Modification by lidocaine of the discharges of primary endings of muscle spindles in cat tenuissimus muscle

Modification by lidocaine of the discharges of primary endings of muscle spindles in cat tenuissimus muscle

European Journal of Pharmacology, 53 (1979) 159--166 159 © Elsevier/North-Holland Biomedical Press MODIFICATION BY LIDOCAINE OF THE DISCHARGES OF P...

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European Journal of Pharmacology, 53 (1979) 159--166

159

© Elsevier/North-Holland Biomedical Press

MODIFICATION BY LIDOCAINE OF THE DISCHARGES OF PRIMARY ENDINGS OF MUSCLE SPINDLES IN CAT TENUISSIMUS MUSCLE HERBERT E. LOWNDES *, BERNARD PAGI~S ** and PAUL BESSOU

Laboratoire de Physiologie, Facult~ de M~decine, Universit~ Paul Sabatier, 133, Route de Narbonne, 31077 Toulouse C~dex, France Received 8 June 1978, revised MS received 8 September 1978, accepted 15 September 1978

H.E. LOWNDES, B. PAG]~S and P. BESSOU, Modification by lidocaine of the discharges of primary endings of muscle spindles in cat tenuissimus muscle, European J. Pharmacol. 53 (1979) 159--166. The effects of lidocaine (1--20/lg/ml) on afferent discharge patterns of primary endings of muscle spindles in cat tenuissimus muscle were investigated. Discharge from the endings, recorded in Ia afferent axons, was evoked by ramp stretch of the muscle, stimulation of single static or dynamic fusimotor axons or by a combination of stretch and fusimotor stimulation. Spontaneous discharge of the endings at the initial length of the muscles was reduced by 2--5/~g/ml and abolished by 10--15 ~g/ml lidocaine. The static but not the dynamic discharge elicited by muscle stretch was blocked by concentrations of 10--15/~g/ml. The same concentrations abolished static and dynamic fusimotor influences on primary ending discharge. However, in one experiment where the spindle was microscopically observed, fusimotor stimulation still resulted in contraction of the intrafusal muscle even when fusimotor stimulation failed to elicit changes in discharge response patterns of the primary endings. These findings indicate that lidocaine interferes with the encoding mechanism prior to block of impulse conduction in either the fusimotor or afferent axons. Muscle spindles

Local anesthetics

Fusimotor axons

1. Introduction The effects of local anesthetics on some aspects of muscle spindle function have been reported. Procaine blocks the encoding (regenerative) mechanism in spindles before impulse conduction in the afferent axons is blocked (Katz, 1950). The generator potential in the primary afferent terminals remains unaffected by concentrations of lidocaine or procaine that abolish afferent activity (Ottoson, 1964). At large doses, procaine more effectively reduces the static (tonic) than the dynamic (phasic) discharges of cat muscle spindles

* Visiting Professor, Address: Department of Pharmacology, CMDNJ, New Jersey Medical School, 100 Bergen Street, Newark, New Jersey, 07103, U.S.A. ** To whom all correspondence should be addressed.

activated by stretch (Grossie and Smith, 1966). The discharge of muscle spindle endings in situ is influenced not only by passive stretch of the muscle, but also by their fusimotor innervation. Primary afferent axons (type Ia) are 6--15 #m at their point of entry into the spindle whereas gamma (~/) fusimotor axons are only 1.2--3.8/~m (Adal and Barker, 1962; Barker et al., 1970). Impulse conduction in small motor axons is blocked prior to conduction in the larger diameter m o t o r axons by local anesthetics (Leksell, 1945; Matthews and Rushworth , 1957) and at lower concentrations (Nathan and Sears, 1961). Therefore, local anesthetics might be expected to block fusimotor influences at concentrations that are without effect on either the encoding mechanism or impulse conduction in the primary afferent axons.

