Search

Boston, Massachusetts Comroe [l] has summed up the contemporary understanding of respiratory centers in man. He reviewed the presence of a medullary center with crudely rhythmic properties and a pair of chemoreceptors (on either side of the medulla) which are affected by changes in pH. The apneustic center in the lower pons from time to time stimulates inspiration only. These impulses are self-limiting through the Hering-Breuer reflex, but the apneustic center is also inhibited by a third respiratory center in the upper third of the pons known as the pneumotaxic center. The location of the respiratory centers in the brain stem was considered to be a strategic one to allow interaction with the reticular activating system. The medullary center received impulses from the cortex, hypothalamus, pons, reticular activating system and sensory stimuli from somatic and visceral tissues. These impulses eventually traveled to the phrenic nerve to activate the diaphragm which played the major role in respiration. Fox [2], Hitchcock and Leece [3] and Pitts [4] have indicated a separation of intercostal and diaphragmatic respiratory pathways. They found different involuntary respiratory fibers deep to the spinothalamic tract and lateral to the anterior horns. Interception of this tract on either side (reticulospinal) caused an immediate reduction of tidal volume from diaphragmatic paralysis. In certain subjects with superimposed respiratory disease this even occasioned apnea and death in deep sleep. These investigators also believed that the motor pathway to the intercostal muscles was situated immediately behind those for the diaphragm. Despite this structured information of respiratory anatomy and physiology, we encountered a patient with a respiratory problem that defied adequate explanation. The patient was able to breathe unaided when awake but became totally apneic when asleep. She From the Tufts Lung Station, Tufts Surgical, Harvard Medical and Neurology Services of Boston City Hospital, Boston, Massachusetts. Requests for reprints should be addressed to Dr. Sadamu Ishikawa, Tufts University School of Medicine, Lemuel Shattuck Hospital, 170 Morton Sheet, Jamaica Plain, Massachusetts 02130. Manuscript accepted November 29. 1973.

The neuroparalytic action of botulinum toxin type A was studied on rat phrenic nervehemidiaphragm preparations. It was determined that the toxin would bind, but would not paralyze, preparations bathed in media that depressed ACh release. The binding was essentially irreversible, but it left the toxin susceptible to inactivation by anti-toxin. The binding process was little affected by temperature (20–35°C), but markedly affected by low ionic strength ( I −2 ) and low pH (4.5). These data are used to propose a model for the interaction between botulinum toxin and cholinergic nerve terminals.

In the endplates of rat phrenic nerve-diaphragm, application of the acetylcholine-like compound, carbachol, causes a marked increase in transmitter release, as measured electrophysiologically using miniature endplate potential frequency. Washing out of carbachol reverses the increase in frequency. The ability of carbachol to increase transmitter release is greatly enhanced by perfusion of the preparation with Ringer solution containing elevated K + . At concentrations of carbachol greater than 30 μM, the onset of the postjunctional blocking action of carbachol is too rapid and obscures the increase in miniature potential frequency. The rate of increase in transmitter release is dependent on the concentration of carbachol applied and can be antagonized by d -tubocurarine (10-60 nM) and other blocking compounds. These findings, in contrast to previous reports, indicate that cholinergic nerve endings, like adrenergic nerve endings, respond to applied acetylcholine-like drugs with measurable increases in transmitter output.

This study shows that (a) the slopes of dose-response curves of bromoacetylcholine (BrACh) and acetylcoline (ACh) were the same; (b) the dose-response curves of BrACh to contract superfused chick biventer cervicis and frog rectus abdominis muscles were shifted to the right by d-tubocurarine in a parallel fashio; and (c) the responses of BrACh were not affected by triethylcholine, which paralyzes cholinergic neurons through inhibition of ACh synthesis. These results indicate that BrACh interacts with cholinergic receptors specifically and directly. When the incubation time was prolonged to 15 min or longer in the organ bath, BrACh (2×10−3 M) produced irreversible blockade of the neuromuscular transmission in chick biventer cervicis and rat diaphragm preparations. The latter blockade was prevented partially by ACh (2×10−2 M) and d-tubocurarine (3.4×10−6 M) (25–38%) and markedly by carbachol (1×10−4 M) and decamethonium (6.2×10−4 M)_(76–79%), indicating agani that BrACh binds specifically and irreversibly to the cholinergic receptor. Therefore, 14C-BrACh is recommended for use in mapping, estimation, and isolation of cholinergic receptors. In order to do such experiments, the biological preparations would have to be pretreated with cold organophosphorus compounds in order to prevent irreversible binding of BrACh to cholinesterases.

