot and inter-spot staining intensities found with the FM-dye was similar in magnitude to that for terminal cross sectional area determined from the electron microscopy. We conclude that the spotty pattern of stained vesicles seen with the optical microscope results primarily from vesicle accumulations associated with terminal varicosities.

Spatial and temporal distribution of nerves, ganglia, and smooth muscle during the early pseudoglandular stage of fetal mouse lung development.

Neural tissue and smooth muscle appear early in the developing fetal lung, but little is known of their origin and subsequent distribution. To investigate the spatial and temporal distribution of nerves, ganglia, and airway smooth muscle during the early pseudoglandular stage, fetal mouse lungs at embryonic days (E) 11 to 14 were immunostained as whole-mounts and imaged by confocal microscopy. At E11, the primordial lung consisted of the future trachea and two budding epithelial tubules that were covered in smooth muscle to the base of the growing buds. The vagus and processes entering the lung were positive for the neural markers PGP 9.5 (protein gene product 9.5) and synapsin but no neurons were stained at this stage. An antibody to p75NTR revealed neural crest cells on the future trachea as well as in the vagus and in processes extending from the vagus to the lung. This finding indicates that even though neuronal precursors are already present at this stage, they are still migrating into the lung. By E12, neural tissue was abundant in the proximal part of the lung and nerves followed the smooth muscle-covered tubules to the base of the growing buds. At E13 and E14, a neural network of interconnected ganglia, innervated by the vagus, covered the trachea. The postganglionic nerves mainly followed the smooth muscle-covered tubules, but some extended out into the mesenchyme beyond the epithelial buds. Furthermore, we show in a model of cultured lung explants that neural tissue and smooth muscle persist and continue to grow and differentiate in vitro. By using fluorescent markers and confocal microscopy, we present the developing lung as a dynamic structure with smooth muscle and neural tissue in a prime position to influence growth and development.

Membrane recycling due to low and high rates of nerve stimulation at release sites in the amphibian (Bufo marinus) neuromuscular junction.

The activity-dependent labelling of motor nerve terminals with the dye FM1-43 has been used to estimate the relative levels of membrane recycling (due to synaptic vesicle exocytosis and recovery) at release sites in response to 1,200 nerve stimulations delivered at either low (0.5 Hz) or high (30 Hz) frequency. Dye in terminals appears as fluorescent spots distributed along the terminal branches; each spot is thought to be a cluster of labelled vesicles associated with a release site. Relative fluorescence in spots was quantified from images obtained with a confocal microscope. Spot intensities varied widely within branches following labelling at both frequencies, but the distribution was highly skewed towards lower intensities at low frequency stimulation; at high frequency, more spots had stronger fluorescence. Both weak and strongly stained spots were uniformly distributed along the length of terminal branches after low frequency stimulation; however, there was a gradual decline in all spot intensities towards the distal end of branches loaded with dye at high frequency stimulation. Antibody staining for synaptic vesicles was, on average, uniformly distributed along the branches. The increase in number of more strongly FM1-43-labelled spots in terminal branches stimulated at high compared with low frequency suggests that more release sites are active at high rates of nerve stimulation. This "recruitment" of release sites at high frequency stimulation occurs mostly in the proximal half of terminal branches and is not related to the abundance of synaptic vesicles in the terminal.

Mapping the innervation of the bronchial tree in fetal and postnatal pig lung using antibodies to PGP 9.5 and SV2.

The extent of the innervation in the adventitial surface of the airway wall is demonstrated by mapping all the nerves and ganglia of the first trimester fetal and postnatal (4-wk-old) pig lung. We used antibodies to two pan-neuronal nerve markers-Protein Gene Product (PGP) 9.5, a nonspecific marker for all nerves, and synaptic vesicle protein 2 (SV2), a marker of synaptic vesicle membranes-to obtain detailed maps of the innervation of the airways. The airway smooth muscle has been stained concurrently with an antibody to alpha-actin. To obtain an overview of the passage of nerves down the bronchial tree, whole mounts of the bronchial tree have been imaged using confocal microscopy. Several conclusions can be drawn from our work: (1) Nerves branch from the main nerve trunks to form an extensive plexus covering the surface of the smooth muscle in fetal pigs but penetrate through the smooth muscle and are more organized in postnatal airways. In general, two main nerve trunks per airway were observed. (2) The antibody to PGP 9.5 in the fetal lung revealed in many ganglia and nerve trunks, the latter exhibiting cell profiles. In the postnatal lung, cell bodies were restricted to ganglia in the central airways. (3) The antibody to SV2, in general, showed varicose staining of the fine nerve fibers at both ages.

