肌梭传入对呼吸的调节作用

实验用６３只麻醉、制动、切断双侧颈迷走神经、人工呼吸的家兔,以延髓呼吸相关神经元（ＲＲＮ）和膈神经放电（Ｐｈｒ．Ｄ）作为呼吸观测指标,观察了股动脉注射琥珀胆碱（Ｓｃｈ）诱发的肌梭传入活动对呼吸的影响。结果显示：（１）股动脉注射Ｓｃｈ可产生明显的呼吸易化作用,主要表现为吸气时程（Ｔｉ）延长、呼气时程（Ｔｅ）缩短不明显,Ｔｉ／Ｔｅ比值增加以及呼吸频率（ＲＦ）变化不在,称为吸气延长效应;或Ｔｅ缩短,Ｔｉ     

Superimposing noise linearizes the responses of primary muscle spindle afferents to sinusoidal muscle stretch

Experiments were conducted in anaesthetized and spinalized cats to measure the extent to which the non-linear response of Ia afferent fibers to sinusoidal muscle stretch as expressed by the peristimulus-time-histograms, PSTHs, can be transformed into a linear one by means of the superposition of random stretch ("mechanical noise"). The gastrocnemius muscles of one hind leg were stretched and the response to sinewave muscle stretch (amplitudes between 0.01 and 4.0 mm, frequencies between 0.1 and 20 Hz) were investigated while band-limited mechanical noise was superimposed on the sinewave stretch. The random stretch upper cut-off frequency was varied between 60 and 300 Hz; the displacements were normally distributed. The noise amplitude sigma, i.e. the standard deviation of the displacement distributions, was varied systematically between 0.002 and 0.4 mm. Mechanical noise was very effective in raising the mean discharge rate. Added to the sinusoidal stretch it prevented the cessation of firing during the release phase of the stretch cycle, or at least reduced the duration of discharge pauses, i.e., a linearization occurred. In general, the larger the noise amplitude, the more the amplitude of the fundamental harmonic component was attenuated and the phase lead reduced. Apart from this rule the particular combination of superimposing small noise (sigma less than 0.02 mm) on small sinewave stretch (A less than 0.02 mm) could enhance the depth of sinusoidal modulation of cycle histograms (compared with responses to pure sinusoids). Linearizing the sinewave response by additional noise allowed the estimation of frequency response characteristics in the otherwise non-linear range of amplitudes (sinewave amplitude 0.5-1.0 mm).(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP-sensitive potassium channels in adult mouse skeletal muscle: Characterization of the ATP-binding site

Summary Single K⁺-selective channels were studied in excised inside-out membrane patches from dissociated mouse toe muscle fibers. Channels of 74 pS conductance in symmetrical 160mm KCl solutions were blocked reversibly by 10 m internal ATP and thus identified as ATP-sensitive K⁺ channels. The channels were also blocked reversibly bymm concentrations of internal adenosine, adenine and thymine, but not by cytosine and uracil. The efficacy of the reversible channel blockers was higher when they were present in internal NaCl instead of KCl solutions. An irreversible inhibition of ATP-sensitive K⁺ channels was observed after application of several sulphydryl-modifying substances in the internal solution: 0.5mm chloramine-T, 50mm hydrogen peroxide or 2mm n-ethylmaleimide (NEM). Largeconductance Ca-activated K⁺ channels were not affected by these reagents. The presence of 1mm internal ATP prevents the irreversible inhibition of ATP-sensitive K⁺ channels by NEM. The results suggest that internal Na⁺ ions increase the affinity of the ATP-sensitive K⁺ channel to ATP and to other reversible channel blockers and that a functionally important SH-group is located at or near the ATP-binding site.     

Observations on phase-locking within the response of primary muscle spindle afferents to pseudo-random stretch