The fractional rate of protein synthesis (ks) in the denervated rat-diaphragm has been measured in vivo by the continuous amino acid infusion technique at 1, 3, 5 and 10 days after nerve section, and compared with the rate determined in normal rats. Similar rates of protein synthesis, 14% per day, were found for both the left and right hemidiaphragms in the control animals. In the denervated rats, the rates of protein synthesis in the contralateral control hemidiaphragms were significantly increased as soon as 1 day after nerve section. This is considered to be evidence of a compensatory synthesis in the control tissues. In the denervated hemidiaphragm, the rate of protein synthesis had doubled by the third day after nerve section, but by the fifth day had fallen slightly to a value some 50% greater than that of the controls, and remained at this level for a further 5 days. Based on these measured values of protein synthetic rate, calculated estimates have been made of the rate of protein degradation necessary to account for the reported (Turner, L.V. and Manchester, K.L. (1972) Biochem. J. 128, 789-801) changes in mass of the denervated tissue. During the first three days after nerve section, the rate constant for degradation increased to more than twice the normal rate for skeletal muscle, and remained at this value throughout the peak of the hypertrophy.

The effect of different types of acidosis on sensitivity to neuromuscular blocking agents was studied in vivo on the tibialis muscle of cats and in vitro in the phrenic nerve diaphragm preparation of the rat. In vivo metabolic acidification was caused by diffusion of weak organic acids, which penetrate easily into the cell and imitate closely the state of metabolic acidosis in man. Respiratory acidosis was caused by inhalation of 10% carbon dioxide in oxygen. In the in vitro experiments, acidification was caused by titration with the same acids as well as hydrochloric acid. pH was lowered to levels just above 7.0. Blank in vivo studies showed that the metabolic acidosis by itself causes no significant depression of indirect muscle contraction, while respiratory acidosis causes only a slight depression. A minor depression was seen in the blank in vitro metabolic acidosis studies. Both succinylcholine and decamethonium blocks are strongly antagonised in vivo and in vitro by metabolic and respiratory acidosis. Pancuronium bromide block was potentiated by metabolic and respiratory acidosis both in the in vivo and in vitro experiments. It is most likely that changes in transmembrane potential are the cause for the changes in sensitivity seen in these experiments. ZUSAMMENFASSUNG Der Einfluss verschiedener Arten von Azidose auf die Empfindlichkeit fur muskelerschlaffende Mittel wurde mit Hilfe von in vivo Experimenten auf den Tibialis-Muskel der Katze und in vitro Experimenten auf das Phrenicus-diaphragma-Praparat der Ratte untersucht. Die metabole Azidose wurde in vivo durch schwache organische Saure hervorgerufen, welche schnell in die Zelle hineintreten und der metabolen Azidose beim Mensch am nachsten stehen. Respiratorische Azidose wurde durch Inhalation von 10% Kohlensaure im Sauerstoff hervorgerufen. In den in vitro Experimenten wurde die Azidose durch Titration der Spulflussigkeit mit den selben Sauren und mit Salzsaure hervorgerufen. Das pH wurde bis gerade oberhalb 7.0 herabgedruckt. Blanke in vivo Experimente zeigten, dass die metabole Azidose selbst keine signifikante Erniedrigung der indirekten Muskelkontraktion ergibt und respiratorische Azidose nur eine leichte. Auch in den in vitro Experimenten wurde eine leichte Depression durch die metabole Azidose festgestellt. Die Muskelerlahmung durch Succinylcholine und Decamethonium wurde durch metabole und respiratorische Azidose in vitro und in vivo starkstens antagonisiert. Muskelerschlaffung durch Pancuronium wurde aber durch metabole und respiratorische Azidose in gleiche Weise in den in vitro und in vivo Experimenten potenziert. Die Anderungen des Ruhepotentials der Zellmembrane werden als verantwortlich fur die Empfindlichkeitsveranderungen angesehen.

