The action of inhibitory drugs on nerve terminals in crayfish muscle

Summary The presynaptic inhibitory effects of -amino-butyric acid (GABA) and related drugs on the excitatory and inhibitory terminal potentials were studied using extracellular microelectrodes.Triphasic conducted action potentials recorded from the excitatory nerve fiber in the vicinity of the terminal were altered to monophasic positive potential changes after application of inhibitory drugs. These drugs thus can block conduction near to the terminal.Diphasic potential changes recorded near to the excitatory terminal (e.n.t.p.s) were made monophasic positive after application of inhibitory drugs. Neural presynaptic inhibition had the same effect. Also during drug induced inhibition therefore the point of block of conduction in the excitatory terminal shifted centrally.The potentials recorded from the inhibitory nerve terminal were not or very little affected by applied GABA.GABA in concentrations that blocked excitatory transmission did not change the frequency of spontaneous excitatory potentials.It is concluded that presynaptic inhibition by GABA and related drugs mimicks in all respects known neural presynaptic inhibition. The probable mechanism of this inhibition is a conductance increase without much potential change in the excitatory nerve terminal.

---

Zusammenfassung Die hemmenden Effekte von GABA und verwandten Drogen auf die Potentiale der erregenden und hemmenden Nervenendigungen wurden mit Hilfe von extracellulären Mikroelektroden-Ableitungen untersucht.Von erregenden Nervenfasern in der Nähe der Endigung wurden triphasische Aktionspotentiale abgeleitet. Nach der Gabe von hemmenden Drogen wurden die Potentiale monophasisch positiv; diese Substanzen könnten also einen Block der Fortleitung des Aktionspotentials herbeiführen.Nahe der erregenden Endigungen abgeleitete diphasische Potentiale (e.n.t.p.) wurden nach Applikation von hemmenden Drogen kleiner und monophasisch positiv. Präsynaptische Hemmung durch Reiz des hemmenden Nerven hatte dieselbe Wirkung. Es verschiebt sich also auch während der Hemmung durch Drogen die Stelle, an der die Fortleitung des Aktionspotentials aufhört, zentralwärts.Von der hemmenden Nervenendigung abgeleitete Potentialänderungen wurden durch GABA nicht oder sehr wenig beeinflußt.GABA in Konzentrationen, bei denen die erregende synaptische Übertragung unterbrochen war, veränderte nicht die Frequenz der spontanen erregenden Potentiale.Die Befunde zeigen, daß die präsynaptische Hemmung durch GABA und verwandte Substanzen von der präsynaptischen Hemmung über den hemmenden Nerven nicht unterschieden werden kann. Wahrscheinlich ist der Mechanismus dieser Hemmung eine Erhöhung der Leitfähigkeit der Zellmembran der erregenden Nervenendigung, ohne daß dabei das Membranpotential sich wesentlich verschiebt.

---

---

With 5 Figures in the Text

---

This investigation was supported by grants from the Deutsche Forschungsgemeinschaft.     

Presynaptic effect of gamma-aminobutyric acid on the inhibitory nerve and nerve terminals in the crayfish neuromuscular junction

Experiments were carried out in voltage-clamped fibres of the opener muscle of the first walking leg or claw of small crayfish. Repetitive discharges in the inhibitory nerve innervating the muscle were induced by adding serotonin (10(-6) mol/l) and forskolin (10(-4) mol/l) to the superfusate. Rates of nerve discharge were determined by recording nerve evoked inhibitory postsynaptic currents (IPSCs) in the voltage-clamped muscle fibre. Subsequently, the effect of gamma-aminobutyric acid (GABA) on the rate of IPSCs in normal and Cl- -deficient superfusate was investigated. In normal superfusate GABA (10(-5) mol/l) abolished the IPSCs whereas in Cl- -deficient superfusate GABA (10(-4) mol/l) enhanced the rate of IPSCs. Moreover, in Cl- -deficient superfusate the rate of asynchronous quantal release of inhibitory transmitter could be enhanced by GABA. The results indicate that in the crayfish neuromuscular junction the inhibitory axon is supplied with GABA receptors which may affect (a) axonal excitation and (b) quantal output at the inhibitory axon terminals.     

