Electrical properties and innervation of fibers in the orbital layer of rat extraocular muscles.

1. The inferior rectus muscle of rat, one of the extraocular muscles, contains two populations of multiply innervated fibers (MIFs): orbital MIFs, located in the orbital layer of the muscle and global MIFs, found in the global layer. The electrical properties and the responses to nerve stimulation of orbital MIFs were studied with single intracellular electrodes and compared with those of twitch fibers of the orbital layer, MIFs of the global layer, and tonic fibers of the frog. 2. About 90% of the orbital MIFs did not produce overshooting action potentials. In these fibers the characteristics and time course of the responses to nerve stimulation varied along the length of the fibers. Within 2 mm of the end-plate band of the muscle, the responses consisted of several small end-plate potentials (EPPs) and a nonovershooting spike. Distal to 2 mm, the responses in most fibers consisted of large and small EPPs with no spiking response. Some fibers produced very small spikes surmounted on large EPPs. 3. Overshooting action potentials were observed in approximately 10% of the orbital MIFs recorded between the end-plate band and 2 mm distal. The presence or absence of action potentials was not related to the magnitude of the resting potential of the fibers. 4. The threshold of nerve stimulated responses in orbital MIFs was the same as that in orbital twitch fibers. A large number of orbital MIFs had latencies equal to those for the orbital twitch fibers recorded at the same distance from the end-plate band, but the average latency was greater in the MIFs. The latency of orbital MIFs was about one-half of that for the MIFs of the global layer. The values for the effective resistance and membrane time constant of orbital MIFs fell between those for orbital twitch fibers on the one hand, and global MIFs and frog tonic fibers on the other. 5. In order to compare electrical properties with innervation patterns, fibers identified electrophysiologically as orbital MIFs were injected with the fluorescent dye Lucifer yellow and then traced in Epon-embedded, serial transverse sections. In addition to numerous superficial endings distributed along the fibers, a single "en plaque" ending was also found in the end-plate band that resembled the end plates of the adjacent orbital twitch fibers. 6. From these results we conclude that the electrical activity of orbital MIFs varies along the length of the fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Dependence of tonic tension on extracellular calcium in rat extraocular muscle.

Ca2+-free saline containing 3.0 mM Mg2+ virtually abolishes the tonic tension evoked by depolarization with a high K+ concentration of the tonic multiply innervated fibers of rat extraocular muscles. The tonic tension abolished by Ca2+ withdrawal is restored when Ca2+ is substituted by Sr2+ but not by Ni2+. The increase of Mg2+ reduces the tonic tension and displaces the tension-log K+ relationship to the right. Cd2+ significantly reduces the tension amplitude but does not shift the tension-log K+ relationship. The organic blocker of Ca2+ channels, nifedipine (1-10 microM), has no effect on the tonic tension. In contrast, diltiazem (20 microM) reduces the amplitude of the responses without changing the tension-log K+ relationship. Both foreign anions NO3- and SCN- potentiate tonic tension without changing the tension-log K+ relationship. SCN- increases the resting tension of the muscle; this effect depends on Ca2+. In conclusion, the disappearance of tonic tension after Ca2+ withdrawal is not due to depolarization of the fibers or inactivation of the contractile responses. It is suggested that entry of extracellular Ca2+, via a voltage-dependent Ca2+ conductance, or specific interactions of Ca2+ with membrane sites involved in the regulation of excitation-contraction coupling play a role in evoking tension in tonic fibers.

Effects of epinephrine on resting and tonic tensions of rat extraocular muscles.

"In vitro" epinephrine (10(-6) to 10(-4) M) stimulates the rat inferior rectus, one of the extraocular muscles (EOMs). When applied to resting muscles, it induces a slow rising tension in about one half of the preparations. The amplitude of this tension is equivalent to about 9% of the maximal tetanic response. This effect is blocked by propranolol (10(-7) to 10(-6) M). When epinephrine is applied prior to a depolarization of the muscle fibers with 30 mM K, the drug consistently induces a significant potentiation of the long lasting tonic tension normally evoked by that elevated K concentration. One possible explanation for these results is that epinephrine induces a slow rising tension by increasing a resting Ca conductance of the sarcolemma of the tonic or Felderstruktur fibers of EOM.