A quantitative study of the segmental distribution of somitic cells in the developing chick limb bud using laser-scanning confocal microscopy.

Our aim was to map the segmental distribution of somitic cells in the limb before and after the fusion of these cells into myotubes. Somitic cells of the brachial somites were labeled by injection of DiI and DiO into the somitoceles of embryonic Day 2 (E2) embryos. The quantitative distribution of dye-labeled cells from injected somites was examined in whole mounts of E4 wing buds and in sections of E5 wing buds using confocal microscopy. Dye derived from cells of anterior brachial somites 16 and 17 was highly concentrated in the anterior half of E4 wings, whereas dye from posterior brachial somites 20 and 21 was concentrated in the posterior half of the wing. Not more than 14% (on average) of the dye in the wing was found outside the preferred half. Dye was equally dispersed in the anterior and posterior halves of the wing when the middle two somites contributing to the wing musculature, numbers 18 and 19, were injected. The total amount of dye in the whole limb at E4 was not significantly different after injection of similar amounts of dye into either pair of brachial somites 16/17, 18/19, or 20/21. In E5 embryos the distribution of labeled cells in the newly formed muscle masses, identified with antibody 2H2 to myosin light chains, was examined in cryostat cross-sections of the wing. Dye was more widely distributed in the wing than at E4. As much as 32% (on average) of the dye was found in the muscle mass outside the preferred half after administration of dye into the anterior or posterior pair of somites. We conclude that the branchial somites contribute similar numbers of cells to the wing musculature and that the segmental origin of these cells is rigidly maintained during their lateral migration into the limb. Cells from adjacent segments then "mix" when they fuse to form myotubes at E5. In addition, we found a significantly greater amount of dye per unit of muscle mass in the proximal compared with distal parts of the limb. We argue that this finding is due to division of myogenic cells during the course of their migration in the wing.

Loss of the position-dependent reinnervation of regenerated toad (Bufo marinus) glutaeus muscle.

The terminations of motor axons in the toad glutaeus muscle show a course dependency on the segmental origins of the axons on the spinal cord. Rostral axons in spinal nerve 8 innervate muscle fibres near the ventral surface of the muscle, while caudal axons in spinal nerve 9 innervate fibres mostly towards the opposing dorsal surface. Axons originating between these extremes tend to innervate the central regions of the muscle. A similar topographic projection is reestablished after denervation and when regenerating axons reinnervate the muscle via entirely novel pathways (Brown and Everett [1991] J. Comp. Neurol. 309:495-506). The findings are compatible with the graded expression of a determinant within the glutaeus muscle that biases the formation of synapses between positionally matched muscle fibres and motor axons. In the present work, we provide strong support for this view by showing that when the muscle is reinnervated by axons arising from only one spinal nerve, they expand their projection and form synapses in the muscle in a topographically appropriate manner. In a second experiment, we tested whether a muscle that had regenerated from its resident myogenic (satellite) cell population would be similarly reinnervated. This experiment was prompted by the work of others (Donoghue et al. [1992] Cell 69:67-77) showing that the myogenic precursor cells in adult muscle are a repository of "positional memory." In our experiments, a glutaeus muscle was removed from adult toads and soaked in bupivacaine for a brief period to destroy the muscle fibres before being sutured back into its normal position in the limb. The distribution of motor units in the muscles was determined by glycogen depletion after allowing 3-4 months for the muscles to regenerate from their satellite cell population and to become reinnervated. We found that muscle fibres belonging to single motor units were dispersed widely in the regenerated muscles and showed no topographic organisation. We conclude that the positional cues that direct topographic map formation are available to motor axons when they reinnervate a denervated mature muscle, but play no role in the reinnervation of a regenerated muscle.

Innervation and function of the distal airways in the developing bronchial tree of fetal pig lung.