A reversible nerve-blocking spin label, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) was used to study the nature of anesthetic-binding sites in nerve membranes as a function of pressure. The nerve-blocking effect of TEMPO is enhanced under pressure. At atmospheric pressure, TEMPO blocks nerve conduction by solubilizing in the apolar region of the nerve membrane. However, the nerve-conduction-block by TEMPO at 150 atm of helium was related to the binding of TEMPO to a pressure-induced high-affinity polar site in the nerve membrane. The new TEMPO-binding site could not be detected in lipid model membranes and, thus, the involvement of membrane protein in the new site was inferred. Pressure may induce a nerve membrane conformation change in the presence of TEMPO. The observation that under different pressure, a single anesthetic, i.e., TEMPO, was capable of blocking nerve conduction by binding to two different sites within the nerve membrane, supports the view that there are multiple anesthetic receptor sites, which differ in chemical composition and location within the nerve membrane. These sites, when occupied by different classes of anesthetics, produce the general phenomenon of nerve-conduction block. The enhancement of nerve-conduction block by pressure may be due to the increased concentration of TEMPO in the new site in the nerve membrane under pressure.

The depressant actions of apomorphine, etorphine, dextromoramide, laevomoramide, naloxone, codeine, morphine and nalorphine have been examined on the rat diaphragm preparation. Their descending order of potency (in the order given above) differed greatly from that published for activity at specific opiate receptors. The depressant action of morphine was not antagonized by naloxone. The stereoisomers dextro- and laevomoramide were equipotent in depressing the preparation. On the transmurally stimulated guinea-pig ileum preparation the depressant action of dextromoramide was antagonized by naloxone. Laevomoramide was 10 000 times less potent than its (+)-isomer, and was not antagonized by naloxone. It is concluded that the effects of narcotic analgesics on transmission at a skeletal neuromuscular junction are not mediated via opiate receptors.

Thirty-four patients with hiatus hernia, divided into two groups, were operated upon by the transthoracic and transdiaphragmatic approach. In half of the patients (group I), the diaphragmatic incisions were made without the guidance of the nerve stimulator. In the other half (group II), the line of diaphragmatic incisions was guided by the nerve stimulator in order to avoid injury to the phrenic nerve. The pre- and postoperative respiratory function, arterial blood gas tensions, and diaphragmatic functional tests were statistically analysed in both groups. In group I, significant reductions in regional ventilation, perfusion and volume of the left lung, particularly the left basal field, were associated with a high incidence of diaphragmatic paralysis (10/17). Application of the nerve stimulator in group II resulted in significant improvement in the regional lung function after surgery and the diaphragmatic paralysis could be almost avoided (1/17). No statistical differences in the spirometric values coul...

Effects of lethal doses of a toxic protein from Actinia equina were studied on anesthetized rats. The electrical activity of the phrenic nerves, the respiratory movements, the mechanical activity of the heart, the ECG, and the arterial blood pressure on intact, vagotomized and artificially respired animals were recorded. Shortly (15–50 sec) after beginning the i.v. injection of equinatoxin, the trains of action potentials in the phrenic nerve disappeared and respiration stopped. Bradycardia was observed, indicating elevated vagal tone. A transient fall of arterial blood pressure in vagotomized and in intact animals was registered for 10–15 sec, simultaneously with the block of respiration; thereafter an elevation of arterial blood pressure was registered for 3–7 min. The elevation of the arterial blood pressure was also observed in artificially respired animals. No spontaneous respiratory movements were registered during the period of elevated arterial blood pressure. The diaphragm remained excitable when stimulated indirectly via the phrenic nerve. Conduction disturbances, ectopic foci, myocardial ischemia, negative inotropic and negative chronotropic actions were followed on the ECG and on the in situ exposed heart.

The in vitro neuromuscular blocking properties of a sea snake ( Enhydrina schistosa ) venom have been investigated in a variety of skeletal muscle preparations and compared with those of d-tubocurarine and diallylbisnortoxiferine. Seven different muscles were examined ranging from the monkey extraocular to the leech dorsal wall. The blocking properties of the venom, presumably due to the presence of neurotoxins, were markedly different from those of d-tubocurarine and diallylbisnortoxiferine which were very similar to each other. This, and other results, were discussed in terms of the possible sites of action of the venom and identification of the nicotinic cholinoceptor.