Induction of action potentials by denervation of tonic fibres in rat extraocular muscles.

The effects of denervation by nerve section on the electrical properties of tonic and twitch fibres of rat extraocular muscles were examined. Normally innervated tonic fibres lack action potentials. Upon direct stimulation they generate graded, voltage-dependent responses or slow peak potentials (s.p.p.s). However, one week after denervation the s.p.p.s are transformed into action potentials which are slower and broader than those of twitch fibres. The action potentials are Na dependent and partially resistant to blockade with 10(-5) M-tetrodotoxin and 10(-6) M-saxitoxin. Changing the holding potential of the fibres from -80 mV to more negative levels increases the maximal rate of rise of the action potential. This effect is not observed on the s.p.p.s of normally innervated fibres. Following denervation the resting potential of tonic and twitch fibres becomes about 10-15 mV less negative. In denervated muscles stimulation with pulses of hyperpolarizing current evokes graded responses in tonic fibres and action potentials in twitch fibres. In normally innervated muscles, these anodal break responses are never observed in tonic fibres and are very rare in twitch fibres. By two weeks after nerve section, reinnervation is present. The action potentials of tonic fibres are still present but stronger stimulation is needed to evoke anodal break responses. By three weeks, direct stimulation of tonic fibres evokes normal s.p.p.s in about 25% of the studied fibres and action potentials in the rest. By four weeks, most tonic fibres have lost the action potential but small anodal break responses can be evoked in most. It is suggested that following denervation a new population of Na channels appears in tonic fibres. The properties of these channels are different from those of the channels normally present in innervated tonic fibres but they are in some ways similar to those of the channels which appear in twitch fibres following denervation.

Cadmium reduces extraocular muscle contractility in vitro and in vivo.

Cadmium, a blocker of calcium channels in various excitable cells, reduces the contractility of extraocular muscles. When applied to rat extraocular muscles in vitro, it reduces the sustained or tonic tension generated by the tonic multiply innervated fibers of the global layer of the muscles. When injected in vivo into rabbit extraocular muscles, it produces a temporary paralysis of the muscles and a deviation of the eye position. These effects are presumed to involve a blockade of the calcium channels of the muscle fibers and of the neuromuscular junctions. It is proposed that, on the basis of these effects, a non-surgical treatment of strabismus could be developed.

Distribution of mitochondrial and lipidic alterations in abnormal extraocular muscle of rat.

An abnormal superior rectus muscle of rat displayed marked differences in the respective distributions of mitochondrial alterations and excessive lipid accumulations, both of which are thought to be indicators of faulty oxidative metabolism. The mitochondrial alterations were widespread, extending over 46% of muscle length. In contrast, the excess lipids extended over but 11% of the muscle length and were virtually confined to the end-plate region. The end plates themselves were essentially normal. These data raise the possibility that the end-plate region may exhibit a locally greater deficit of oxidative metabolism, due to a possibly higher metabolic requirement needed to support the localized end-plate potential activity.

Intermitochondrial junctions in the extraocular muscle of the rat.

Intermitochondrial junctions with a spacing of 17-21 nm were observed in the superior rectus muscle of a rat. Periodic rounded densities are aligned midway between the apposed outer mitochondrial membranes at some of these junctions. Such densities have a diameter of about 8-10 nm and a center-to-center spacing of about 26-30 nm. These junctions occur in cases where one mitochondrial profile is enclosed within another or where two profiles are interlocked so that their combined overall form has a smoothly contoured profile. Intermitochondrial junctions seem not to have been previously described in muscle, but have been reported in other kinds of tissues. In agreement with those previous reports, the presently observed intermitochondrial junctions usually involve mitochondria that display atypical features indicative of tissue abnormality or stress. Such junctions were never observed in normal extraocular muscle.