We investigated whether the airways of the lungs from 8 to 10 g fetal pigs (the pseudoglandular phase) have a nerve supply, are functionally innervated and narrow in response to electrical field stimulation and to agonists. Measurements of airway narrowing were made by real-time video imaging of intact isolated bronchial tree freed of associated parenchyma and vasculature. The distal (100 to 300 microns lumen diameter) and terminal (approximately 25 to 50 microns lumen diameter) airways narrowed strongly to acetylcholine and histamine, to within 50 to 80 microns of the base of the epithelial buds. Electrical field stimulation produced rapid narrowing followed by relaxation, and responses were blocked by atropine and tetrodotoxin, indicating a functional cholinergic innervation. Transient periods (3 to 5 min) of spontaneous narrowing were seen in localized regions of the bronchial tree which moved the lung liquid to and fro. An extensive nerve supply to the bronchial tree was seen after immunocytochemical staining of tissue whole mounts with anti-neurofilament. Nerves supplied the distal and terminal airways with fine branches penetrating the smooth muscle of the airway wall. A few fibers extended to the growing edge of the smooth muscle at the very base of the epithelial bud. The smooth muscle was cylindrically arranged around the airway wall all the way to the epithelial bud. We propose that onset of function of smooth muscle and its innervation occurs shortly after differentiation of the smooth muscle at the growing tips of the airways.

Retinal ganglion cell survival in vitro maintained by a chondroitin sulfate proteoglycan from the superior colliculus carrying the HNK-1 epitope.

We recently reported evidence implicating a superior colliculus-derived chondroitin sulfate proteoglycan (SCCP) in the trophic support of cultured retinal ganglion cells (Schulz et al., 1990). In the present work we show preparations of the SCCP to be reactive with an antibody (CS-56) to chondroitin sulfate types A and C and with the HNK-1 antibody. Reaction with the HNK-1 antibody allowed us partially to purify the native proteoglycan by immunoaffinity chromatography. HNK-1 reactive material was further processed by a combination of molecular sieve chromatography in the presence of 4M guanidine HCL followed by anion exchange chromatography to yield a product that migrated electrophoretically as a single band in polyacrylamide gel with an apparent molecular weight of not less than 400 k. The SCCP, when added to a fully defined culture medium, maintained the survival of the vast majority (80%) of the ganglion cells over a 16 hr culture period with 86% of these cells showing a profusion of processes; few ganglion cells (10%) survived in the absence of the proteoglycan. Electrophoretic analysis of nonreduced preparations of the molecule did not reveal any low molecular weight silver stained components that may have remained associated with the molecule after guanidine HCL treatment. However, two bands corresponding to molecular weights of around 60 and 80 k were reproducibly observed on polyacrylamide gels following electrophoresis of the molecule in the presence of beta-mercaptoethanol. Our findings provide further evidence suggesting a role for a chondroitin sulfate proteoglycan carrying the HNK-1 epitope in the trophic support of central neurones.

Uncoupling of actin-activated myosin ATPase activity from actin binding by a monoclonal antibody directed against the N-terminus of myosin light chain 1.

The role of the N-terminal region of myosin light chain 1 (LC1) in actomyosin interaction was investigated using an IgG monoclonal antibody (2H2) directed against the N-terminal region of LC1. We defined the binding site of 2H2 by examining its cross-reactivity with myosin light chains from a variety of species and with synthetic oligopeptides. Our findings suggest that 2H2 is directed against the N-terminal region of LC1 which includes the trimethylated alanine residue at the N-terminus. In the presence of 2H2, the rate of actomyosin superprecipitation was reduced, although the extent was not. 2H2 caused a reduction in the Vmax of both myosin and chymotryptic S1(A1) actin-activated ATPase activity, while the Km appeared to be unaltered. The Mg(2+)-ATPase activity of myosin alone was also unaffected. Binding studies revealed that 2H2 did not prevent the formation of acto-S1 complex, either in the presence or in the absence of ATP, nor did it affect the ability of ATP to dissociate S1 from F-actin. Our findings suggest that the N-terminal region of LC1 is not essential for actin binding but is involved in modulating actin-activated ATPase activity of myosin.

Evidence for resegmentation in the formation of the vertebral column using the novel approach of retroviral-mediated gene transfer.

We have examined the somitic cell contribution to the vertebral column of the chick by genetic labeling of sclerotomal cells in early development. Single somites of embryonic Day 2 embryos were filled with retroviral particles containing the lacZ transducing vector BAG. After a further 14 or 17 days of incubation the embryos were fixed and the vertebral column was sectioned and stained histochemically for the lacZ gene product beta-galactosidase. Cells staining for the enzyme were found exclusively on the injected side of two vertebral segments; the staining was largely restricted, however, to the caudal half of the more rostral segment and the rostral half of the next more caudal segment. No embryos were observed with labeling in less than two vertebral segments. Moreover, labeled cells were not uniformly distributed within the labeled region of each vertebra; the neural arch, for example, usually contained a higher proportion of labeled cells than did the centrum. These observations support the concept of resegmentation, whereby a vertebra forms from sclerotomal cells derived from two consecutive somites resulting in a vertebral column shifted by one half segment with respect to the segmented boundaries of the somites. The quantitative distribution of labeled cells in the vertebrae also suggests that sclerotomal cells populate the region of a future vertebral segment in an orderly fashion dependent on when the cells migrate from the somite.