The influence of gamma-aminobutyric acid on the motor responses of the normal and denervated adductor muscle of the crayfish

Summary An investigation was made of the effect produced by gamma-aminobutyric acid (GABA) on the contraction of the normal and denervated adductor muscle of the claw ofAstacus Astacus and ofAstacus Leptodactylus. GABA was shown to exert an inhibitory effect on normal muscular contraction, a result which agreed with published reports. In simulating the action of the inhibitory nerve, GABA evidently acts on the nerve endings to promote the appearance of the mediator. There was no GABA effect on the muscular contraction after section of the inhibitory nerve alone, or after total denervation. The muscle was sensitive to GABA after division of the motor nerves and retention of the inhibitory innervation. Low GABA concentrations selectively blocked the slow component of this reaction, in both normal and in denervated muscle.(Presented by Active Member AMN SSSR S. V. Anichkov) Translated from Byulleten' Éksperimental'noi Biologii i Medisiny, Vol. 52, No. 12, pp. 8–12, December, 1961     

Proprioceptive fields of crayfish claw motor neurons.

1. Action potentials of crayfish claw motor neurons were recorded during both imposed constant-velocity displacements and imposed alternating sequences of opening and closing step movements of the dactyl. 2. Peristimulus time (PST) histograms show that the firing probabilities of two neurons, the opener inhibitor (OI) and the slow closer excitor (CE) consistently increased during opening ramp movements and declined during closing ramp movements. Hyperpolarizing synaptic potentials were observed in both cells during closing movements. 3. The proprioceptive field organizations of OI and CE were analyzed with response planes and contour planes. Each PST histogram in a plane displays the firing probability of the neuron as a function of time following step displacements at a given position. A relatively uniform early primary response followed each successive opening step. The probability of occurrence of later activity, when present, usually became more pronounced as the joint angle increased. Often both cells were silent during closing steps; when the cells were active, their firing probabilities were highest at the more open joint angles. 4. When both OI and CE were active, their spike trains were usually temporally correlated. 5. The other claw efferents did not respond to imposed movements in a consistent manner. When CE was active it was most likely to respond to closing movements near the closed position. 6. It is concluded that OI and CE are strongly and similarly influenced by proprioceptive reflexes. The responses of the two cells to imposed dactyl movements change as a function of joint angle, time after movement, and direction of movement.     

Effect of lithium on veratridine-induced quantal and non-quantal release from inhibitory nerve terminals in crayfish muscle

Muscle fibres of small crayfish were voltage clamped and superfused for about 10 min with Li⁺ saline (Na⁺ replaced by Li⁺) which contained 5 mmol/l glutamate to desensitize excitatory postsynaptic receptors. Then 100 mol/l veratridine were added to the superfusate which caused strong asynchronous quantal release of inhibitory transmitter. However, in the presence of Li⁺ strong inhibitory quantal release was only transient. It could be activated a second time by removal of Li⁺ and readministration of Na⁺. From the total of 0.7 to 1.1 million quanta released by veratridine only about 30–35% could be released in Li⁺ saline. The voltage clamp DC-currents recorded during veratridine-induced quantal release suggested that a nonquantal release component is additionally involved. This non-quantal release component was most prominent during the period of quantal release in Li⁺ superfusate while it was less obvious during the second enhancement of quantal release in normal saline. Together with previous results (Martin and Finger 1988) it may be concluded that quantal release, but not non-quantal release, is decreased by Li⁺ in the nerve terminals.This investigation was supported by the Deutsche Forschungsgemeinschaft, SFB 220     

Projectin is an invertebrate connectin (titin): Isolation from crayfish claw muscle and localization in crayfish claw muscle and insect flight muscle

Summary A filamentous protein was isolated from crayfish claw muscle. This protein had physiochemical properties very similar to vertebrate skeletal muscle connectin (titin), although its apparent molecular mass ( 1200 kDa) was considerably lower than that of connectin ( 3000 kDa). Polyclonal as well as monoclonal antibodies against chicken skeletal muscle connectin reacted with the 1200 kDa protein from crayfish claw muscle. Conversely, polyclonal antibodies against crayfish 1200 kDa protein crossreacted with chicken connectin. Circular dichroic spectra indicated the abundance of-sheet structure ( 60 %). Low-angle shadowed images showed filamentous structures (0.2 0.5m) by electron microscopy. Proteolysis of the 1200 kDa protein by -chymotrypsin or V8 protease rapidly resulted in formation of 1000 kDa or 1100 and 800 kDa peptides. The amino acid composition was very similar to those of vertebrate connectins and of honeybee flight muscle projectin. Based on the molecular weight and amino acid composition, the 1200 kDa protein is regarded to be crayfish projectin.Immunofluorescence and immunoelectron microscopy revealed that crayfish projectin was localized in the A/I junction area and A-band except for its centre region in crayfish claw muscles. Polyclonal antibodies against crayfish claw muscle projectin reacted with 1200 kDa projectin of honeybee and beetle flight muscle. A monoclonal antibody against chicken skeletal muscle connectin also reacted with honeybee and beetle projectin. Immunoelectron microscopic observations revealed that anti-crayfish projectin antibodies bound the connecting filaments linking the Z-line and the thick filaments up to the M-line of honeybee muscle sarcomere. Anti-crayfish projectin antibodies bound the I-band region near the Z-line of beetle flight muscle.It is concluded that the 1200 kDa projectin from crayfish claw muscle is an invertebrate connectin (titin). Recent work with locust flight muscle mini-titin (Nave & Weber, 1990) is in good agreement with the present study, except that the isolated minititin estimated as 600 kDa appears to be a proteolytic product ( 1100 kDa) of the parent molecule ( 1200 kDa).     