In order to uncover encoder properties of primary muscle spindle afferent fibers, time coupling (phase-locking) of action potentials on cyclic muscle stretch was studied by means of pseudo-random noise. In cats Ia action potentials were recorded from dorsal root filaments and the gastrocnemius muscles of one hind leg were stretched. The stimulus time course was a determined sequence of randomly varying muscle length which could be applied repeatedly (sequence duration 0.6 or 20 s). The noise amplitude (standard deviation of displacements) was varied between 5 and 300 m, the upper cut-off frequency of noise f _c was varied between 20 and 100 Hz. The responses to the consecutive pseudo-random noise cycles were displayed as raster diagrams and cycle histograms. Phaselocking characterized the responses at all noise amplitudes outside the near threshold range (>10 m). The higher and f _c , the stronger was the phase-locking of impulses on the stretch. When and f _c were selected to achieve high mean stretch velocities of about 500 mm/s, phase-locking was as precise as 0.15 ms, measured as the variability of spike occurrences with respect to stretch. The rasters obtained with low noise amplitudes (<40 m) showed a loose phase-locking and this gave insight into underlying mechanisms: The elicitation of action potentials caused by dynamic stretch can be prevented by a post-spike depression of excitability. This disfacilitation was very effective in counteracting weak stretch components within the random sequence and less effective or even missing when relatively strong stretch components could force the spike elicitation. This led to the reestablishment of phase-locked patterns. The results were discussed in relation to the known encoder models.     

Computer simulation of the impulse pattern of muscle spindle afferents under static and dynamic conditions

Summary A simple model has been employed to describe and interprete measurements from deefferented muscle spindle afferents with static and dynamic stimulation; the model simulates the generator potential of the spindle and the time dependent change of sensitivity at the impulse generating membrane. The properties of the model in transforming the steady and time dependent analogue signals into impulse patterns are demonstrated, and the influence of the various parameters on the response characteristics have been investigated. Results from simulations are compared with experimental data, and it is shown that the impulse patterns of secondary muscle spindle afferents can be simulated quantitatively. The frequency distributions of impulse intervals and different sequential dependencies within the impulse patterns are analysed.

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Zusammenfassung Zur Beschreibung und Deutung von Messungen an deefferentierten Muskelspindelafferenzen unter statischer und dynamischer Reizung wird ein einfaches Modell verwendet, das haupts?chlich das Generatorpotential des Mechanoreceptors und die zeitabh?ngige ?nderung der Empfindlichkeit an der impulserzeugenden Membran simuliert. Die Eigenschaften des Modells bei der Transformation konstanter und zeitabh?ngiger Analogsignale in Impulsfolgen werden dargestellt und die Einflüsse der verschiedenen Modellparameter untersucht. Im Vergleich der Simulationsergebnisse mit experimentellen Daten wird gezeigt, da? die Impulsmuster sekund?rer Muskelspindelafferenzen quantitativ simuliert werden k?nnen. Die dabei verwendeten Parameter werden angegeben. Analysisert werden die H?ufigkeitsverteilungen der Impulsintervalle und verschiedene sequentielle Abh?ngigkeiten innerhalb der Impulsfolgen.

     

Characterization of silent afferents in the pelvic and splanchnic innervations of the mouse colorectum

Hypersensitivity in inflammatory/irritable bowel syndrome is contributed to in part by changes in the receptive properties of colorectal afferent endings, likely including mechanically insensitive afferents (MIAs; silent afferents) that have the ability to acquire mechanosensitivity. The proportion and attributes of colorectal MIAs, however, have not previously been characterized. The distal ～3 cm of colorectum with either pelvic (PN) or lumbar splanchnic (LSN) nerve attached was removed, opened longitudinally, pinned flat in a recording chamber, and perfused with oxygenated Krebs solution. Colorectal receptive endings were located by electrical stimulation and characterized as mechanosensitive or not by blunt probing, mucosal stroking, and circumferential stretch. MIA endings were tested for response to and acquisition of mechanosensitivity by localized exposure to an inflammatory soup (IS). Colorectal afferents were also tested with twin-pulse and repetitive electrical stimulation paradigms. PN MIAs represented 23% of 211 afferents studied, 71% (30/42) of which acquired mechanosensitivity after application of IS to their receptive ending. LSN MIAs represented 33% of 156 afferents studied, only 23% (11/48) of which acquired mechanosensitivity after IS exposure. Mechanosensitive PN endings uniformly exhibited significant twin-pulse slowing whereas LSN endings showed no significant twin-pulse difference. PN MIAs displayed significantly greater activity-dependent slowing than LSN MIAs. In conclusion, significant proportions of MIAs are present in the colorectal innervation; significantly more in the PN than LSN acquire mechanosensitivity in an inflammatory environment. This knowledge contributes to our understanding of the possible roles of MIAs in colon-related disorders like inflammatory/irritable bowel syndrome.     