Position- and fibre type-dependent selectivity by regenerating motor axons in reformation of the topographical projection to the glutaeus muscle in the adult toad (Bufo marinus).

The toad glutaeus magnus muscle receives a topographical innervation dependent on the rostrocaudal origins of glutaeus motor axons on the spinal cord. The same projection is re-established in adult animals after cutting the glutaeus muscle nerve and allowing regenerating axons to reinnervate the muscle (Brown and Everett (1990) J. Comp. Neurol. 292:363-372). To determine whether the topographically selective reinnervation of the glutaeus muscle comes about because of genuine positional qualities associated with the pre- and post-synaptic tissues, we undertook two different experimental procedures: first, the nerve supply to the muscle was cut and redirected to ensure that regenerating axons entered the muscle via entirely novel pathways; and second, the triceps femoris nerve was cut so that the glutaeus muscle was reinnervated from a large pool of motor axons which were mostly foreign but segmentally appropriate. The motor projection to the muscle was determined by the glycogen depletion procedure; single glutaeus motor axons were repetitively stimulated in vitro to deplete muscle fibres of their stores of glycogen. The spatial location of fibres belonging to single motor units could then be determined by staining sections of the muscle histochemically for glycogen. A similar to normal projection was re-established after both experiments in that rostrally located motor axons innervated mostly the ventral portion of the muscle and caudal axons mostly the dorsal portion of the muscle. "Intermediate" axons synapsed predominantly with fibres in the middle of the muscle. Furthermore, the arrangement of fibre types in the muscle could not fully account for the reformation of the topographical projection by a type matching mechanism, although a secondary role for fibre types in the subsequent refinement of the projection was clearly suggested by our findings. Our results provide evidence for a distinction between at least two determinants that influence the reinnervation of muscle: positional cues and fibre types, with the former taking precedence over the latter.

Ezrin immunoreactivity in neuron subpopulations: cellular distribution in relation to cytoskeletal proteins in sensory neurons.

Ezrin was first identified as a low-abundance phosphoprotein associated with the cytoskeletal core of microvilli, where it may function as a regulatory protein. We report immunocytochemical evidence for expression of ezrin, or an ezrin-like protein of molecular mass near 80 KD, confined to select populations of neurons, including sensory, motor, and autonomic, during chick embryonic development. We have compared the distribution of anti-ezrin staining with that of other major cytoskeletal proteins in sensory neurons in an effort to identify a possible association of the neural homologue of ezrin with the neuronal cytoskeleton. The diffuse distribution of anti-ezrin staining in the cell soma bore little resemblance to the filamentous staining observed with antibodies to the 68 KD neurofilament protein and alpha-tubulin. F-actin staining with fluorescein-conjugated phalloidin was indistinguishable from the anti-ezrin staining pattern in the soma of cultured neurons, including a peak in staining intensity around the periphery of the cell. Microfilaments in growth cones did not stain with the ezrin antibody. A close correspondence between the anti-ezrin and anti-spectrin staining patterns was found on cryostat sections of dorsal root ganglia, but the anti-spectrin staining was weak and could not be demonstrated in culture. Our findings, primarily from cultured neurons, are not inconsistent with ezrin associating with F-actin, although not with microfilaments found in motile structures such as growth cones.

Different ratio of myosin heavy chain isoforms in arterial smooth muscle of spontaneously hypertensive rats.

The relative proportion of the two putative heavy chains of smooth muscle myosin (MHC1 and MHC2) was determined in the caudal and femoral arteries of spontaneously hypertensive rats (SHR) and normotensive (WKY) rats at 16 weeks of age. The heavy chain polypeptides with Mr 204,000 and 200,000 were resolved electrophoretically under denaturing conditions in porous polyacrylamide gels. Both proteins reacted strongly with a monoclonal antibody (2C4) to smooth muscle MHC. In caudal arteries the ratio of MHC1/MHC2 was 3.1:1 in SHR rats compared with 1.8:1 in WKY rats (p less than 0.005) and similarly in femoral arteries, 2.8:1 vs 1.5:1 (p less than 0.001). In the portal vein there was no significant difference, 1.7:1 vs 1.5:1. The possibility that the higher MHC ratio in the SHR is the genetically mediated defect in arterial smooth muscle cells leading to the hypertension is discussed as an alternative to the elevated systemic blood pressure causing the altered MHC ratio.