Isolation and characterization of a kettin-like protein from crayfish claw muscle

Summary A 540 kDa protein was isolated from crayfish claw muscle (closer). The secondary structure mainly consisted of -sheet (70%). The rotary shadowed images were long filaments, 300–360 nm long. It is localized in the sides of the Z-lines extending to the I band and elongatable upon stretch of muscle. Immunological crossreactivities strongly suggested that this protein corresponds to kettin (500–700 kDa) of insect striated muscle. In view of molecular shape and secondary structure, and immunological crossreactivities, it is suggested that this kettin-like protein belongs to connectin/titin family of striated muscle.     

Connectin,giant elastic protein,in giant sarcomeres of crayfish claw muscle

Summary In the giant sarcomeres (sarcomere length, 10 m at rest) of crayfish claw muscle, 3000 kDa connectin-like protein but not projectin (mini-titin) appears to be responsible for passive tension generation. Proteolysis of crayfish connectin in skinned fibres was parallel with disappearance of resting tension. Immunofluorescence observations using the antiserum to crayfish connectin showed that crayfish connectin linked the A band to the Z line ina giant sarcomere. It appears that crayfish connectin exerts a centering force on the A band in a sarcomere. Very thin filaments in the I band were visualized after the actin filaments had been removed by the treatment with plasma gelsolin. Crayfish connectin was partially purified and its rotary shadowed image was a very long filament. Projectin was localized on the A band of crayfish giant sarcomeres and remained unmoved during stretch or contraction. However, on dissolution of myosin filaments, projectin moved to the Z line together with crayfish connectin. It seems that projectin binds to connectin on the myosin filament. In regular size of sarcomeres (sarcomere lengths, 3–4 m at rest) of crayfish stretcher muscle, projectin linked the A band to the Z line, as in insect flight muscle.     

Synaptic integration in excitatory and inhibitory crayfish motoneurons

The passive integrative properties of two crayfish abdominal motoneurons, the fast flexor inhibitor (FI) and a posterior, ipsilateral fast flexor excitor (FE), were studied electrophysiologically and through simulations with multicompartment models of their electrotonic structures. Responses of the models to simulated giant neuron input were quite similar to the motoneurons' responses to giant neuron stimulation, which suggests that differences in the electrotonic structures and the sites of synaptic input to the two cells can account in large part for differences in their responses to a common input. A full action potential created in the initial axon compartment of the FI model produced attenuated potentials in the adjacent integrating segment compartment and contralateral soma compartment. These potentials are similar in amplitude and time course to attenuated antidromic action potentials recorded in the corresponding regions of the FI neuron. A location of the spike initiation zone of the FI at the initial axon segment is consistent with this result. The responses of FI to ipsi- and contralateral inputs are different. Shock of a single abdominal second root produced a larger, faster rising excitatory postsynaptic potential in the ipsilateral FI soma than in the contralateral soma. Second root shock also caused the contralateral FI to produce an action potential either alone or before the ipsilateral FI neuron. Responses of the FI model to ipsilateral and contralateral inputs differ in the same way as the cell's responses. Inputs to the FI model that are ipsilateral to the soma compartment produce larger responses there than do contralateral inputs. Conversely, those contralateral inputs produce larger responses in the initial axon compartment than do ipsilateral inputs. This difference results from the long integrating segment that connects the soma compartment to the initial axon compartment. These results can account for the FI responses to lateralized inputs. Unlike the responses of FIs, the soma responses of contralaterally homologous FEs to ipsilateral and contralateral second root shocks were similar in waveform and amplitude, with the ipsilateral root producing the larger response. This result is consistent with theoretical results from the FE model simulations. We conclude that a smaller size, larger input resistance and shorter membrane time constant allow the FE to respond to giant neuron input before the FI, and so help to achieve the proper timing of flexor contraction and relaxation during a tailflip.(ABSTRACT TRUNCATED AT 400 WORDS)     