Characterization of in vitro motility assays using smooth muscle and cytoplasmic myosins

We have used two in vitro motility assays to study the relative movement of actin and myosin from turkey gizzards (smooth muscle) and human platelets. In the Nitella-based in vitro motility assay, myosin-coated polymer beads move over a fixed substratum of actin bundles derived from dissection of the alga, Nitella, whereas in the sliding actin filament assay fluorescently labeled actin filaments slide over myosin molecules adhered to a glass surface. Both assay systems yielded similar relative velocities using smooth muscle myosin and actin under our standard conditions. We have studied the effects of ATP, ionic strength, magnesium, and tropomyosin on the velocity and found that with the exception of the dependence on MgCl2, the two assays gave very similar results. Calcium over a concentration of pCa 8 to 4 had no effect on the velocity of actin filaments. Phosphorylated smooth muscle myosin propelled filaments of smooth muscle and skeletal muscle actin at the same rate. Phosphorylated smooth muscle and cytoplasmic myosin monomers also moved actin filaments, demonstrating that filament formation is not required for movement.     

Characterization of the mRNA for mouse muscle acetylcholine receptor alpha subunit by quantitative translation in vitro

The nicotinic acetylcholine receptor of mammalian skeletal muscle is a multisubunit membrane glycoprotein whose synthesis is regulated by developmental and physiological cues. We report here the identification and characterization of the primary translation product of alpha subunit mRNA. The alpha subunit synthesized in rabbit reticulocyte lysate is approximately 2000 larger in apparent molecular weight than the native alpha subunit polypeptide found in acetylcholine receptor. Evidence from peptide maps and the effect of co-translational incubation with dog pancreas microsomes suggests that the in vitro product differs in two ways from native alpha subunit: 1) it is synthesized with an NH2-terminal signal peptide which is removed in vivo, and 2) the in vitro product is not glycosylated. We have characterized the alpha subunit mRNA activity by using a quantitative the membrane-bound polysome fraction. It is poly(A+) and approximately 2000 nucleotides long. Finally, we have shown that in BC3H-1 cells, alpha subunit mRNA is regulated developmentally. We detected a 10-fold increase in the relative abundance of alpha subunit mRNA in cells which had undergone the transition from log phase growth to differentiated myoblast.     

Characterization of an adult muscle acetylcholine receptor subunit by expression in fibroblasts.

To study the functional and structural roles of the epsilon subunit in adult muscle acetylcholine receptor (AChR), we have co-expressed the alpha and epsilon subunits of the mouse receptor in transfected fibroblasts. Ligand binding studies suggest that association of epsilon with alpha subunit results in a lower association rate constant for 125I-labeled alpha-bungarotoxin binding than that of the unassembled alpha subunit, approaching that for toxin binding to the AChR. Furthermore, alpha epsilon complexes contain high affinity binding sites for competitive antagonists and agonists not present in the unassembled alpha subunit, but similar to one of the two nonequivalent binding sites in the adult AChR. Structural analysis of alpha epsilon complexes by sucrose gradient velocity centrifugation suggests that some of the complexes formed are trimers or tetramers of alpha and epsilon subunits. Comparison of these data with those previously obtained for alpha gamma complexes suggests that gamma and epsilon have homologous functional roles and identical structural positions in the fetal and adult AChRs, respectively.     

Fusimotor reflexes influencing secondary muscle spindle afferents from flexor and extensor muscles in the hind limb of the cat.

The purpose of this study was to investigate secondary muscle spindle afferents from the triceps-plantaris (GS) and posterior biceps and semitendinosus (PBSt) muscles with respect to their fusimotor reflex control from different types of peripheral nerves and receptors. The activity of single secondary muscle spindle afferents was recorded from dissected and cut dorsal root filaments in alpha-chloralose anaesthetized cats. Both single spindle afferents and sets of simultaneously recorded units (2-3) were investigated. The modulation and mean rate of firing of the afferent response to sinusoidal stretching of the GS and PBSts muscle were determined. Control measurements were performed in the absence of any reflex stimulation, while test measurements were made during reflex stimulation. The reflex stimuli consisted of manually performed movements of the contralateral hind limb, muscle stretches, ligament tractions and electrical stimulations of cutaneous afferents. Altogether 21 secondary spindle afferents were investigated and 20 different reflex stimuli were employed. The general responsiveness (i.e. number of significant reflex effects/number of control-test series) was 52.4%, but a considerable variation between different stimuli was found, with the highest (89.9%) for contralateral whole limb extension and the lowest (25.0%) for stretch of the contralateral GS muscle. The size of the response to a given stimulus varied considerably between different afferents, and, in the same afferent, different reflex stimuli produced effects of varying size. Most responses were characterized by an increase in mean rate of discharge combined with a decrease in modulation, indicative of static fusimotor drive (Cussons et al., 1977). Since the secondary muscle spindle afferents are part of a positive feedback loop, projecting back to both static and dynamic fusimotor neurones (Appelberg Et al., 1892 a, 1983 b; Appelberg et al., 1986), it is suggested that the activity in the loop may work like an amplified which, during some circumstances, enhance the effect of other reflex inputs to the system (Johansson et al., 1991 b).     