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2. Materials and methods

2.1. Surgical preparation o f the in situ muscle spindle preparation All experiments were performed on adult cats under sodium pentobarbital anesthesia (40 mg/kg i.p. followed b y small amounts i.v. when necessary). The tenuissimus muscle was dissected free of surrounding tissue, retaining its blood supply and innervation intact (see Bessou and PagSs, 1967; 1972). The freed distal portion of the muscle was then placed in a 50 ml tissue bath containing circulating Tyrode solution without glucose (composition in mM/l: NaC1 136; KC1 2.68; CaC12 1.8; MgC12" 6H20 1.05; NaHCO3 11.9; NaH2PO4 0.42 bubbled with a mixture of 95% 02--5% CO2) and maintained at 37°C. The remaining hindlimb and hip muscles were extensively denervated. Dorsal and ventral roots L7 and $1 were exposed b y lumbar laminectomy.

2.2. Isolation and identification o f spindle innerva tion Single group Ia axons connected to a muscle spindle in the distal third of the tenuissimus muscles were isolated in filaments of the dorsal roots. As many as possible single fusim o t o r axons innervating the muscle spindle were separated from ventral roots and indentified as dynamic (TD) or static (~s) by the effects their stimulation (100 Hz) exerted on the responses of the primary endings to ramp stretch (Matthews, 1962). It was frequently possible to obtain, in the same experiment, b o t h dynamic and static fusimotor axons innervating the same muscle spindle.

2. 3. Experimental procedures The discharge of each primary spindle ending was recorded from single type Ia axons isolated in dorsal roots, amplified and displayed on an oscilloscope as the instantaneous discharge frequency. Permanent records were

H.E. L O W N D E S E T AL.

made on moving film and on magnetic tape. The responses of each ending to stretch were then determined by subjecting the tenuissimus muscle to a ramp and hold stretch (3 mm at 12 mm/sec). For each ramp stretch the following were determined: the spontaneous discharge frequency of the ending at the initial length of the muscle (approximately 5 mm longer than the minimal physiological length), the discharge frequency of the dynamic response of the ending at the end of the ramp stretch (peak dynamic frequency) and the frequency of discharge 0.5 sec after the end of the ramp stretch (static frequency). The effects of fusimotor stimulation were then studied by stimulating, in turn, each of the isolated and identified fusimotor axons with trains of stimuli at 1 0 0 H z . During the period of fusimotor stimulation the muscle was again ramp stretched. This permitted the determination of both the effects of fusimotor stimulation on the spontaneous discharge of the ending and on the dynamic response and the following static response. Lidocaine (Xylocaine, Laboratoires Roger Bellon) was added to the solution bathing the muscle to achieve final concentrations of 1--20 pg/ml, expressed in terms of the salt (hydrochloride). Spindle responses to stretch, fusimotor stimulation and both stretch and fusimotor stimulation were then recorded after 5 min exposure to each new concentration of lidocaine. Similarly, responses were recorded at 5 min intervals after the lidocainecontaining solution had been replaced b y drug-free solution.

2.4. Visualization of intrafusal muscle contraction during fusimotor stimulation In one experiment, the spindle under study was visualized microscopically b y removing overlying extrafusal muscle fibres while leaving the spindle capsule and its innervation undisturbed (Bessou and PagSs, 1975). This permitted observation of intrafusal muscle contraction when a single ~/ fusimotor axon was stimulated.

LIDOCAINE ON MUSCLE SPINDLES

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3. Results

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Seven primary endings were evaluated for their responses to stretch alone. In untreated muscles, the endings at their initial lengths discharged tonically at frequencies of 5--10 Hz and responded to ramp stretch with a peak dynamic frequency of 137 + 10.6 Hz (average -+S.E.M.). A sample response is illustrated in fig. 1A. Addition of increasing concentrations of lidocaine to the tissue bath resulted in abolition of both spontaneous and static discharge (fig. IB,C). Tests were performed 5 min after each addition of the drug to the bath but its effects, particularly on tonic discharge, became evident 1--3 min after it was added to the bath. Spontaneous and static discharges were reduced by 2--5/~g/ml and totally abolished by 15 pg/ml lidocaine (fig. 2). The discharge frequencies returned to their pre-

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3.2. Effects on primary ending discharge evoked by fusimotor stimulation

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Fig. 1. Sample record of the effect of lidocaine on the instantaneous discharge frequency of a primary ending of a muscle spindle in cat tenuissimns muscle. Spindle discharge was evoked by ramp stretch (3 m m at 12 mm/sec) alone. Response of an untreated ending (A) and after 5 min in 5 (B) and 15 (C)/lg/ml lidocaine and after 10 (D) and 25 (E) rain washing in drug-free solution.

