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Effect of iontophoretically applied 5-hydroxytryptamine on the excitability of single primary afferent C- and A-fibers in the cat spinal cord
Brain Research, 220 (1981) 151-158
151
Elsevier/North-Holland Biomedical Press
E F F E C T OF I O N T O P H O R E T I C A L L Y A P P L I E D 5 - H Y D R O X Y T R Y P T A M I N E O N T H E E X C I T A B I L I T Y OF S I N G L E P R I M A R Y A F F E R E N T C- A N D AFIBERS I N T H E CAT S P I N A L C O R D
E. CARSTENS*, D. KLUMPP, M. RANDI(~** and M. ZIMMERMANN I!. Physiologisches lnstitut der Universitiit, D-69 Heidelberg, Im Neuenheimer Feld 326 ( G.F.R.)
(Accepted January 15th, 1981) Key words: 5-hydroxytryptamine --excitability testing - - C-fiber - - A-fiber - - presynaptic inhibi-
tion
SUMMARY Excitability testing of single sural afferent C- and A-fibers was employed to study possible presynaptic effects of 5-hydroxytryptamine (5-HT) applied iontophoretically at the intraspinal point of lowest threshold for their antidromic activation in anesthetized or decerebrate spinalized cats. Threshold for single fibers recorded in the sural nerve was measured prior to and during iontophoretic application of 5-HT through a micropipette positioned in close proximity to the intraspinal stimulating electrode. 5H T produced dose-related increases in threshold for antidromic activation in 21 or 30 C-fibers. Six of 9 A ~-, and 4 of 7 Aft-fibers were similarly affected.
1NTRODUCTION 5-HT generally has a depressant action when applied iontophoretically near spinal dorsal horn neurons1,10,14, 21. This spinal action of 5-HT has been associated with descending inhibitory effects on spinal neurons produced by stimulation in brain stem areas known to contain serotonergic neurons1,2,5, s. Some evidence exists for both pre- and postsynaptic inhibitory actions of 5-HT 14,19,21. To more directly evaluate a possible presynaptic action of 5-HT, we have used a modification4,11,17,~2,za, 25 of Wall's 26 method to measure excitability changes at the central terminals of single sural
*Present address and address for correspondence: Department of Animal Physiology, University of California, Davis, Calif. 95616, U.S.A. **Present address: Department of Veterinary Physiology and Pharmacology, Iowa State University, Ames, Iowa 50011, U.S.A. 0006-8993/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
152 C- a n d A-fibers d u r i n g i o n t o p h o r e t i c a p p l i c a t i o n o f 5 - H T near these terminals. Excitability changes are t h o u g h t to reflect changes in the terminal m e m b r a n e potential, which in turn influences the a m o u n t o f t r a n s m i t t e r released16, 24. A p r e l i m i n a r y r e p o r t was made% MATERIALS AND METHODS T w e n t y - t w o cats (2.6-3.2 kg) were anesthetized with s o d i u m p e n t o b a r b i t a l ( N e m b u t a l , 40 m g / k g i.p.). Anesthesia was m a i n t a i n e d by slow intravenous infusion o f N e m b u t a l (3 m g / k g / h ) . A n i m a l s were i m m o b i l i z e d ( p a n c u r o n i u m bromide, 0.4 m g / k g / h ) a n d artificially ventilated. A r t e r i a l b l o o d pressure, end-tidal pCO2, a n d rectal t e m p e r a t u r e were m o n i t o r e d a n d m a i n t a i n e d within n o r m a l limits. A l u m b a r l a m i n e c t o m y was done, a n d the spinal cord was left intact in all but one cat, which was spinalized at the L1 level. T w o a d d i t i o n a l cats anesthetized with h a l o t h a n e were dec e r e b r a t e d by occlusion o f b o t h c o m m o n c a r o t i d a n d vertebral arteries a n d spinalized at the CI level. The left sural nerve was dissected free a n d placed on a dissection p l a t f o r m . Small filaments were cut peripherally and placed on a m o n p o l a r silver wire (0.2 m m d i a m e t e r ) r e c o r d i n g electrode (Fig. I A). Single sural fibers were identified by recording u n i t a r y action potentials elicited a n t i d r o m i c a l l y by surface stimulation o f
excttability testing
A s~ngle fiber ..... record,ng
~
a
** A-fibers'
67pA
, ,..i
---
C~
/~~LI
2215pA
/
.'m
-____ ~ "\ ~._~.~_~
22 ~A ~g- , , 2t'-~se c
50 rose(
~
k... sp,no/cord
/
Fig. 1. Single fiber excitability testing method. A : experimental set-up. Small filaments of sural nerve were recorded peripherally. Single afferent fibers in filament were antidromically activated by intraspinal stimulation with excitability testing electrode. Drugs were iontophoretically applied through a micropipette whose tip was close ( < 10 pm) to that of excitability testing electrode. B: oscilloscope traces of antidromically evoked A-fiber action potential (indicated by arrow). Intraspinal stimulus current strength (given to left) was suprathreshold in upper- and lower-most pairs of traces. Middle pair of traces shows absence of action potential when current strength was just subthreshold. This current strength was defined as threshold. C: antidromically evoked action potentials in a Cfiber (arrow). lntraspinal stimulus strength (to left) was lowered in successive traces until action potential could not be evoked (threshold; middle pair of traces). Strength was again raised (lower 2 traces) and threshold determination repeated.