Mineralization of adult mouse bone marrow in vitro

Murine adult bone marrow exhibits mineralizing capacity in vitro as is demonstrated by the new in vitro assay we report here. In less than 2 weeks after the onset of the cultures, mineralization is obtained in more than 80% of the marrow cultures. Moreover, morphological studies reveal that during incubation phenotypic changes related to osteogenic differentiation occur at the extracellular matrix as well within cell populations. Well banded collagen is synthesized. Matrix vesicles and needles of hydroxy-apatite crystals are observed via transmission electron microscopy. Osteoblast-like cells are present with membrane-associated alkaline phosphatase activity. The mineralization is specific for cultured bone marrow and is not observed in cultured spleen fragments as is shown via 85Sr uptake, calcein uptake and histomorphology. No inducing agent is added to the tissue culture medium except for 10% fetal calf serum, beta-glycerophosphate (10(-2) M) and ascorbic acid. However, the prerequisite for obtaining mineralization is the three-dimensional structure of the marrow in culture. The in vitro organ culture we developed may provide the opportunity to identify which marrow cells have osteogenic potential and to investigate the mechanisms triggering differentiation towards osteogenesis.     

Different fusimotor reflexes from the ipsi- and contralateral hind limbs of the cat assessed in the same primary muscle spindle afferents

Experiments were performed in forty-five cats anaesthetized with alpha-chloralose. The aim of the study was to investigate a sample of primary muscle spindle afferents from triceps muscle with respect to their fusimotor reflex control from ipsi- as well as contralateral hind limb. Primary muscle spindle afferents of the triceps surae muscle were recorded from the mean rate of firing and the modulation of the afferent response to sinusoidal stretching of the triceps surae muscle was determined. Test measurements were made during tonic stretch of the ipsilateral PBSt, contralateral PBSt, contralateral triceps muscle or during extension of the intact contralateral hind limb. Control measurements were made with ipsi- and contralateral PBSt as well as contralateral triceps muscles relaxed and with contralateral hind limb in resting position. The occurrence and types of fusimotor effects were assessed by comparing test to control responses. The main finding of the present investigation was the great variability in type and size of the fusimotor effects evoked by different ipsi- and contralateral reflex stimuli. Both ipsi- and contralateral stimulations gave rise to predominantly dynamic, predominantly static or mixed static and dynamic fusimotor reflexes. In the same preparation, a given reflex stimulus often caused different reflex responses in different triceps surae primary spindle afferents. In the same afferent unit, different reflex stimuli usually produced fusimotor effects which differed from each other in type and/or size. In general, contralateral whole limb extension and stretch of contralateral PBSt muscles were more potent as reflex stimuli than stretch of the ipsilateral PBSt muscle. Stretch of the contralateral triceps surae muscle was, but for a few afferent units, ineffective as reflexogenic stimulus. It is concluded that the individualized receptive profiles of the primary muscle spindle afferents, which have been postulated in earlier investigations where the effects of different stimuli have been investigated on different cell populations, still seems to hold good when the stimuli are tested on the same units. The individuality of the receptive profiles of gamma-motoneurones is discussed in relation to different motor control hypotheses.     

Fusimotor reflex profiles of individual triceps surae primary muscle spindle afferents assessed with multi-afferent recording technique.