Fig. 3A (left-hand portion) illustrates sample responses obtained from a Ia axon when the spindle was activated by stimulation of a single dynamic fusimotor axon (TD). This fusimotor influence caused untreated primary endings to discharge at an average frequency of 42 + 5.2 Hz (fig. 5 -- average of 9 7D axons studied on 7 primary endings). Lidocaine (5 #g/ml) significantly reduced this discharge frequency and the effects of 7D stimulation were totally negated by concentrations of 10--15/~g/ml. The remaining discharge observed upon beginning ~/D stimulation (fig. 3C,

162

H.E. LOWNDES ET AL.

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Fig. 3. Sample record o f the effects of cumulative concentrations oflidocaine on the discharge characteristics o f a primary ending in a muscle spindle. The discharge was evoked by combined stimulation of a dynamic (~/D) fusimotor axon (100 Hz during bar) and ramp stretch (3 m m at 12 mm/sec). Fusimotor stimulation was c o m m e n c e d about 0.75 sec prior to the stretch. Pre-drug responses (A) and responses after 5 rain in 5 (B) or 15 (C) pg/ml lid•cain•. D and E after 10 and 15 rain washing in drug-free solution.

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left-hand portion) is an acceleration response consisting of 1--3 impulses when stimulation was initiated. It remained even in the presence of 15--20 #g/ml lidocaine. Stimulation of 17 single static fusimotor axons innervating 7 muscle spindles resulted in primary ending discharge frequencies of 53.7 + 7.3 Hz (fig. 5). A sample is illustrated by the left-hand portion of fig. 4A. Static fusimotor stimulation has been reported to exert either a greater influence on primary ending discharge rates than dynamic fusimotor stimulation (Andersson et al., 1968) or an equally powerful action (Crowe and Matthews, 1964; Brown et al., 1965). In a manner analogous to its actions on responses elicited by 7D stimulation, lidocaine reduced and then abolished the influence of h's stimulation on primary ending discharge (figs. 4, left-hand portion and 5). The EDs0 for reduction of 3's influences was comparable to that for reduction of ~fD influences. A small acceleration response was seen upon initiation of 3's stimulation which was not affected by lidocaine (fig. 4C).

LIDOCAINE ON MUSCLE SPINDLES

163

Washing of the muscle in drug-free solution completely reversed the effects of lidocaine on both ~D and 7s stimulation. Discharge frequencies returned to pre-drug levels within 15-25 min. of termination of drug exposure (fig. 5). 3.3. Effects on primary endings subjected to stretch and fusimotor stimulation The right-hand portions of figs. 3 and 4 illustrate sample responses obtained from a primary ending when the tenuissimus muscle was stretched 3 mm at 12 mm/sec during YD {fig. 3) or 7s (fig. 4) fusimotor stimulation at 100 Hz. In normal spindles, 7D stimulation exerted a much greater effect on discharge during stretch. Peak dynamic frequencies approaching 250 Hz were observed upon 9'D stimulation while ~s stimulation seldom evoked discharge rates in excess of 170 Hz. The static discharge frequencies measured 0.5 sec after application of stretch were considerably enhanced by fusimotor stimulation, being ca. 70.5 + 6.3 and 63.8 + 7.5 Hz for 7D and 7s stimulation respectively. Lidocaine, as a function of dose, reduced and then abolished the static discharge responses evoked by both ~D and 7s stimulation (fig. 6). This action was reversed b y washing the muscle. The peak dynamic discharge evoked b y combined stretch and fusimotor stimulation was reduced by increasing concentrations of lidocaine (fig. 6). However, even 20/~g/ml lidocaine failed to abolish this response completely, for the endings still fired at frequencies of 120--140 Hz in its presence. It is significant to point o u t that the dynamic frequencies were reduced to values identical to those seen with stretch alone (fig. 2). 3.4. Effects on con traction o f in trafusal muscle A muscle spindle was prepared for visualization of the intrafusal fibres upon stimulation, in this case, of a single static fusimotor axon. The afferent discharge was recorded simul-

