susceptibility of either the axons in the dorsal and ventral roots, or the intramedullary

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1 213 J. Physiol. (31958) I40, 2I3-2I9 THE SITE OF ACTION OF PROCAINE ON THE ISOLATED SPINAL CORD OF THE FROG BY M. HARMEL AND J. L. MALCOLM From the Department of Physiology, State University of New York, College of Medicine at New York City, Brooklyn, New York, U.S.A., and the National Institute for Medical Research, Mill Hill, London, N. W. 7 (Received 2 August 1957) It is frequently reported that, in man, the onset of spinal anaesthesia by the intrathecal injection of such local anaesthetics as procaine or allied compounds produces a selective blocking of different types of sensation and reveals a difference in the susceptibility of motor and sensory functions. This selective action of spinal anaesthetics could be due to differences in the susceptibility of either the axons in the dorsal and ventral roots, or the intramedullary branches of these axons and their associated neurones. The experiments described in this paper were designed to determine whether there was a preferred site of action for procaine on intramedullary structures. For such an investigation, the preparation of the cat's spinal cord in situ has the disadvantage that, with it, it is difficult to ensure that the local anaesthetic solution completely surrounds the cord, and that it is not being constantly diluted by the cerebrospinal fluid. The isolated frog's spinal cord provides a simpler and more satisfactory preparation, as it can be completely itmersed in the test solution, while the dorsal and ventral roots are kept clear for stimulating and recording. METHODS The spinal cord of a frog, below the first cervical segment, was removed complete with the dorsal and ventral roots of the 9th and 10th segments, and placed in a Perspex bath (Fig. 1) through which circulated Ringer's solution equilibrated with 95% 02 +5% C02. The composition of the Ringer's solution was: NaCl, 0 7; KCI, 0-014; CaCi,, 0-012; NaHCO3, 0-02 g/100 ml. H20. The dorsal and ventral roots were separated and lifted on to platinum electrodes in the covering layer of paraffin for stimulating and recording. The Ringer's solution was earthed close to the spinal cord. Procaine HCI was added to the Ringer's solution to the concentration required and the effects recorded min later. The temperature of the bath was maintained at from 17 to 200 C. Stimuli were obtained from a Grass square-wave stimulator at a frequency of approximately once every 10 sec. Records were made on 35 mm film from a Cossor oscilloscope after d.c. amplification.

2 214 M. HARMEL AND J. L. MALCOLM Earth Electrodes II 11 I hiiii II IIII 111 Fig. 1. Ringer out Ringer in Cross-sectional view of Perspex bath used to maintain isolated frog spinal cord. Rate of flow of Ringer's solution, 100 ml./hr. t RESULTS Fig. 2 illustrates the results obtained in a typical experiment. The records in column A are the responses of the 10th ventral root to a stimulus applied to the 10th dorsal root on the same side. The only consistent effect recorded with concentrations of 1 x 104 and 3 x 104, as used to obtain the second and third records, is a slight delay in the onset ofthe polysynaptic portion of the response, indicated by the changes in the inflexion on the rising phase of the ventral root response. This inflexion is marked by an arrow in column A. The slight reduction in height in the third record is within the limits of experimental error. Procaine in a concentration of 1 x 10-3 (bottom record) is required in order to reduce the height of the response to a height significantly less than the height of the control (top record). The responses recorded from the 9th dorsal root after the same stimulus to the 10th dorsal root (column B) are more susceptible to procaine. A concentration of 1 x 104 procaine, which had no effect on the height of the ventral root response, definitely diminished the dorsal root response (2nd record), and there is further diminution by higher concentrations (3rd and 4th records, column B). The most susceptible root response, however, is that of the dorsal root to an antidromic volley in the ventral root of the same side and segment (column C). Procaine at a concentration of 1 x 104 markedly diminishes the response (2nd record) and at a concentration of 3 x 104 abolishes it (3rd record). Because this response to an antidromic ventral root volley seemed the most susceptible to procaine, the spread of this volley over the ventral horn cells and their dendrites was further examined by a method of surface recording similar to that used by Lloyd (1951) in the spinal cord of the cat. Fig. 3 shows the records from six points on the surface of the cord between the dorsal root