153 L7 or $1 dorsal rootlets. A glass-coated platinum wire (20 /~m diameter, exposed tip < 15 # m in length) microstimulating electrode was then introduced at a 10° or 25 ° angle into the cord just rostral to the dorsal rootlet and the single sural fiber(s) reidentified by intraspinal stimuli (0.2 msec constant current cathodal square pulses) delivered every 3.5 sec. The intraspinal site of lowest threshold for antidromic activation of single sural fibers was determined systematically. This position was verified to be close to the terminals of A-fibers by recording transient threshold reductions (primary afferent depolarization) following electrical stimulation of the posterior tibial nerve. Classification of fibers was based on calculated conduction velocity (Fig. 1B). Threshold determinations were made by reducing stimulus strength in 0.5 ~ decrements until the fiber did not respond to 2 consecutive stimuli (Fig. 1B). Thresholds ranged from 5.7 to 26.6 #A (mean 13.7 #A) for C-fibers and 4.9-17 #A for A-fibers (mean 8 and 9.7 #A for Aft- and A6-fibers, respectively). Stimulation sites were usually 800-1200 # m below the cord surface, corresponding to the lateral substantia gelatinosa. To test 5-HT, a compound electrode, consisting of a microstimulating electrode (as above) glued to an angled single or multi-barrel micropipette (tip separation < 10 #m), was inserted into the cord at the same location and the fiber(s) re-identified. Drug solutions for iontophoresis were: 5-HT HC1 or creatinine sulphate (50 mM, pH 4-5, in water or 165 mM NaCI, Sigma, Serva), creatinine sulphate (50 mM, pH 4-5, Sigma) and NaCI (165 mM, pH 4-5). Drug-filled pipettes had resistances of 15-50 Mf~. A constant retaining current of 10 nA was used for all drugs. Drug application was begun when threshold for antidromic activation did not vary by more than 5-10 ~ over a period of 6 min, and the mean of these values was taken as the 100K baseline. All threshold values before and during drug application were normalized to this level. Only values during the first 6 min of drug application were used. Current balancing procedures were used in several experiments. RESULTS Results were obtained from 30 C-fibers (conduction velocity < 1.5 m/sec), 9 A6-fibers (5-20 m/sec) and 7 Aft-fibers ( > 4 0 m/sec). Iontophoretic application of 5H T (25-200 nA, for periods up to 6 min) resulted in an increase in threshold for antidromic activation of most (31/46) fibers (Table I). For each fiber class, threshold TABLE
I
Effects o f 5 - H T (25-200 nA, up to 6 min) on threshoM for antidromic activation o f sural afferent fibers Type o f fiber
Number o f fibers
Threshold Increase
Decrease
Biphasic
No effect
C
30
21
3
0
6
A6 Aft
9 7
6 4
1 0
1 0
1 3
46
31
4
1
10
Total
154 A
C-fiber 5-HT 50
C-fiber
B
No I00
m
5-HT 200
m
m
120
Creat-SO 4 200 m
-~ 12o
cp
IiO E
o
~ IOO
I00
-o o
90
~c o
90
I lOmm
I0
rnin
Fig. 2. Effect of 5-HT on threshold for antidromic C-fiber activation. A: normalized antidromic threshold measurements in a single sural C-fiber prior to and during iontophoretic application of 5HT (50 nA) and NaC1 (100 nA). Durations of ejections indicated by black bars. B: normalized antidromic threshold values for another C-fiber during iontophoretic application of 5-HT (200 nA) and creatine sulphate (200 nA).
values (in/~A) during the 6-min period before, and again during 5-HT application, were pooled. The mean threshold increase for C-fibers during 5-HT application (to 109 % ~ 1.7 S.E.M. of control) was statistically significant (P < 0.005, F-test), while mean threshold increases for the A-fiber groups were not statistically significant.