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Fig. 6. Effects of lidocaine on the dynamic (DYNAMIC) and static (STATIC) discharge frequencies (-+S.E.M.) of primary endings of muscle spindles activated by ramp stretch in the presence of dynamic (7D STIM) or static (Ts STIM) fusimotor stimulation. Static discharge frequencies were measured 0.5 sec after application of ramp stretch. Number of observations with each type of fusimotor axon stimulation indicated at the top. CONT.: pre-drug controls.

taneously with observation of spindle contraction. Static y stimulation is known to cause strong contraction of some intrafusal muscle fibres while dynamic fusimotor influences result in only weak local contractions of one intrafusal fibre (Bessou and Pages, 1975). ~fs stimulation evoked contraction of the intrafusal muscle which was indistinguishable from that in normal spindles even in the presence of 20 #g/ml lidocaine.

164 4. Discussion These experiments report the effects of lidocaine on primary endings of muscle spindles activated by passive elongation of the muscle (stretch alone), b y active contraction of the intrafusal muscle (fusimotor stimulation alone) and b y passive stretch of intrafusal muscles already in a state of contraction evoked by fusimotor stimulation. Activation of de-efferented muscle spindles b y passive stretch does not involve the fusim o t o r innervation. Lidocaine, in a dosedependent fashion, reduced and then abolished b o t h the spontaneous and the static discharge evoked by maintained stretch of the muscle (figs. 1 and 2). However, the peak dynamic discharge was not altered even at the highest concentrations of lidocaine investigated. This is contrary to the findings of Grossie and Smith (1966) who observed a depression of b o t h tonic and phasic firing rates of spindles exposed to procaine. However, the observations of Grossie and Smith (1966) are n o t strictly comparable as they observed the effects of intravenous injections of procaine and the concentration of procaine at the receptor were therefore not known in their experiments. Tonic discharge was more attenuated than phasic discharge when high doses of procaine were employed. The presence of a dynamic response concomitant with abolished static response indicates that the drug did not interfere with transmission of high frequency (up to 250 Hz) trains of impulses in the Ia fibres (i.e. no Wedensky inhibition was observed). It is unlikely that any of the effects of lidocaine observed represent an action of the drug on the primary afferent axon per se. These observations are consistent with some of the observations of O t t o s o n (1964) on the frog's muscle spindle. For example in fig. 5b of this paper it can be seen that lidocaine in concentration of 50/~g/ ml the dynamic c o m p o n e n t and the peak frequency of discharge were hardly altered although the static response was abolished. The steps involved in the transduction of a

H.E. LOWNDES ET AL. mechanical event into sensory afferent discharge are: mechanoreceptor deformation, receptor terminal deformation, initiation of a generator or receptor potential and pacemaker firing (Matthews, p. 265, 1972). Paintal referred to the pacemaker as the regenerative region (1971). The drug would not be expected to alter the first two steps in the transduction process. The most susceptible step is the regenerative region or pacemaker (Paintal, 1964; 1971). Local anesthetics block the pacemaker at concentrations which do not affect the generator potential in both muscle spindles (Katz, 1950; Ottoson, 1964) and pacinian corpuscles (Diamond et al., 1958; Sato and Ozeki, 1963; Sato et al., 1968) probably by depressing sodium conductances. Conversely, drugs such as aconitine or veratridine, which enhance sensory receptor discharge, enhance sodium conductances (Wellhoner, 1968; Ulbright, 1969). While sodium fluxes are involved in both the generator potential and the regenerative encoding process, the sodium carrier may n o t be the same in the two processes (Ottoson, 1964). The observation that static responses were abolished while dynamic responses remained could result from several possibilities. Lidocaine may elevate the threshold of the sensory ending thus requiring a greater input to activate it. On the other hand, there is no necessity to postulate that the same mechanism is involved in generating both the static and dynamic responses at the same ending. Several possible sites of impulse generation exist within the afferent terminal of muscle spindles (Ito, 1970; Kuroda and Ito, 1972; Brokensha and Westbury, 1973; Ito et al., 1974; Brokensha and Westbury, 1978). The observed afferent discharge results from a mixing process at these multiple sites (Brokensha and Westbury, 1973, 1974; Eagles and Purple, 1974; Brokensha and Westbury, 1978). Perhaps entirely different sites on the afferent terminal give rise to static and dynamic discharges. Lidocaine abolished the former b u t was without effect on either of the manifestations of the latter, that is on