Compartmental and topographical specificity of reinnervation of the glutaeus muscle in the adult toad (Bufo marinus).

The extent to which the compartmental and topographical innervation patterns were re-established in the mature glutaeus muscle after nerve cut was determined by HRP retrograde labelling procedures and single motor unit glycogen depletion experiments. Glutaeus muscle axons normally cluster together along the length of the sciatic nerve and enter the triceps femoris nerve along with axons to the cruralis and tensor fasciae latae muscles. Glutaeus axons did not specifically reinnervate the glutaeus muscle after cutting the triceps femoris nerve. Axons within the glutaeus muscle nerve are grouped according to the primary nerve branch, and therefore muscle compartment, for which they are destined. When the glutaeus nerve was cut, regenerating axons lost their compartmental organization within the glutaeus nerve and nonspecifically reinnervated both glutaeus muscle compartments. In marked contrast, the topographical projection of spinal motoneurones to the ventral and dorsal halves of the glutaeus muscle was largely re-established after glutaeus nerve cut; that is, rostral motoneurones mostly reinnervated ventral muscle fibres, whereas caudal motoneurones reinnervated dorsally located muscle fibres. The relatively confined caudal projection was closely related to the sharp fibre type divisions within the glutaeus muscle. The results suggest that factor(s) present during development that determine the correct innervation of a muscle and its compartments no longer operate in the adult, although cues that direct motor terminals to their appropriate muscle region do, and these are most likely related to the topographical distribution of fibre types.

Compartmental and topographical distributions of axons in nerves to the amphibian (Bufo marinus) glutaeus muscle.

The present work seeks to determine if axons to an amphibian muscle are segregated in nerve trunks between the spinal cord and muscle according to their primary nerve destination or their topographical projection in the muscle. The distribution of axons to different compartments and subcompartments of the amphibian (Bufo marinus) glutaeus muscle has been determined in transverse sections of spinal and limb nerves after retrogradely labelling the axons with horseradish peroxidase. Glutaeus axons were dispersed widely through spinal nerves 8 and 9 but loosely gathered together in one quadrant of the sciatic nerve after passing through the lumbar plexus. Glutaeus axons became tightly clustered to the exclusion of other axons along the length of the triceps femoris nerve after it divides from the sciatic nerve. Furthermore, axons destined for one of the two glutaeus primary nerve branches segregate from those of the other branch at the level of the triceps femoris nerve before the glutaeus nerve forms. On the other hand, motoneurones that subserve a primary branch are not segregated, but are found throughout the rostrocaudal extent of the glutaeus motoneurone pool. Injection of horseradish peroxidase under the epimysium of either the ventral or the dorsal surfaces of the glutaeus muscle labelled motoneurones preferentially in either the rostral or caudal part of the motoneurone pool, respectively. This confirms studies that have shown a topographical projection from the spinal motoneurone pool onto the glutaeus muscle. However, there was no segregation of dorsally projecting axons in the glutaeus and primary nerve branches. Thus, glutaeus axons segregate according to their muscle compartmental projections well before entering the muscle, but they show no organization in nerves with respect to their topographical projections within a compartment.

The development of topographical maps and fibre types in toad (Bufo marinus) glutaeus muscle during synapse elimination.