C-fibers Threshold increases in 21 C-fibers during 5-HT application ranged from 106 to 139 % of control, while thresholds were unaffected in 6 and reduced in 3 C-fibers (88-95 %) (Table I). Threshold increases were seen in 3 of 4 C-fibers from spinalized cats. The threshold increases usually began within 30-60 sec following the start of 5-
/
%
A
B lZ.0
5 C- fibers 120
120
.c-
u E o
£ ~ 100 E 2
100 5-HT B0 -6
-3
'
0
100nA '
3
min
v '
6
80
t
-6
i
3
200 n A
5 - HT
i
i
0
3
J
mm
6
Fig. 3. Dose-related antidromic threshold increases induced by 5-HT in C-fibers. A: composite of normalized antidromic threshold values 6 min before and 6 min after the start of iontophoretic application of 100 nA 5-HT. Each curve corresponds to one drug application (7 drug applications in 5 C-fibers). B: same as in A, except that strength of 5-HT ejection current was 200 nA. Eight drug applications in 5 C-fibers.
155 H T iontophoresis, reached a maximum within 3-5 min, and recovered within 1-3 min after the end of 5-HT application (Fig. 2). The degree of threshold increase during 5H T application appeared to be dose-related. Fig. 3 shows composite plots of normalized individual thresholds for several C-fibers before and during 5-HT application at 100 nA (A) and 200 nA (B). The mean threshold increase produced by 5-HT was greater at 200 nA (119~o q- 11.6 S.D., n = 8) than at 100 nA (106.4~ 4- 6, n = 7). In one experiment, we attempted to determine whether the threshold increase induced by 5-HT represents a selective action at the fiber terminal, or a generalized effect on the axonal membrane. When the compound electrode was positioned near the C-fiber axon in the dorsal rootlet, 5-HT had no effect on threshold. When the electrode was positioned intraspinally near the terminal of the same C-fiber, 5-HT (200 nA) produced a marked threshold increase (to 119 ~).
A-fibers Iontophoretic application of 5-HT (25-200 nA) produced threshold increases in 6/9 AO-fibers (range: 111-128 ~ ) and in 4/7 Aft-fibers (range: 106-128 ~o) (Table I). Thresholds of the remaining A-fibers were unaffected or reduced (in one case with biphasic effects).
Current controls Current controls (ejection of Na + or creatinine ions through a separate barrel) sometimes produced small threshold changes in C- and A-fibers tested, but in each case the effect was smaller than that produced by 5-HT (Fig. 2).
Primary afferent depolarization (PAD) Threshold changes were not produced in any of 7 C-fibers tested by conditioning stimulation of the posterior tibial nerve at a strength supramaximal for C-fibers (30 V, 1 msec, conditioning test interval 250 msec), either before or during 5-HT application. Threshold reductions of up to 80 ~o of control (i.e. PAD) were observed in 3 A6- and 3 Aft-fibers following tibial nerve stimulation at A-fiber strength (2 V, 0.1 msec), and this PAD was unaffected during 5-HT application. DISCUSSION Iontophoretic application of 5-HT resulted in increases in threshold for antidromic activation of about 67 ~o of C- and A-fibers. In discussing this effect in terms of a possible presynaptic action of 5-HT, it is important to critically consider other factors which might contribute to such threshold changes. For example, iontophoretic application of a compound might induce changes in extracellular ionic concentration, leading to osmotic changes and changes in tissue volume. Tissue swelling (threshold increase) or shrinkage (threshold reduction) might occur. Ejection of Na + ions had relatively minor effects on fiber threshold, but we have not yet tested other ions which might produce changes in tissue permeability. The small effects of Na + and creatinine
156 application on fiber thresholds also argue against non-specific current or pH effects. It cannot be determined with this method whether 5-HT acts directly at the fiber terminal, or indirectly, e.g. via inhibition of interneurons mediating tonic PAD at the fiber terminals. However, the intraspinal antidromic thresholds of some polymodal nociceptors and high-threshold mechanoreceptors with afferent C-fibers were recently shown to be raised during electrical stimulation of the brain stem nucleus raphe magnus (NRM, 11 ) in which 5-HT-containing neurons are located 5. N R M stimulation is also associated with a reduction in excitability of spinal dorsal horn neurons ~,9,2s, suggesting that its effect on C-fibers might reflect a presynaptic inhibitory mechanism. 