LIDOCAINE ON MUSCLE SPINDLES the dynamic discharge signalling velocity o f muscle stretch (the first derivative o f discharge frequency) and on t he acceleration response (the second derivative). T he existence o f discrete sites mediating these responses might explain the differential sensitivity of the responses to lidocaine. Activation o f primary endings by fusim o t o r stimulation was negated by increasing concentrations o f lidocaine. This observation, taken in concert with that o f unaltered impulse transmission in the Ia afferents, suggests that fusimotor influences m a y be lost due to block of impulse c o n d u c t i o n in the fusimotor axons. However, in one spindle dissected for visualization, fusimotor stimulation still resulted in c o n t r a c t i o n o f t he intrafusai muscle fibres even in t he presence of 20 ~g/ml lidocaine. This observation suggests t hat there was no blocking effect o f lidocaine on fusim o t o r axons at this concentration, but possibly one observation is n o t enough t o rule o u t such a blocking action for all spindles. It is likely that this apparent loss o f fusimotor influence is merely a not he r manifestation o f .the effects o f th e local anesthetic on the regenerative region of the afferent termination (vide supra). Stimulation o f a dynamic fusimotor axon results in a small localized contraction o f one intrafusal fibre alone (hag t ype) while static fusimotor activation causes greater contractions o f several intrafusal muscle fibres (bag a n d / o r chain types) (Bessou and Pages, 1975). It was anticipated t h a t dynamic fusimotor influences might be m or e sensitive t o the actions o f lidocaine but examination o f the responses o f the spindle endings to VD and Vs stimulation indicate t h a t b o t h were equally attenuated b y the drug. T he EDs0s for block were the same (ca. 8 #g/ml). Local anesthetics have been r epor t e d to exert an effect on neuromuscular transmission in skeletal muscles. T h e y subtly alter neuromuscular transmission (Straughan, 1961; Inoue and Frank, 1962) and depress the repetitive capacity o f the m o t o r nerve terminals (Usubiaga and Standaert, 1967; Lowndes and

165

Johnson, 1971) by altering the ionic conductances underlying end-plate potentials (Maeno, 1966; Steinbach, 1968). It is possible that lidocaine has some actions at the neuromusculax junctions on intrafusal muscle fibres but if such alterations occurred they were not sufficient to compromise fusimotor activation of intrafusai muscle fibres as shown by observations of spindles treated with lidocaine. References Adal, M.N. and D. Barker, 1962, Intramuscular diameters of afferent nerve fibres in the rectus femoris muscle of the cat, in: Symposium on Muscle Receptors, ed. D. Barker (Hong Kong University Press) p. 249. Andersson, B.E., G. Lennerstrand and U. Thoden, 1968, Response characteristic of muscle spindle endings at constant length to variations in fusimotor activation, Acta Physiol. Scand. 74, 301. Barker, D., M.J. Stacey and M.N. Adal, 1970, Fusimotor innervation in the cat, Phil. Trans. B 258, 315. Bessou, P. and B. Pages, 1967, Enregistrement de mouvements de fuseaux neuromusculaires partiellement diss~qu~s cons~cutifs ~ la stimulation de fibres fusimotrices statiques chez le chat, C.R. Acad. Sci., Paris 265, 351. Bessou, P. and B. Pages, 1972, Intracellular potentials from intrafusal muscle fibres evoked by stimulation of static and dynamic fusimotor axons in the cat, J. Physiol. 227,709. Bessou, P. and B. Pages, 1975, Cinematographic analysis of contractile events produced in intrafusal muscle fibre by stimulation of static and dynamic fusimotor axons, J. Physiol. 252, 397. Brokensha, G. and D.R. Westbury, 1973, Evidence from the adaptation of discharges of the frog muscle spindle for the participation of multiple spike generators, J. Physiol. 232, 25. Brokensha, G. and D.R. Westbury, 1974, Adaptation of the discharge of frog muscle spindles following a stretch, J. Physiol. 242, 383. Brokensha, G. and D.R. Westbury, 1978, Modification by previous afferent discharge of the adaptation of frog muscle spindles following an extension, J. Physiol. 274, 397. Brown, M.C., A. Crowe and P.B.C. Matthews, 1965, Observations on the fusimotor fibres of the tibialis posterior muscle in the cat, J. Physiol. 177,140. Crowe, A. and P.B.C. Matthews, 1964, Further studies of static and dynamic fusimotor fibres, J. Physiol. 175, 132.