The perfusion of the diaphragm, the interchondral portion of the intercostals and the scaleni have been calculated from measurements of cardiac output and its fractional distribution in rabbits. At every level of ventilation the diaphragm is more perfused than the interchondral portion of intercostal muscles or scaleni. During quiet breathing the diaphragmatic blood flow is 39.5 ml · min −1 · 100 g −1 . During maximal CO 2 induced hyperventilation the blood flow reaches 81.4 ml · min −1 · 100 g −1 . During eleetrophrenic respiration the blood flow is higher than during hyperventilation. During hyperventilation the increase in blood flow is not significant in the extradiaphragmatic inspiratory muscles.

Although most mammalian muscles begin to atrophy shortly after losing their innervation, the denervated hemidiaphragm of the rat undergoes a gross hypertrophy that persists for approximately 1 mo. This transient hypertrophy has been attributed to the repetitive, unopposed stretching of the denervated hemidiaphragm resulting from the rhythmic contractions of the intact, contralateral hemidiaphragm. The latter situation affords a unique opportunity for ascertaining the effects of stretch, in the absence of innervation, on the several types of skeletal muscle fibers commonly present in the diaphragm. Samples of the denervated hemidiaphragm were studied histochemically for succinic dehydrogenase (SDH) and “myofibrillar” adenosine triphosphatase (ATPase) at successive intervals after unilateral section of the phrenic nerve. The three principal fiber types exhibited maximum, though differential hypertrophy 1 wk after surgery. The responsiveness of the musculature varied from locale to locale, with regions demonstrating pronounced hypertrophy alternating with those manifesting little or no change. The small, SDH-high: ATPase-low fibers hypertrophied to the greatest degree and for the longest duration. The large, SDH-low: ATPase-high fibers hypertrophied the least and for the briefest interval. The small, SDH-high: ATPase-high fibers responded in an intermediate fashion. One week or more after denervation other intramuscular changes were also evident. These included the apparent conversion of some fibers to the SDH-high: ATPase-low profile, as well as an apparent hyperplasia of the musculature as a result of satellite formation and/or fiber splitting. The results exemplify the differences in stretch- and innervation-dependence of the three fiber types within the diaphragm, and support the contention that stretch is a major factor determining the histochemical, as well as histological constitution of mammalian skeletal muscle.

In 2 dogs, 5 cats and 30 human cadavers the diaphragmatic innervation has been studied macroscopically and microscopically. General findings of previous authors regarding phrenic and intercostal nerve contributions have been confirmed and subdiaphragmatic connections from the coeliac plexus to the diaphragm have been clearly demonstrated. The presence of this viscerosomatic interconnection in half of the human subjects studied, highlights the possible importance of autonomic control of diaphragmatic musculature. The finding of comparable autonomic connections in dogs raises the possibility of pursuing the question experimentally by carrying out nerve degeneration studies. In addition the matter of surgical incisions in the diaphragm has been reconsidered and the suggested placements have been illustrated.

Arterial pressure, heart rate, preganglionic discharges of the cervical sympathetic nerve and action potentials of the phrenic nerve were recorded in 10 cats under four different conditions: fully conscious (artificial respiration with air, relaxation with suxamethonium), in analgesia (artificial respiration with 66.6% nitrous oxide in oxygen) and in chloralose-urethan anaesthesia (40 mg/kg and 200 mg/kg i.v., respectively) alone or in combination with nitrous oxide. In 10 control experiments cats received nitrous oxide in oxygen during a period of 160 min. In addition, 11 experiments were performed to compare the effects of 66.6% with those of 75% nitrous oxide. The above parameters were recorded both during rest and during acoustic stimulation or asphyxia.