5-HT and morphine4, 2a have similar effects on C-fiber thresholds, and both of these compounds generally reduce spinal neuronal excitabilityS, 20. Therefore, threshold increases may reflect a presynaptic inhibitory mechanism at C-fiber terminals, in contrast to large A-fibers in which presynaptic inhibition is generally thought to be associated with PAD (threshold reductiona6,24). Several mechanisms for the threshold increase have been suggested4,11,16,22, 23. One is that it reflects primary afferent hyperpolarization (PAH). PAH is thought to be associated with presynaptic facilitation, which would be at variance with generally depressant actions of N R M stimulation, opiates and 5-HT. However, recent evidence from invertebrate preparations indicates that presynaptic inhibition is associated with PAH which may be mediated by an increase in chloride conductance7,15. Certain myenteric ~3 and nodose ganglion ~2 neurons are hyperpolarized by 5-HT via an apparent calcium-dependent increase in potassium conductance 1~. An increase in membrane conductance would create a current shunt, which in the present experiments would necessitate a greater stimulus current density to antidromically excite the fiber. It should be noted, however, that 5H T generally has a predominantly depolarizing action on neural membranes ~2,13,27 including non-myelinated nerve fibers is. Any depolarizing action of 5-HT at intraspinal terminals would have to be outweighed by other membrane effects to account for the threshold increases which we commonly observed. PAD (threshold reduction) was consistently produced by N R M stimulation in identified cutaneous A-fiber afferents 17. It is possible that this PAD is mediated by a mechanism other than the spinal release of 5-HT, since the latter more often produced threshold increases in C- and A-fibers in the present experiments. KCANOWLEDGEMENTS The authors are most grateful to Fr. A. Giner for excellent technical assistance, to Fr. A. Manisali for graphics, and to Prof. L. Vyklicky for helpful comments on an earlier version of the manuscript. M.R. was supported by grants from the National Science Foundation (BNF 23871) and the Deutscher Akademischer Austauschdienst. E.C. was recipient of a postdoctoral fellowship from the National Institutes of Health (NINCDS~). This work was supported by a grant (Zi 110) from the Deutsche Forschungsgemeinschaft.
157 REFERENCES 1 Belcher, G., Ryall, R. W. and Schaffner, R., The differential effects of 5-hydroxytryptamine, noradrenaline and raphe stimulation on nociceptive dorsal horn interneurones in the cat, Brain Research, 151 (1978)307-321. 2 Carstens, E., Fraunhoffer, M. and Zimmermann, M., Serotonerglc mediation of descending inhibition from midbrain periaqueductal gray, but not reticular formation, of spinal nociceptive transmission in the cat, Pain, in press. 3 Carstens, E., Klumpp, D., Randi~, M. and Zimmermann, M., Excitability changes at intraspinal terminals of cutaneous afferent C- and A-fibers induced by iontophoretic application of 5-hydroxytryptamine and morphine, Pfliigers Arch.-Europ. J. Physiol., 382 (suppl.) (1979) R50. 4 Carstens, E., Tulloch, I., Zieglg~insberger, W. and Zimmermann, M., Presynaptic excitability changes induced by morphine in single cutaneous afferent C- and A-fibers, Pfliigers Arch. ges. Physiol., 379 (1979) 143-147. 5 Fields, H. L. and Basbaum, A. I., Brainstem control of spinal pain - - transmission neurons, Ann. Rev. Physiol., 40 (1978) 217-248. 6 Fields, H. L., Basbaum, A. I., Clanton, C. H. and Anderson, S. D., Nucleus raphe magnus inhibition of spinal cord dorsal horn neurons, Brain Research, 126 (1977) 441-453. 7 Fuchs, P. A. and Getting, P. A., Ionic basis of presynaptic inhibitory potentials at crayfish claw opener, J. Neurophysiol., 43 (1980) 1547-1557. 8 Guilbaud, G., Besson, J.-M., Oliveras, J. L. and Liebeskind, J. C., Suppression by LSD of the inhibitory effect exerted by dorsal raphe stimulation on certain spinal cord interneurons in the cat, Brain Research, 61 (1973) 417-422. 9 Guilbaud, G., Oliveras, J. L., Giesler, G. and Besson, J.-M., Effects induced by stimulation of the centralis inferior nucleus of the raphe on dorsal horn interneurons in cat's spinal cord, Brain Research, 126 (1977) 355-360. 