Morphologic and electrophysiologic identification of multiply innervated fibers in rat extraocular muscles.

Mammalian extraocular muscles comprise singly and multiply innervated fibers (SIFs, MIFs) that are distributed in both the global and orbital layers of the muscles. Previously it has been concluded solely on the basis of electrophysiologic analysis that in rat, MIFs of the global layer show graded responses or slow peak potentials instead of the action potentials exhibited by SIFs. To confirm directly this conclusion, a protocol that allows the combined electrophysiologic-morphologic characterization of muscle fibers has been developed. It is based on electrophysiological analysis of the muscle fibers followed by intracellular labeling with Lucifer yellow and staining of nerve endings with a modified Koelle stain for cholinesterase activity. Labeled fibers have been recognized and followed in sequential 20-micron cross-sections of the whole muscle.

Calcium action potentials in rat fast-twitch and slow-twitch muscle fibres.

Slow action potentials were evoked in twitch fibres of rat extensor digitorum longus (e.d.l.) and soleus muscles after drastically reducing the Cl and K conductances of the muscle fibres. Cl conductance was eliminated by exposing the muscles to a Cl-free saline in which methanesulphonate replaced Cl. K conductance was reduced by adding tetraethylammonium (TEA) and 3,4-diaminopyridine (3,4-DAP) to the Cl-free saline or by overnight incubation of the muscles in a saline containing Cs and TEA. The delayed rectifier was markedly blocked by TEA and 3,4-DAP. In contrast, the inward rectifier was blocked only by TEA. Depolarization with pulses of increasing amplitude triggered slow responses which had a threshold of -30 to -10 mV and a peak amplitude of 50-60 mV. In e.d.l. muscles the time course of the response was sustained for the duration of the pulses and was not affected by repeated stimulation. In soleus muscles the first evoked response was sustained in about 60% of the fibres and transient in the rest. Transient responses reached a peak amplitude and were followed by a hyperpolarization. Repeated stimulation irreversibly transformed the sustained responses of soleus fibres into transient ones. The responses were blocked when the Ca in saline was replaced by Mg (10 mM) or Co (5 mM) or by the addition of Cd (0.1-1.0 mM) or nifedipine (5-6 microM). Tetrodotoxin did not affect the responses. These results strongly suggest that Ca is the main carrier of current during the response. Nifedipine blocked both the Ca response and the subsequent hyperpolarization, suggesting that the latter is due to the activation of a Ca-dependent K conductance.

Effect of calcium withdrawal on mechanical threshold in skeletal muscle fibres of the frog.

1. Voltage-clamp experiments were performed on frog skeletal muscle fibres using two intracellular micro-electrodes. The threshold for the Na current and the strength-duration curve for mechanical threshold were determined. 2. The change in threshold for the Na current was studied as a function of the external Ca and Mg concentrations which ranged from 0.1 to 50 mM. 3. The resting potential, effective resistance and threshold for the Na current were unchanged when 1.8 mM-Ca was replaced by 3 mM-Mg, indicating that the surface potential and the electrical properties of the fibres were not modified. The additon of 5 mM-EGTA did not affect these parameters. 4. In Ca-free saline (3 mM-Mg and 5 mM-EGTA) the mechanical threshold was significantly increased for short pulses (less than or equal to 20 msec.). In isolated single muscle fibres this effect was observed shortly after applying the Ca-free saline, and was rapidly reversed upon the return to control saline. 5. In isotonic EGTA (85 mM-EGTA) the muscle fibres were depolarized and were unable to contract even if they were hyperpolarized to --90 mV for 12 min prior to stimulation. If 3 mM-Mg was added, most fibres contracted locally. 6. In single muscle fibres caffeine contractures were unmodified after a 30 min exposure to Ca-free saline. 7. It can be concluded that external Ca withdrawal impairs Ca release from the sarcoplasmic reticulum and that external Ca is not essential for triggering contraction.