The action of native and mono- 3 H-acetylated-α-bungarotoxin ( 3 H-BuTX) was studied at the acetylcholine (ACh) receptors of innervated and of chronically denervated diaphragm and soleus muscles of the mouse. The 3 H-BuTX was similar in potency to the native toxin, and both toxins produced complete blockade of endplate potentials of innervated muscles and of ACh sensitivity of chronically denervated muscles. When the innervated muscles were washed for 4–7 hr after exposure to either toxin (at 1–5 μg/ml), recovery to an endplate potential value of 0.5–1 mV was recorded in most of the endplate regions of the surface fibers. Parallel experiments on the chronically denervated muscle after a 4–7 hr wash showed a much larger fraction of reversibility; i.e., while in control denervated preparations the ACh sensitivity was 50–75 mV/nC, after the toxin treatment and washing the values were 5–10 mV/nC. When d -tubocurarine ( d -TC, 28 × 10 −6 m ) was present to protect against the blockade of the toxin at the endplates, a complete recovery of neuromuscular transmission could always be obtained upon washing. In the chronically denervated muscles, however, much less protection from the 3 H-BuTX blockade was observed. Uptake of 3 H-BuTX at the endplates was measured by radioactivity analyses; these revealed that only about 60% of the endplate ACh receptors are protected at saturating d -TC levels. Kinetic analyses of the uptake confirmed this. The observations can be interpreted in terms of two types of sites at the endplate; the two are equally reactive with α-bungarotoxin, but only one of them binds d -TC firmly. Similar observations were made with d -TC protection in the denervated muscles; the results showed much lower affinity of d -TC in these muscles. The changes in d -TC effectiveness in the receptors after denervation are all parallel, when measured in several muscles by three approaches—blockade of ACh sensitivity, prevention of α-bungarotoxin blockade of ACh sensitivity, and inhibition of uptake of 3 H-BuTX. We conclude that the ACh receptor molecules themselves, when induced by denervation, are different from normal receptors in that they interact much less strongly with d -TC.

Anastomoses of the split vagus nerve to the adductor division of the recurrent nerve intralaryngeally and, simultaneously, the phrenic nerve to the abductor division of the recurrent nerve extralaryngeally were carried out with meticulous technique under the operating microscope. Each suture line was protected by silicone elastomer tubing. The rationale behind these simultaneous separate nerve anastomoses was to attempt to avert crossed reinnervation and dyssynergia that often results from other nerve anastomosis techniques. Results were evaluated by follow-up of the animals with electromyographic studies and 16-mm motion pictures. All experimental animals regained normal and symmetrical electromyographic function, a high degree of voluntary synchronous adduction, and to a somewhat lesser extent, spontaneous synchronous abduction, at 12 to 14 months postoperatively. Results indicate that simultaneous separate nerve anastomosis offers promise for active reinnervation of the paralyzed larynx.

On the sciatic nerve-gastrocnemius-soleus muscle preparation of the cat, angiotensin (1–5 μg/kg, i.v.) potentiated the twitch response to maximal and submaximal stimulation of the sciatic nerve and produced partial reversal of an incomplete tubocurarine blockade. These actions could not be explained in terms of increased acetylcholine release since they were not seen in isolated motor nerve-striated muscle preparations and were probably secondary to the cardiovascular actions of angiotensin. Blockade of conduction in the postganglionic cholinergic nerves in the guinea-pig isolated ileum preparation by cooling or anoxia antagonized the response of this tissue to angiotensin. These procedures left the response to exogenous acetylcholine unchanged though they removed the cholinergic component of the response to angiotensin which is known to be present in this tissue. No evidence of increase in catecholamine output could be found in preparations of guinea-pig and rabbit vasa deferentia or rabbit duodenum responding to submaximal stimulation of their adrenergic nerves. It is concluded that angiotensin has no direct action on the stores of neurohumoral transmitter at motor, postganglionic cholinergic or postganglionic adrenergic nerves and that its known acetylcholine releasing action in the isolated ileum results from stimulation of the ganglia.

Heterogeneous synapses in the superior cervical ganglion of the dog were obtained by regeneration of phrenic nerve fibres, after cutting and degeneration of the normal preganglionic fibres. The shape of the terminal of the regenerated fibres and the ultrastructural features of the newly formed synapses are very similar to those present in the normal ganglion. Thus, the fundamental role of the postsynaptic element in the determination of the cytological characteristics of these synapses is evident. A subsynaptic apparatus, in the shape of a layer of dense patches, was observed in a certain number of normal and of regenerated synapses. A special type of small vesicles, 150 A in diameter, was found to be present in the vicinity of the dense projections of many active zones; their functional role is, so far, not established.