10 Headley, P. M., Duggan, A. W. and Griersmith, B. T., Selective reduction by noradrenaline and 5-hydroxytryptamine of nociceptive responses of cat dorsal horn neurones, Brain Research, 145 (1978) 185-189. 11 Hentali, I. D. and Fields, H. L., Segmental and descending influences on the thresholds of single intraspinal C-fibers, J. NeurophysioL, 42 (1979) 1527-1537. 12 Higashi, H., 5-hydroxytryptamine receptors on visceral primary afferent neurons in the nodose ganglion of the rabbit, Nature (Lond.), 267 (1977) 448-450. 13 Johnson, S. M., Katayama, Y. and North, R. A., Multiple actions of 5-hydroxytryptamine on myenteric neurones of the guinea-pig ileum, J. Physiol. (Lond.), 304 (1980) 459-470. 14 Jordan, L. M., Kenshalo, D. R., Martin, R. F., Haber, L. H. and Willis, W. D., Depression of primate spinothalamic tract neurons by iontophoretic application of 5-hydroxytryptamine, Pain, 5 (1978) 135-142. 15 Kawai, N. and Niwa, A., Hyperpolarization of the excitatory nerve terminals by inhibitory nerve stimulation in lobster, Brain Research, 137 (1977) 365-368. 16 Levy, R. A., Presynaptic control of input to the central nervous system, Canad. J. PhysioL Pharmacol., 58 (1980) 751-765. 17 Martin, R. F., Haber, L. H. and Willis, W. D., Primary afferent depolarization of identified cutaneous fibers following stimulation in medial brain stem, J. NeurophysioL, 42 (1979) 779-790. 18 Neto, F. R., The depolarizing action of 5-HT on mammalian non-myelinated nerve fibres, Europ. J. PharrnacoL, 49 (1978) 351-356. 19 Proudfit, H. K. and Anderson, E. G., New long latency bulbospinal evoked potentials blocked by serotonin antagonists, Brain Research, 65 (1974) 542-546. 20 Randi6, M. and Miletic, V., Depressant actions of methionine-enkephatin and somatostatin in cat dorsal horn neurones activated by noxious stimuli, Brain Research, 152 (1978) 196-202. 21 Randi6, M. and Yu, H. H., Effects of 5-hydroxytryptamine and bradykinin in cat dorsal horn neurones activated by noxious stimuli, Brain Research, 111 (1975) 197-203. 22 Sastry, B. R., Morphine and met-enkephalin effects on sural At$ afferent terminal excitability, Europ. J. PharmacoL, 50 (1978) 269-273. 23 Sastry, B. R., Presynaptic effects of morphine and methionine enkephalin in feline spinal cord, Neuropharmacology, 18 (1979) 367-375. 24 Schmidt, R. F., Presynaptic inhibition in the vertebrate central nervous system, Rev. PhysioL Biochem. PharmacoL, 63 (1971) 21-101.
158 25 Schmidt, R. F., Senges, J. and Zimmermann, M., Excitability measurements at the central terminals of single mechanoreceptor afferents during slow potential changes, Exp. Brain Res., 3 (1967) 220-233. 26 Wall, P. D., Excitability changes in afferent fibre terminations and their relation to slow potentials, J. Physiol. (Lond.), 142 (1958) 1-12. 27 Wallis, D. I. and Woodward, B., Membrane potential changes induced by 5-hydroxytryptamine in the rabbit superior cervical ganglion, Brit. J. Pharmacol., 55 (1975) 199-212. 28 Willis,W. D., Haber, L. H. and Martin, R. F., Inhibition of spinothalamic tract cells and interneurons by brain stem stimulation in the monkey, J. NeurophysioL, 40 (1977) 968-981.
151
Elsevier/North-Holland Biomedical Press
E F F E C T OF I O N T O P H O R E T I C A L L Y A P P L I E D 5 - H Y D R O X Y T R Y P T A M I N E O N T H E E X C I T A B I L I T Y OF S I N G L E P R I M A R Y A F F E R E N T C- A N D AFIBERS I N T H E CAT S P I N A L C O R D
E. CARSTENS*, D. KLUMPP, M. RANDI(~** and M. ZIMMERMANN I!. Physiologisches lnstitut der Universitiit, D-69 Heidelberg, Im Neuenheimer Feld 326 ( G.F.R.)
(Accepted January 15th, 1981) Key words: 5-hydroxytryptamine --excitability testing - - C-fiber - - A-fiber - - presynaptic inhibi-
tion
SUMMARY Excitability testing of single sural afferent C- and A-fibers was employed to study possible presynaptic effects of 5-hydroxytryptamine (5-HT) applied iontophoretically at the intraspinal point of lowest threshold for their antidromic activation in anesthetized or decerebrate spinalized cats. Threshold for single fibers recorded in the sural nerve was measured prior to and during iontophoretic application of 5-HT through a micropipette positioned in close proximity to the intraspinal stimulating electrode. 5H T produced dose-related increases in threshold for antidromic activation in 21 or 30 C-fibers. Six of 9 A ~-, and 4 of 7 Aft-fibers were similarly affected.