Curare-like effect of propranolol on rat extraocular muscles.

1 The effects were studied of the beta-adrenoceptor blocking drug, (+/-)-propranolol and its (+)- and (-)-isomers on contractility of rat isolated diaphragm and inferior rectus muscles. 2 Propranolol (10(-5) M) did not modify the resting tension nor the electrically-induced twitch contraction in diaphragm and extraocular muscles, nor the tonic tension evoked in the latter by high K concentrations. 3 In inferior rectus preparations (+/-)-propranolol (5 x 10(-6) M) reduced significantly tensions evoked by succinylcholine (SCh). Cumulative dose-response curves to SCh were shifted to the right in a nearly parallel manner by (+)- and (-)-propranolol which were about equiactive. 4 It is concluded that the depressant action of propranolol is independent of its adrenoceptor blocking potency and it is not due to an alteration of the contraction mechanism. Propranolol appears to depress SCh-evoked tensions through a curare-like effect on cholinoceptors.

Intracellular marking of skeletal muscle cells with horseradish peroxidase in combination with a stain for cholinesterase.

A procedure is described by which muscle fibers can be electrophysiologically studied, intracellulary marked with horseradish peroxidase (HRP), and stained for acetylcholinesterase (AChE) activity to identify nerve endings. Following electrophysiological characterization, 4% HRP is injected through the recording microelectrode with small depolarizing pulses. Muscles are fixed, washed, and stained with a Koelle procedure for AChE modified by extending the incubation period to 15 hours to identify small nerve endings. The HRP is visualized with 3,3'-diaminobenzidine tetrahydrochloride or benzidine dihydrochloride. Stained muscles are embedded in Epon and sectioned. Lucifer yellow dye is also demonstrated to be an excellent intracellular marker of muscle fibers but it cannot be combined with AChE staining.

Ionic basis for electrical properties of tonic fibres in rat extraocular muscles.

1. The ionic conductances underlying some of the electrophysiological properties of multiply innervated or tonic fibres of rat extraocular muscles were examined in vitro with double-barrelled micro-electrodes.2. Exposure of the muscle to a Cl-free saline did not change the effective resistance (R(eff)) of tonic fibres which was 5.14 +/- 0.45 MOmega (n = 7) in control saline and 4.78 +/- 0.45 MOmega (n = 12) in Cl-free saline (P > 0.1). In contrast, in singly innervated or twitch fibres Cl removal increased R(eff) from 1.77 +/- 0.21 MOmega (n = 19) to 2.69 +/- 0.12 MOmega (n = 22) (P < 0.001).3. Tonic fibres with membrane potentials restored to - 80 mV by injecting current responded to intracellular depolarizing pulses with a brief, slow response (slow peak potential) which added to the rising phase of the electrotonic potential. The slow peak potential began at a membrane potential of - 40 to - 35 mV and was graded. Increasing depolarizations evoked faster and larger responses which did not over-shoot the zero level of membrane potential.4. The slow peak potential was not blocked by 10 muM-D-600 hydrochloride but was markedly reduced by the absence of Na and by 10 muM-tetrodotoxin. The response was broadened about five times by 25 mM-tetraethylammonium.5. Raising bath temperature from 21-25 degrees C to 37 degrees C reversibly depressed and shortened the slow peak potential but did not transform it into an action potential.6. It is concluded that the characteristic high R(eff) of tonic fibres results from a lack of a membrane conductance to Cl and that the slow peak potential involves the transient activation of Na and K channels which are pharmacologically similar to the respective channels of twitch fibres.

Electrophysiological identification of two types of fibres in rat extraocular muscles.