The experiments were performed on 21 cats anaesthetized with alpha-chloralose. The aim of the study was to investigate sets of simultaneously recorded spindle afferents (2-4 in each set) from the triceps surae muscle (GS) with respect to the pattern of fusimotor reflex effects evoked by different types of ipsi- and contralateral reflex stimulation. The afferents' responses to sinusoidal stretching of the GS muscle were determined and the fusimotor reflex effects were assessed by comparing the afferent responses (i.e. the mean rate of firing and the depth of modulation) elicited during reflex stimulation with those evoked in absence of any reflex stimulus. Natural of electrical activations of ipsi- and contralateral muscle, skin and joint receptor afferents were used as reflex stimuli. The spindle afferents were influenced by several modalities and from wide areas, with a majority responding to both ipsi- and contralateral stimuli. A particular reflex stimulus often caused different effects on different afferents, and the various reflex stimuli seldom gave similar effects on a particular afferent. Multivariate analysis revealed that the variation in response profiles among simultaneously recorded afferents were as great as between afferents recorded on different occasions. This suggests that the individualized response prifiles, observed in earlier investigations, represent a very diversified reflex control of the spindle primary afferents, and are not a reflection of changes in the setting of the spinal interneuronal network, occurring during the time interval between the recordings of different units. Also, there was no relation between the conduction velocity of the afferents and the reflex profiles of the afferents, but non-linear relations were found between effects elicited by different types of stimuli. Indications were also found that it may be possible to separate the population of GS muscle spindles into subgroups, according to the fusimotor effects exhibited by activation of various categories of ipsi- and contralateral receptor afferents. It is concluded that one possible way of making the very complex reflex system controlling the muscle spindles intelligible may be a combination of multiple simultaneous recordings of spindle afferents and multivariate analysis.     

Characterization of thymic progenitors in adult mouse bone marrow

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1(-)) (Thy1.1(-) progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1(-) progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1(-) population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.     

Evaluation of an in vitro muscle contraction model in mouse primary cultured myotubes

To construct an in vitro contraction model with the primary cultured myotubes, we isolated satellite cells from the mouse extensor digitorum longus. Differentiated myotubes possessed a greater number of sarcomere assemblies and higher expression levels of myosin heavy chain, cytochrome c oxidase IV, and myoglobin than in C2C12 myotubes. In agreement with these results regarding the sarcomere assemblies and protein expressions, the primary myotubes showed higher contractile activity stimulated by the electric pulses than that in the C2C12 myotubes. These data suggest that mouse primary myotubes will be a valuable research tool as an in vitro muscle contraction model.     

Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse

To generate temporally-controlled targeted somatic mutations selectively and efficiently in smooth muscles, we have established a transgenic SMA-Cre-ER(T2) mouse line in which the expression of the Tamoxifen-dependent Cre-ER(T2) recombinase is under the control of a large genomic DNA segment of the mouse smooth muscle alpha actin (SMA) gene, contained in a Bacterial artificial chromosome (Bac). In this transgenic mouse line, Cre-ER(T2)-mediated recombination of LoxP-flanked target DNA is strictly Tamoxifen-dependent, and efficient in both vascular and visceral smooth muscle cells. Moreover, with the exception of few cardiomyocytes, LoxP-flanked DNA excision is restricted to smooth muscle cells. Thus, SMA-Cre-ER(T2) mice should be of great value to analyze gene function in smooth muscles, and to establish new animal models of human smooth muscle disorders.     

Parvalbumin in mouse muscle in vivo and in vitro

Parvalbumin is a cytosolic calcium-binding protein found in adult fast-twitch mammalian muscle. Using an antibody to paravalbumin, we have shown that its distribution in adult mouse muscles is associated with certain fibre types. It is absent from slow-twitch type 1 fibres, is absent or at low levels in fast-twitch type 2A fibres, but is present at moderate or high levels in fast-twitch type 2B fibres. When adult mouse muscle is cultured with embryonic mouse spinal cord, the regenerated fibres become innervated, express the adult fast isoform of myosin heavy chain and appear histochemically as fast-twitch fibres. We therefore investigated whether these apparently mature fibres also contained parvalbumin. Parvalbumin was not found in any fibres of twenty mature cultures, suggesting that neurotrophic activity in the absence of specific adult nerve activity patterns was insufficient to cause the expression of parvalbumin in the cultures.     

Characterization of Sgo1 expression in developing and adult mouse

SGO1 has been characterized in its function in correct cell division and its role in centrosome cohesion in the nucleus. However, its organ-specific maturation-related expression pattern in vivo remains largely uncharacterized. Here, we show clear SGO1 expression in post-developmental neuronal cells and cytoplasmic localisation in nucleated cells with a transgenic mice model and immunohistochemistry of wild type mice. We demonstrate extranuclear expression of Sgo1 in the developing heart and gut, which have been shown to be dysregulated in humans with homozygous SGO1 mutation. Additionally, we show Sgo1 expression in select population of retinal cells in developing and post-developmental retina. Our expression analysis strongly suggests that the function of SGO1 goes beyond its well characterized role in cell division.