Presynaptic and postsynaptic effects of inhibitory drugs on the crayfish neuromuscular junction

Summary -aminobutyric acid (GABA) and related drugs reduce the size of the excitatory postsynaptic potential (e.p.s.p.) in the crayfish neuromuscular preparation. The effective inhibitory concentrations of these drugs were compared and the sites of action—postsynaptic and presynaptic—were determined.GABA, -amino--hydroxy-butyric acid, guanidino acetic acid, -alanine and others were found to increase the membrane conductance of the muscle fibers. These drugs also shift the membrane potential in the same direction as the inhibitory postsynaptic potential. It is concluded that this group of drugs imitates the postsynaptic action of the inhibitory transmitter substance.-guanidino-propionic acid (GP), -guanidino-butyric acid and others did not affect the membrane conductance of the muscle fibers. These drugs therefore do not inhibit through the conductance type of postsynaptic inhibition.Both groups of drugs mentioned above (GABA and GP) did reduce the amount of transmitter released from the excitatory nerve terminal per stimulus, they thus have a presynaptic inhibitory effect similar to neural inhibition. This was shown by analysis of the quantum content of the excitatory postsynaptic potential: The inhibitory drugs reduced the number of quanta liberated per stimulus, the size of the quantum remaining constant.Although GP has no direct effects on the membrane resistance of the muscle fiber, it reduces the inhibitory postsynaptic potential and diminishes the action of applied GABA on the membrane conductance. GABA and GP seem to compete for the inhibitory receptor site on the muscle membrane.

---

Zusammenfassung -amino-Buttersäure (GABA) und verwandte Substanzen verkleinern das postsynaptische erregende Potential (e.p.s.p.) des Nerv-Muskel-Präparates des Krebses. Die wirksamen hemmenden Konzentrationen dieser Drogen wurden verglichen, und es wurde versucht, Art und Ort der Hemmungswirkungen — postsynaptisch oder präsynaptisch — festzustellen.GABA, -amino--hydroxy-Buttersäure, guanidino-Essigsäure, -Alanin und andere erhöhten die Leitfähigkeit der Muskelzellmembran. Bei Gabe dieser Drogen verschiebt sich auch das Membranpotential in derselben Richtung wie das hemmende postsynaptische Potential. Folglich hat diese Gruppe von Drogen dieselbe Wirkung auf die Muskel-Zell-Membran wie der hemmende neurale Überträgerstoff.-guanidino-Propionsäure (GP), -guanidino-Buttersäure und andere Guanidinosäuren haben keinen Einfluß auf die Membranleitfähigkeit der Muskelzellen. Der hemmende Effekt dieser Drogen ist also nicht vom Typ der postsynaptischen Leitfähigkeitshemmung.Beide oben erwähnten Gruppen von Substanzen (GABA und GP) setzen die Menge des pro Reiz von der erregenden Nervenendigung freigesetzten Überträgerstoffes herab, sie hemmen also präsynaptisch ebenso wie die neurale Hemmung. Diese präsynaptische Hemmung wurde bewiesen durch Analyse des Quantengehalts der erregenden postsynaptischen Potentiale: Die hemmenden Drogen verkleinerten die Zahl der pro Reiz ausgeschütteten Quanten, ohne die Größe des Quantums zu verändern.Obgleich GP keine direkten Effekte auf den Membranwiderstand der Muskelfasern hat, verkleinert es das postsynaptische hemmende Potential und setzt die Wirkung von GABA auf die Leitfähigkeit der Muskelzellmembran herab. GABA und GP verdrängen sich gegenseitig am Hemmungs-Receptor der Muskelmembran, so daß GP den Effekt von GABA kompetitiv hemmt.

---

---

With 5 Figures in the Text

---

This work was supported by the Deutsche Forschungsgemeinschaft.     