1NTRODUCTION 5-HT generally has a depressant action when applied iontophoretically near spinal dorsal horn neurons1,10,14, 21. This spinal action of 5-HT has been associated with descending inhibitory effects on spinal neurons produced by stimulation in brain stem areas known to contain serotonergic neurons1,2,5, s. Some evidence exists for both pre- and postsynaptic inhibitory actions of 5-HT 14,19,21. To more directly evaluate a possible presynaptic action of 5-HT, we have used a modification4,11,17,~2,za, 25 of Wall's 26 method to measure excitability changes at the central terminals of single sural
*Present address and address for correspondence: Department of Animal Physiology, University of California, Davis, Calif. 95616, U.S.A. **Present address: Department of Veterinary Physiology and Pharmacology, Iowa State University, Ames, Iowa 50011, U.S.A. 0006-8993/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
152 C- a n d A-fibers d u r i n g i o n t o p h o r e t i c a p p l i c a t i o n o f 5 - H T near these terminals. Excitability changes are t h o u g h t to reflect changes in the terminal m e m b r a n e potential, which in turn influences the a m o u n t o f t r a n s m i t t e r released16, 24. A p r e l i m i n a r y r e p o r t was made% MATERIALS AND METHODS T w e n t y - t w o cats (2.6-3.2 kg) were anesthetized with s o d i u m p e n t o b a r b i t a l ( N e m b u t a l , 40 m g / k g i.p.). Anesthesia was m a i n t a i n e d by slow intravenous infusion o f N e m b u t a l (3 m g / k g / h ) . A n i m a l s were i m m o b i l i z e d ( p a n c u r o n i u m bromide, 0.4 m g / k g / h ) a n d artificially ventilated. A r t e r i a l b l o o d pressure, end-tidal pCO2, a n d rectal t e m p e r a t u r e were m o n i t o r e d a n d m a i n t a i n e d within n o r m a l limits. A l u m b a r l a m i n e c t o m y was done, a n d the spinal cord was left intact in all but one cat, which was spinalized at the L1 level. T w o a d d i t i o n a l cats anesthetized with h a l o t h a n e were dec e r e b r a t e d by occlusion o f b o t h c o m m o n c a r o t i d a n d vertebral arteries a n d spinalized at the CI level. The left sural nerve was dissected free a n d placed on a dissection p l a t f o r m . Small filaments were cut peripherally and placed on a m o n p o l a r silver wire (0.2 m m d i a m e t e r ) r e c o r d i n g electrode (Fig. I A). Single sural fibers were identified by recording u n i t a r y action potentials elicited a n t i d r o m i c a l l y by surface stimulation o f
excttability testing
A s~ngle fiber ..... record,ng
~
a
** A-fibers'
67pA
, ,..i
---
C~
/~~LI
2215pA
/
.'m
-____ ~ "\ ~._~.~_~
22 ~A ~g- , , 2t'-~se c
50 rose(
~
k... sp,no/cord
/
Fig. 1. Single fiber excitability testing method. A : experimental set-up. Small filaments of sural nerve were recorded peripherally. Single afferent fibers in filament were antidromically activated by intraspinal stimulation with excitability testing electrode. Drugs were iontophoretically applied through a micropipette whose tip was close ( < 10 pm) to that of excitability testing electrode. B: oscilloscope traces of antidromically evoked A-fiber action potential (indicated by arrow). Intraspinal stimulus current strength (given to left) was suprathreshold in upper- and lower-most pairs of traces. Middle pair of traces shows absence of action potential when current strength was just subthreshold. This current strength was defined as threshold. C: antidromically evoked action potentials in a Cfiber (arrow). lntraspinal stimulus strength (to left) was lowered in successive traces until action potential could not be evoked (threshold; middle pair of traces). Strength was again raised (lower 2 traces) and threshold determination repeated.