1. The toad glutaeus muscle consists of two muscle compartments. A study has been made of the topographical distribution of motor units in these compartments, in relation to the fibre types which arise during different stages of development. 2. Monoclonal antibodies to myosin allowed the distribution of fibre types to be determined. In mature muscles (from toads of greater than 30 g body weight) clusters of type 5 (tonic) fibres were found exclusively at the dorsal surface of the muscle, surrounded by a layer of type 3 (slow-twitch) fibres. A homogeneous layer of type 2 (fast-twitch red) fibres was found beneath this dorsal rind of slow and tonic fibres. The rest of the muscle, including the ventral surface, consisted of a mosaic of type 1 (fast-twitch white) and type 2 fibres. 3. Glycogen-depletion methods, together with the myosin antibodies, allowed the distribution of single motor units and their fibre types to be determined. In mature muscles, axons originating from rostral spinal cord possessed muscle units located in a band extending from the ventral surface to beyond the middle of the muscle; these units consisted of 78% type 1 and 22% type 2 fibres found amongst the mosaic of type 1 and type 2 fibres. Intermediate axons possessed muscle units located primarily in the middle and dorsal half of the muscle. These units consisted mostly of type 2 fibres (29% type 1, 71% type 2) also found amongst the mosaic of type 1 and type 2 fibres. Thus rostral and intermediate units were of mixed fibre type, with type 1 fibres predominating in the former units and type 2 in the latter. Caudal axons possessed muscle units located mostly in the homogeneous layer of type 2 fibres, beneath the dorsal rind of tonic fibres; these units were almost always composed entirely of type 2 fibres. 4. The distribution of single motor units and their fibre types were determined for the caudal axons during development. In juvenile animals (toads of about 10 g body weight) the dorsal rind of tonic and slow fibres, together with the underlying homogeneous layer of type 2 fibres, were still present, but the rest of the muscle to the ventral surface consisted almost entirely of type 1 fibres. Caudal axons innervated the type 2 fibre layer at the dorsal surface as they do in mature animals. 5. The glutaeus in post-metamorphic toads (0.15 g body weight) had only a small number of tonic and slow-twitch fibres in the very dorsal layer of cells; the muscle was largely type 1.(ABSTRACT TRUNCATED AT 400 WORDS)

Transient appearance of a fast myosin heavy chain epitope in slow-type muscle fibres during stretch hypertrophy of the anterior latissimus dorsi muscle in the adult chicken.

Myosin expression during hypertrophy of the chicken anterior latissimus dorsi (ALD) muscle was investigated by immunocytochemical procedures using monoclonal antibodies to the fast and slow isoforms of the myosin heavy chain (myosin HC). Antifast antibody 1F9 bound to the adult fast HC of pectoralis muscle and cross-reacted with the HC found in early developing muscle. Antislow antibody 3D1 bound exclusively to the HC of slow myosin 2 (SM2). Stretch hypertrophy of the ALD was produced by attaching a weight to the wing; there was no evidence for a change in fibre number in the muscle. Between 4 and 6 days of stretch there appeared a dramatic increase in the number of fibres staining with the antifast antibody which reached a peak between 12 and 19 days. By this time between 28 and 52% of the fibres in the stretched ALD stained to varying degrees with the antifast antibody compared with much less than 1% in the unstretched control ALD. Most antifast-stained fibres in the stretched muscle also stained with the antislow antibody; the contralateral control muscle showed mostly antislow staining except for the very small number of strongly antifast-stained fibres. By 50 days in some birds and by 80 days in all birds antifast staining had returned to normal. Analysis of the isomyosin composition of the ALD by native gel electrophoresis did not reveal a significant increase in fast myosin content of the hypertrophied muscle even though immunocytochemical staining may have suggested otherwise.

Expression of myosin heavy chains during thyroid hormone-induced cardiac growth.

The expression of mRNAs for two cardiac myosins has been studied in the ventricles of hypo- and hyperthyroid rabbits by using cloned cDNA sequences corresponding to the mRNAs of the alpha- and beta-myosin heavy chains (HCs). The temporal change in relative levels of the alpha and beta HC mRNAs after triiodothyronine (T3) treatment of hypothyroid rabbits was determined by nuclease S1 mapping. In the hypothyroid state, only NC beta-mRNA was expressed in the ventricles. The HC alpha-mRNA was first detectable 4 h after administration of T3 (200 micrograms/kg) to hypothyroid animals. By 12, 24, and 72 h, HC alpha-mRNA represented 20, 50, and 90% of total myosin mRNA. The relationship between the relative mRNA levels and relative synthesis rates of myosin HCs was evaluated in 5- to 6-wk-old normal and thyrotoxic rabbits. Myosin synthesis rates were determined by labeling of protein in vivo with [2H]leucine. The V1 (HC alpha) and V3 (HC beta) isomyosins were separated by immune affinity chromatography and the HCs were isolated electrophoretically. In a normal euthyroid group of animals and in animals 12 and 24 h after administration of 200 micrograms of thyroxine, the relative mRNA levels and relative synthesis rates of the alpha and beta HCs were not significantly different. Our results show that, first, thyroid hormone causes a rapid accumulation of HC alpha-mRNA and loss of HC alpha-mRNA, and second, in normal and thyrotoxic rabbits, the relative synthesis rates of HC alpha and HC beta reflect the relative abundance of their respective mRNAs. These data are consistent with the thyroid hormones regulating synthesis of ventricular myosin at steps that precede translation of its message.