3 ACTION OF PROCAINE ON FROG SPINAL CORD 215 entry and the ventral root exit, before (column A), and after (column B), bathing the cord in procaine at a concentration of 3 x 104. The pattern of the surface responses is interpreted as follows. The first wave (after the stimulus artifact) marked a is always positive at all points on the surface of the cord, and negative only when the recording electrode is placed on the ventral root. Hence it signals the presence of the incoming volley in the ventral root axons. This a-wave is followed bya short wave marked s that is negative at points on the ventral region of the lateral surface of the cord (points 4-6, Fig. 3) and positive at points on the dorsal region (points 1 and 2, Fig. 3). Such a distribution of positive and negative waves strongly suggests that it signals the invasion of the soma of the ventral horn cells. A B C 4s 0 X~'2x 20 c/s Fig. 2. Records of root potentials of an isolated frog spinal oord. Diagrams at the top of each column indicate the position of stimulating electrodes (S) and recording electrodes (B). Column A, records obtained from the 10th ventral root by stimulating the 10th dorsal root. Column B, records obtained from the 9th dorsal root by stimulating the 10th dorsal root. Column C, records obtained from the 10th dorsal root by stimulating the 10th ventral root. First record in each column obtained with Ringer's solution only bathing the special cord. Second record in each column, after bathing for 30 min in procaine 1 x Third record, after 30 mm in procaine 3 x 10-'. Lowest record, after 30 min in procaine 1 x Arrows on records in column A indicate points of commencement of the polysynaptic component of the ventral root response. The third wave, marked d, is always negative and its characteristics suggest that it signals the invasion of dendrites. Its appearance is increasingly delayed as the recording point is moved away from the region of the ventral horn cells. (Compare d in the records from points 1 and 6, Fig. 3, column A.) At the more distant points (e.g., point 1, Fig. 3), this negative d-wave is preceded by a positive wave that is coincident with the negative d-wave recorded at points adjacent to the ventral horn. The more distant branches of

4 216 M. HARMEL AND J. L. MALCOLM the dendritic tree evidently act as 'sources' of current to the 'sinks' in the proximal branches, before they in turn are invaded, become 'sinks' and give rise to a delayed negative deflexion. The presence of a negative d-wave in all the records in column A, Fig. 3, indicates that some dendrites close to these points on the surface were invaded by the antidromic volley. A d as B d -IUU CIS asd as d Fig. 3. Records obtained from points at mid-segmental level on the surface of the cord as indicated on the diagrammatic section on the left. The cord and a proximal part of the ventral root were fully immersed in the Ringer's solution. Stimulus applied, at S, to the ventral root to initiate an antidromic volley. Column A before, column B after, 30 min in procaine 3 x Stimulus artifact indicated by dot and line. a-, s-, and d-waves, see text. The records in column B, Fig. 3, were taken 20 min after procaine 3 x 10-4 had been added to the perfusing fluid. In this experiment, unlike that illustrated in Fig. 2, such a concentration abolished the dorsal root response to an antidromic volley, but had no effect on either the dorsal or ventral root responses to an orthodromic dorsal root volley. The records from the surface of the cord, however, show that the d-wave is markedly decreased while the a- and s-waves are not significantly altered. The diminution of the d-wave in the regions proximal to the cell bodies (records 5 and 6, Fig. 3) coincides with a decrease in the positive wave in the records from the more distant regions (records 1 and 2, Fig. 3). The positive wave recorded at these more distant

5 ACTION OF PROCAINE ON FROG SPINAL CORD 217 points is followed by only a minute negative wave. This shows that, as a result of the action of procaine, the more distant branches of the dendritic tree can still act as a 'source' to the smaller 'sink' in the proximal branches, while very few of the more distant branches are themselves invaded. Procaine interferes with the normal facilitatory action of a dorsal root volley on an antidromic ventral root volley. This facilitation is confined to the dendritic component as illustrated by Fig. 4. Record A, in Fig. 4, is the normal response to an antidromic volley recorded at a point corresponding to position 4 in Fig. 3, while B is the response to the same volley at the same point 35 msec Cb t1 t IT t a s d a s d Fig. 4. The response of a point in the middle of the lateral surface of a frog's spinal cord to an antidromic volley in the ventral root, Records A and B before, C and D after, bathing the cord in procaine 1 x 10-4 for 30 min. Records A and C without, B and D with, a facilitating dorsal root volley initiated 35 msec earlier. a, s and d, as in Fig. 3 and in text. after a maximal dorsal root volley. The a- and s-waves are unchanged, but the d-wave is greatly increased, i.e. the antidromic volley has now spread into many more branches of the dendritic tree. After bathing the cord in procaine 1 X 10-4 for 30 min the antidromic response alone is slightly depressed (Fig. 3 C) and the extent to which the d-wave can be facilitated greatly diminished (Fig. 3D). At this concentration of procaine, the conditioning facilitating volley was unaffected. 14 PHYSIO. CXL