153 L7 or $1 dorsal rootlets. A glass-coated platinum wire (20 /~m diameter, exposed tip < 15 # m in length) microstimulating electrode was then introduced at a 10° or 25 ° angle into the cord just rostral to the dorsal rootlet and the single sural fiber(s) reidentified by intraspinal stimuli (0.2 msec constant current cathodal square pulses) delivered every 3.5 sec. The intraspinal site of lowest threshold for antidromic activation of single sural fibers was determined systematically. This position was verified to be close to the terminals of A-fibers by recording transient threshold reductions (primary afferent depolarization) following electrical stimulation of the posterior tibial nerve. Classification of fibers was based on calculated conduction velocity (Fig. 1B). Threshold determinations were made by reducing stimulus strength in 0.5 ~ decrements until the fiber did not respond to 2 consecutive stimuli (Fig. 1B). Thresholds ranged from 5.7 to 26.6 #A (mean 13.7 #A) for C-fibers and 4.9-17 #A for A-fibers (mean 8 and 9.7 #A for Aft- and A6-fibers, respectively). Stimulation sites were usually 800-1200 # m below the cord surface, corresponding to the lateral substantia gelatinosa. To test 5-HT, a compound electrode, consisting of a microstimulating electrode (as above) glued to an angled single or multi-barrel micropipette (tip separation < 10 #m), was inserted into the cord at the same location and the fiber(s) re-identified. Drug solutions for iontophoresis were: 5-HT HC1 or creatinine sulphate (50 mM, pH 4-5, in water or 165 mM NaCI, Sigma, Serva), creatinine sulphate (50 mM, pH 4-5, Sigma) and NaCI (165 mM, pH 4-5). Drug-filled pipettes had resistances of 15-50 Mf~. A constant retaining current of 10 nA was used for all drugs. Drug application was begun when threshold for antidromic activation did not vary by more than 5-10 ~ over a period of 6 min, and the mean of these values was taken as the 100K baseline. All threshold values before and during drug application were normalized to this level. Only values during the first 6 min of drug application were used. Current balancing procedures were used in several experiments. RESULTS Results were obtained from 30 C-fibers (conduction velocity < 1.5 m/sec), 9 A6-fibers (5-20 m/sec) and 7 Aft-fibers ( > 4 0 m/sec). Iontophoretic application of 5H T (25-200 nA, for periods up to 6 min) resulted in an increase in threshold for antidromic activation of most (31/46) fibers (Table I). For each fiber class, threshold TABLE
I
Effects o f 5 - H T (25-200 nA, up to 6 min) on threshoM for antidromic activation o f sural afferent fibers Type o f fiber
Number o f fibers
Threshold Increase
Decrease
Biphasic
No effect
C
30
21
3
0
6
A6 Aft
9 7
6 4
1 0
1 0
1 3
46
31
4
1
10
Total
154 A
C-fiber 5-HT 50
C-fiber
B
No I00
m
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Fig. 2. Effect of 5-HT on threshold for antidromic C-fiber activation. A: normalized antidromic threshold measurements in a single sural C-fiber prior to and during iontophoretic application of 5HT (50 nA) and NaC1 (100 nA). Durations of ejections indicated by black bars. B: normalized antidromic threshold values for another C-fiber during iontophoretic application of 5-HT (200 nA) and creatine sulphate (200 nA).
values (in/~A) during the 6-min period before, and again during 5-HT application, were pooled. The mean threshold increase for C-fibers during 5-HT application (to 109 % ~ 1.7 S.E.M. of control) was statistically significant (P < 0.005, F-test), while mean threshold increases for the A-fiber groups were not statistically significant.
C-fibers Threshold increases in 21 C-fibers during 5-HT application ranged from 106 to 139 % of control, while thresholds were unaffected in 6 and reduced in 3 C-fibers (88-95 %) (Table I). Threshold increases were seen in 3 of 4 C-fibers from spinalized cats. The threshold increases usually began within 30-60 sec following the start of 5-
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6
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5 - HT
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i
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Fig. 3. Dose-related antidromic threshold increases induced by 5-HT in C-fibers. A: composite of normalized antidromic threshold values 6 min before and 6 min after the start of iontophoretic application of 100 nA 5-HT. Each curve corresponds to one drug application (7 drug applications in 5 C-fibers). B: same as in A, except that strength of 5-HT ejection current was 200 nA. Eight drug applications in 5 C-fibers.
155 H T iontophoresis, reached a maximum within 3-5 min, and recovered within 1-3 min after the end of 5-HT application (Fig. 2). The degree of threshold increase during 5H T application appeared to be dose-related. Fig. 3 shows composite plots of normalized individual thresholds for several C-fibers before and during 5-HT application at 100 nA (A) and 200 nA (B). The mean threshold increase produced by 5-HT was greater at 200 nA (119~o q- 11.6 S.D., n = 8) than at 100 nA (106.4~ 4- 6, n = 7). In one experiment, we attempted to determine whether the threshold increase induced by 5-HT represents a selective action at the fiber terminal, or a generalized effect on the axonal membrane. When the compound electrode was positioned near the C-fiber axon in the dorsal rootlet, 5-HT had no effect on threshold. When the electrode was positioned intraspinally near the terminal of the same C-fiber, 5-HT (200 nA) produced a marked threshold increase (to 119 ~).