166 Diamond, J., J.A.B. Gray and D.R. Inman, 1958, The relation between receptor potentials and the concentration of sodium ions, J. Physiol. 142, 382. Eagles, J.P. and R.L. Purple, 1974, Afferent fibres with multiple encoding sites, Brain Res. 77, 187. Grossie, J. and C.M. Smith, 1966, Depression of afferent activity originating in muscle spindles by mephenesin, procaine and caramiphen, Arch. Intern. Pharmacodyn. 159, 288. Inoue, F. and G.B. Frank, 1962, Action of procaine on frog skeletal muscle, J. Pharmacol. Exptl. Therap. 136, 190. Ito, F. 1970, Effects of polarising currents on long lasting discharges in the frog muscle spindle, Jap. J. Physiol. 20, 697. Ito, F., N. Kanamori and H. Kuroda, 1974, Structural and functional asymmetries of myelinated branches in the frog muscle spindle, J. Physiol. 241, 389. Katz, B., 1950, Depolarization of sensory terminals and the initiation of impulses in the muscle spindle, J. Physiol. 111,261. Kuroda, H. and F. Ito, 1972, Functional differentiation of sensory terminal branches in the frog muscle spindle, Proc. Jap. Acad. 48, 206. Leksell, L., 1945, The action potentials and excitatory effects of the small ventral root fibres to skeletal muscle, Acta Physiol. Scand. 10, Suppl. 31, 1. Lowndes, H.E. and D.D. Johnson, 1971, The effect of lidocaine on twitch potentiation and antidromic produced by soman and neostigmine, Can. J. Physiol. Pharmacol. 49, 464. Maeno, T., 1966, Analysis of sodium and potassium conductances in the procaine end-plate, J. Physiol. 183, 592. Matthews, P.B.C., 1962, The differentiation of two types of fnsimotor fibres by their effects on the dynamic response of muscle spindle primary endings, Quart. J. Exp. Physiol. 4 7 , 3 2 4 . Matthews, P.B.C., 1972, Mammalian Muscle Recep-

H.E. LOWNDES ET AL. tors and their Central Actions (Williams and Wilkins Co. Baltimore) p. 630. Matthews, P.B.C. and G. Rushworth, 1957, The relative sensitivity of muscle nerve fibres to procaine, J. Physiol. 135, 263. Nathan, P.W. and P.A. Sears, 1961, Some factors concerned in differential nerve block by local anesthetics, J. Physiol. 157,565. Ottoson, D., 1964, The effects of sodium dificiency on the response of the isolated muscle spindle, J. Physiol. 171,109. Paintal, A.S., 1964, Effects of drugs on vertebrate mechanoreceptors, Pharmacol. Rev. 16, 341. Paintal, A.S., 1971, Action of drugs on sensory nerve endings, Ann. Rev. Pharmacol. 11,231. Sato, M. and M. Ozeki, 1963, Response of the nonmyelinated nerve terminal in pacinian corpuscles to mechanical and antidromic stimulation and the effects of procaine, choline and cooling, Jap. J. Physiol. 13, 564. Sato, M., M. Ozeki and K. Nishi, 1968, Changes produced by sodium-free condition in the receptor potential of the non-myelinated terminal in pacinian corpuscles, Jap. J. Physiol. 18, 232. Steinbach, A.B., 1968, Alterations by Xylocaine (lidocaine) and its derivatives on the time course of the end-plate potential, J. Gen. Physiol. 52, 144. Straughan, D.W., 1961, The action of procaine at the neuromuscular junction, J. Pharm. Pharmacol. 13, 49. Ulbright, W., 1969, The effect of veratridine on excitable membranes of nerve and muscle, Ergeb. Physiol. 61, 18. Usubiaga, J.E. and J.G. Standaert, 1967, The effects of local anesthetics on motor nerve terminals, J. Pharmacol. Exptl. Therap. 159, 353. Wellhoner, H.-H., 1968, Effects of aconitine on the slowly adapting stretch receptor neurone of the crayfish, Pfliiger's Arch. Ges. Physiol. 304, 104.