1. The synaptic potentials and electrical properties of rat inferior rectus muscles were examined in vitro. 2. In most fibres the spontaneous synaptic activity consisted of typical miniature end-plate potentials which had a normal distribution of amplitudes and rather uniform time courses. Suprathreshold and maximal nerve stimulation evoked unitary end-plate potentials (e.p.p.s). The synaptic activity of these fibres could be recorded only in the innervation zone of the muscle. These fibres were identified as being focally innervated. 3. Focally innervated fibres gave action potentials upon direct and indirect stimulation. They had an effective resistance (Reff) of 1.62 +/- 0.22 M omega (mean +/- S.E., twenty-two fibres) and a time constant (tau m) of 3.8 +/- 0.4 msec (twenty-one fibres). Voltage-current curves in control saline were linear between membrane potentials of -50 to -140 mV. 4. In a small number of fibres the spontaneous synaptic activity consisted of miniature small-nerve junction potentials which had a skewed distribution of amplitudes with predominance of smaller voltages and time courses with a wide range of variation. Nerve stimulation evoked composite small-nerve junction potentials (s.j.p.s) which could be resolved into unitary components by varying the strength of stimulation. S.j.p.s had a higher threshold than e.p.p.s. Synaptic potentials could be recorded outside the innervation zone, at various sites along the muscle length. These fibres were recognized as being multiply innervated with polyneuronal innervation. 5. Multiply innervated fibres lacked action potentials had a large Reff of 6.0 +/- 1.1 M omega (six fibres) and a prolonged tau m of 29.8 +/- 4.8 msec. Reff show a moderate decrease to hyperpolarization and a rather large decrease to depolarization which denote, respectively, the presence of anomalous and delayed reactification. 6. It is concluded that rat extraocular muscles contain at least two populations of muscle fibres that in terms of synaptic activity and electrical properties are comparable to twitch fibres of other mammalian muscles and to slow or tonic fibres of amphibians.

Activation of two types of fibres in rat extraocular muscles.

1. The contractile responses of the inferior rectus, one of the extraocular muscles of the rat, to a depolarization induced by an elevation of the potassium concentration in the external medium ([K]O) have been studied 'in vitro'. 2. The elevation of [K]O to 20 and 30 mM-K produced contractures that consisted of a sustained or tonic tension. When [K]O was increased to 50 mM or more a well-defined transient or phasic tension appeared before the tonic response. The increment of [K]O above 50 mM enhanced the phasic component and depressed the tonic tension. The maximal tonic tension, usually evoked by 50 mM-K, is about 50% of the tetanic tension, shows a gradual decline with time and lasts for hours. Control experiments performed in diaphragm showed that this muscle only responds with phasic tensions. 3. The difference in the repriming of the phasic and tonic responses when tensions were induced with salines containing low or normal [Cl] suggests that the muscle fibres responsible for the tonic tension are poorly permeable to Cl-. 4. The amplitude of the tonic tension was reduced by Ca deprivation and by an elevation of [Ca] in the saline to 10 mM. 5. It is concluded that in rat extraocular muscles, an increase in [K]O activates two types of muscle fibres: singly and multiply innervated. These appear to be functionally equivalent to the twitch and slow fibres of amphibian and avian muscle and would give rise to the phasic and tonic components of the contracture, respectively.

Twitch potentiation by potassium contractures in single muscle fibres of the frog.

1. The modifications of isometric twitches after K contractures have been studied in single muscle fibres from frog muscles.2. The twitch was greatly enhanced and somewhat prolonged after a conditioning K contracture. This potentiating effect was more evident in fibres having low values of the ratio control twitch tension/tetanus tension (P/P(0)).3. In most of the fibres, the time course of the decay of the potentiation phenomenon could be represented by the sum of three negative exponentials with average half-times of 9.1, 40 and 485 sec.4. Between 3 and 10 min after the onset of the twitch potentiation the resting potential and action potential were unchanged.5. Mn blocked the K contractures and prevented the onset of the twitch potentiation.6. 40-140 sec after inducing a K contracture with 30 or 40 mM-K, a second exposure to the same [K] evoked a tension equivalent only to 57% of the first one. This partial refractoriness of the fibres is not seen when both contractures were elicited with 190 mM-K, and contrasts with the full recovery and potentiation of the twitch after a K contracture.