A-fibers Iontophoretic application of 5-HT (25-200 nA) produced threshold increases in 6/9 AO-fibers (range: 111-128 ~ ) and in 4/7 Aft-fibers (range: 106-128 ~o) (Table I). Thresholds of the remaining A-fibers were unaffected or reduced (in one case with biphasic effects).
Current controls Current controls (ejection of Na + or creatinine ions through a separate barrel) sometimes produced small threshold changes in C- and A-fibers tested, but in each case the effect was smaller than that produced by 5-HT (Fig. 2).
Primary afferent depolarization (PAD) Threshold changes were not produced in any of 7 C-fibers tested by conditioning stimulation of the posterior tibial nerve at a strength supramaximal for C-fibers (30 V, 1 msec, conditioning test interval 250 msec), either before or during 5-HT application. Threshold reductions of up to 80 ~o of control (i.e. PAD) were observed in 3 A6- and 3 Aft-fibers following tibial nerve stimulation at A-fiber strength (2 V, 0.1 msec), and this PAD was unaffected during 5-HT application. DISCUSSION Iontophoretic application of 5-HT resulted in increases in threshold for antidromic activation of about 67 ~o of C- and A-fibers. In discussing this effect in terms of a possible presynaptic action of 5-HT, it is important to critically consider other factors which might contribute to such threshold changes. For example, iontophoretic application of a compound might induce changes in extracellular ionic concentration, leading to osmotic changes and changes in tissue volume. Tissue swelling (threshold increase) or shrinkage (threshold reduction) might occur. Ejection of Na + ions had relatively minor effects on fiber threshold, but we have not yet tested other ions which might produce changes in tissue permeability. The small effects of Na + and creatinine
156 application on fiber thresholds also argue against non-specific current or pH effects. It cannot be determined with this method whether 5-HT acts directly at the fiber terminal, or indirectly, e.g. via inhibition of interneurons mediating tonic PAD at the fiber terminals. However, the intraspinal antidromic thresholds of some polymodal nociceptors and high-threshold mechanoreceptors with afferent C-fibers were recently shown to be raised during electrical stimulation of the brain stem nucleus raphe magnus (NRM, 11 ) in which 5-HT-containing neurons are located 5. N R M stimulation is also associated with a reduction in excitability of spinal dorsal horn neurons ~,9,2s, suggesting that its effect on C-fibers might reflect a presynaptic inhibitory mechanism. 5-HT and morphine4, 2a have similar effects on C-fiber thresholds, and both of these compounds generally reduce spinal neuronal excitabilityS, 20. Therefore, threshold increases may reflect a presynaptic inhibitory mechanism at C-fiber terminals, in contrast to large A-fibers in which presynaptic inhibition is generally thought to be associated with PAD (threshold reductiona6,24). Several mechanisms for the threshold increase have been suggested4,11,16,22, 23. One is that it reflects primary afferent hyperpolarization (PAH). PAH is thought to be associated with presynaptic facilitation, which would be at variance with generally depressant actions of N R M stimulation, opiates and 5-HT. However, recent evidence from invertebrate preparations indicates that presynaptic inhibition is associated with PAH which may be mediated by an increase in chloride conductance7,15. Certain myenteric ~3 and nodose ganglion ~2 neurons are hyperpolarized by 5-HT via an apparent calcium-dependent increase in potassium conductance 1~. An increase in membrane conductance would create a current shunt, which in the present experiments would necessitate a greater stimulus current density to antidromically excite the fiber. It should be noted, however, that 5H T generally has a predominantly depolarizing action on neural membranes ~2,13,27 including non-myelinated nerve fibers is. Any depolarizing action of 5-HT at intraspinal terminals would have to be outweighed by other membrane effects to account for the threshold increases which we commonly observed. PAD (threshold reduction) was consistently produced by N R M stimulation in identified cutaneous A-fiber afferents 17. It is possible that this PAD is mediated by a mechanism other than the spinal release of 5-HT, since the latter more often produced threshold increases in C- and A-fibers in the present experiments. KCANOWLEDGEMENTS The authors are most grateful to Fr. A. Giner for excellent technical assistance, to Fr. A. Manisali for graphics, and to Prof. L. Vyklicky for helpful comments on an earlier version of the manuscript. M.R. was supported by grants from the National Science Foundation (BNF 23871) and the Deutscher Akademischer Austauschdienst. E.C. was recipient of a postdoctoral fellowship from the National Institutes of Health (NINCDS~). This work was supported by a grant (Zi 110) from the Deutsche Forschungsgemeinschaft.
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