Several central depressant and other drugs have been examined for their effects upon acetylcholine release from the stimulated, perfused cat superior cervical ganglion and rat isolated phrenic nerve-diaphragm preparations. The acetylcholine released was assayed biologically. Amylobarbitone sodium, chloral hydrate, trichloroethanol, methylpentynol, methylpentynol carbamate, paraldehyde, procaine hydrochloride and troxidone reduced the presynaptic release of acetylcholine from the ganglion. They also exhibited a postsynaptic blocking action, this component of depressant activity being particularly prominent with paraldehyde and troxidone. Closely analogous findings were obtained at the neuromuscular junction with methylpentynol and its carbamate, paraldehyde, procaine hydrochloride, trichloroethanol and troxidone. At both sites the drug-induced depression, both of transmission and of acetylcholine output, was reversible. Whereas hexamethonium regularly blocked ganglionic transmission with no effect upon acetylcholine release, tetraethylammonium not only completely blocked ganglionic transmission but concomitantly augmented acetylcholine output. These results are discussed in relation to the electrophysiological and metabolic events associated with neuro-effector transmission.

Since Fourneau and Bovet (1) reported 2-isopropyl-5-methyl phenoxy-ethyl-diethylamine as a potent anti-histaminic, many publications on the central action of anti-histaminics have appeared during the last three decades. However, no report on the action of anti-histaminics to the neuromuscular junction has appeared, despite the fact that listlessness of the limbs occurs when a small dose of anti-histaminics is administered, as in the case of succinylcholine administration. In the present paper, the neuromuscular blocking property of several anti-histaminics was first screened, and then its mechanism of action was studied using rat phrenicdiaphragm preparation.

1. Unit activity was recorded from single and few fibre preparations in a cardiac branch of the right vagus nerve of the cat. 2. Increases in blood pressure mediated solely by the right carotid sinus nerve produced bradycardia when all other nerves to the heart had been cut. Myelinated fibres in the cardiac branch of the right vagus nerve were reflexly activated by the same procedure. 3. The fibres were silent when blood pressure was below 140–150 mm Hg. As the pressure began to rise, they discharged phasically with the cardiac cycle. At pressures greater than 180 mm Hg, the discharge was continuous attaining maximum rates of 40/sec. 4. Stimulation of carotid body chemoreceptors also reflexly excited these fibres, as did stimulation of baroreceptors in both the left carotid sinus and aortic arch. Afferent fibres in the left vagus discharging in response to changes in blood pressure reflexly excited the cardiac efferent fibres. Increases in phrenic motoneurone discharge coincided with inhibition of these fibres. Electrical stimulation of the glossopharyngeal nerve also produced inhibition.

Summary. 1) An investigation of the effect of temperature on the neuromuscular block caused by magnesium in the isolated phrenic nerve diaphragm preparation from the rat has been carried out. The effect of tetraethylammonium (TEA) on the neuromuscular depression produced by magnesium has been investigated in the rat diaphragm preparation as well as in nerve muscle preparations in situ in rabbits and cats. 2) A concentration of 6 mM magnesium in the bath just produced complete neuromuscular block of the responses to single nerve shocks at 37°C. At 22° C 10 mM magnesium was needed. In the presence of a neuromuscular block due to magnesium at 37° C, lowering the temperature to 22° C restored transmission. 3) TEA in a concentration of 1 mM in the bath partly restored neuromuscular transmission depressed by magnesium. The β-hydroxy-derivative of TEA and choline only increased the magnesium block. In the rabbit and cat TEA in a dose of 4 to 7 mg per kg body weight injected intravenously restored neuromuscular transmission strongly depressed by continuous infusion of magnesium. In spite of continuous magnesium infusion, the restoration of muscle contractions obtained with the single dose of TEA was far more sustained than the blood pressure fall caused by TEA. 4) The sensitivity of the motor end plate to externally applied acetylcholine was not increased after TEA had restored transmission in the magnesium-blocked rat diaphragm. 5) The end plate potential obtained under a magnesium block was increased in amplitude without significant change in time course, when TEA was added. It is concluded that TEA like calcium antagonizes the neuromuscular block caused by magnesium by effecting an increased output of acetylcholine from the presynaptic nerve terminals.