隐神经收肌管段的应用解剖学

在80侧成人下肢标本上观察了隐神经及其髌下支穿Hunter's管(收肌管)前壁的类型。其中隐神经除有93.8％穿该管前壁腱板浅出外,尚有6.3％未穿该管前壁腱板。神经穿出处之裂孔按形态分为3种:裂隙形(隐神经为74.7％;髌下支为65.0％)、近圆孔形(隐神经为14.7％;髌下支为20.0％)、狭窄形(隐神经为为10.7％;髌下支为15.0％。并测量了神经穿出点的位置距Hunter's管前壁腱板上缘的长度、裂孔的长度和横径,为临床探讨隐神经卡压症的病因和治疗提供应用解剖学资料。     

Relaxation after a voltage step of inhibitory synaptic current elicited by nerve stimulation (crayfish neuromuscular junction)

The clamped membrane potential of small crayfish muscle fibers was shifted in rapid steps between potentials of about –60 and –120 mV, and the clamp currents measured after de-and hyperpolarizing steps were averaged. In addition, the inhibitory nerver fiber was stimulated either synchronously or asynchronously with the averaging. Synchronous stimulation yielded the usual IPSCs, and asynchronous stimulation a steady state inhibitory current which relaxed to a new level after a voltage step. Fast relaxations were observed in all fibers. Their time constants =15 to 20 ms at –60 mV (10°C) decreased on hyperpolarization and agreed with those of the decay of the IPSC at the respective potential. The relaxations could be described quantitatively by a model in which the synaptic current depends on membrane potential due to (1) the potential dependence of the life time of a synaptic channel, (2) to a constant channel permeability, and (3) to the potential difference from the equilibrium potentialE _Cl.In many fibers slow relaxations of the inhibitory synaptic current were observed also, with time constants to 150 ms at –60 mV (10°C), which decreased at more negative potentials. These slow relaxations correspond to a reported slow current noise component induced by GABA. Interpretations of this slow synaptic current component are discussed.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Interactions among an ensemble of chordotonal organ receptors and motor neurons of the crayfish claw.

1. Action potentials of crayfish propodite-dactyl (PD) chordotonal organ receptors and two claw motor neurons, the opener inhibitor (OI) and slow closer excitor CE) were simultaneously monitored during imposed step and ramp movements of the dactyl or while the dactyl was held at various positions. 2. The activities of the cells during imposed displacements were analyzed using peristimulus time histograms and response and contour planes. The proprioceptive fields (PFs) of individual receptors resemble components of the more complex motor neuron PFs. Some receptors are briefly active after each successive opening step, while others do not respond to steps near the closed position but respond as the joint angle increases, becoming active when the claw is held open. Another type of receptor responds to closing movements. 3. Interactions among the various types of receptors and the two motor neurons were detected and analyzed by various statistical methods and intracellular recording techniques. The results indicate that receptors activated during opening movements and when the dactyl is held at open positions excite OI and CE via divergent functional connections. The efficacies of the connections made by a receptor may differ. Receptors activated by closing movements produce hyperpolarizing synaptic potentials in both efferents, possible directly or via interneurons. 4. It is concluded that several types of chordotonal organ receptors form an ensemble of parallel input channels, which modulates the activities of OI and CE and contributes to the generation of the spatial-temporal nonuniformities of their proprioceptive reflex responses.     

A study of the action of picrotoxin on the inhibitory neuromuscular junction of the crayfish

1. The effect of picrotoxin on the neuromuscular junction of the crayfish (Cambarus clarkii) was investigated. The potential changes were recorded intracellularly and extracellularly with micro-electrodes. The membrane conductance of the muscle fibre was also measured.2. Picrotoxin depressed the amplitudes of the inhibitory junctional potentials and the potential changes produced by iontophoretically applied gamma-aminobutyric acid (GABA), but had no appreciable effect on the excitatory junctional potentials and the potential changes produced by L-glutamate.3. The presynaptic action of GABA and the neural transmitter was depressed by picrotoxin. The presynaptic action of beta-guanidinopropionic acid was also depressed by picrotoxin.4. The increase in the membrane conductance produced by the addition of GABA in the bath fluid was depressed by picrotoxin. The dose-response relation showed that picrotoxin depressed the conductance increase produced by GABA in a non-competitive manner. The action of picrotoxin on the conductance increase produced by GABA was more effective in low Cl- solution.5. The analysis of the dose-response curves showed that the action of picrotoxin was well expressed by the Michaelis-Menten equation, but the slope of the dose-response curve of GABA was steeper than this relation. It is proposed that the conductance of the junctional membrane was increased by the combination of two molecules of GABA with a receptor, and the attachment of one molecule of picrotoxin to a specific site depressed the conductance increase.