6 218 M. HARMEL AND J. L. MALCOLM DISCUSSION The effects of low concentrations of procaine are primarily on the dendrites of the ventral horn cell. This is clearly indicated by the block of an antidromic volley before it spreads very far beyond the soma, and the inability of an orthodromic volley, apparently unaffected, to facilitate this spread. More weight is given to this hypothesis by the finding that a dorsal root response to a neighbouring dorsal root volley is little affected by concentrations of procaine that block the dorsal root response to an antidromic ventral root volley. Presumably the terminal ramifications of the afferent fibres are less susceptible than the dendrites of the ventral horn cells. It is not possible to assess the role played by interneurones in these experiments. If they provide the preferential site of action for low concentrations of procaine one would expect that blocking their activity would diminish the spread of the antidromic volley in the ventral root only if they normally maintained a continuous background of facilitating bombardment. The cessation of such a background of facilitation would be at once obvious on the normal ventral root response to a dorsal root volley if the ventral root discharge were dependent on the integrity of the whole motoneurone. This, however, is probably not the case as a nearly normal ventral root discharge can be obtained when the dendrites cannot be antidromically activated. A large factor of safety is seemingly maintained so that activation by the afferent volley of soma and proximal dendrites alone is sufficient to produce a normal response. The slight delay in the initiation of the later, polysynaptic, portion of the response suggests that the presynaptic volleys are not quite so adequate as normally. It is not possible, therefore, to say whether the depression of dendritic activity is due to direct action of procaine on the dendrites or indirect via diminished interneurone activity. It seems unlikely, however, that interneurones would show much continuous activity in such an isolated preparation as used here, especially as the tension of CO2 provided from the perfusing fluid is higher than that normally found in the frog's spinal cord and would be expected therefore to depress the over-all activity of interneurones. The results of these experiments therefore strongly suggest that the dendrites of the motoneurones are the most susceptible to the action of procaine. The reason for this susceptibility does not necessarily lie in a specific chemical difference between dendrites and other structures. Two other possible explanations should be borne in mind. One is that the differences in susceptibility depend upon variations in the safety factor for conduction of nerve volleys in different units. The excitation process, normally, may be only just adequate for conduction over dendrites, whereas for firing a motoneurone it may be many times that needed to reach threshold. In these circumstances,

7 ACTION OF PROCAINE ON FROG SPINAL CORD 219 the procaine would be more effective on dendritic conduction though equally distributed over all the elements in the cord. The other possible explanation is that the procaine diffuses unequally throughout the cord and hence units that ramify over the widest field are the ones most likely to be affected by the procaine. This would account for the greater susceptibility of the motoneurone dendrites which ramify extensively through several segments of the cord. SUMMARY 1. The sensitivity to procaine of different parts of the isolated frog's spinal cord was observed by recording the electrical responses of dorsal and ventral roots and the spinal cord to orthodromic and antidromic nerve volleys. 2. The most sensitive responses were: (a) the dendritic response of the motoneurone to an antidromic, ventral root volley; (b) the dendritic response of the motoneurone to an antidromic volley after facilitation by an orthodromic, dorsal root volley; (c) the dorsal root response to an antidromic, ventral root volley. 3. The most sensitive responses depend primarily on the integrity of the motoneurone dendritic tree, though the possibility of the failure of interneurones, if active, playing some part in the recorded changes cannot be excluded. REFERENCE LLOYD, D. P. C. (1951). Electrical signs of impulse conduction in spinal motor neurones. J. gen. Phy8iol. 35,