Brain Research, 88 (1975) 109-114

109

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Correlation between the topographical distribution of [=H]GABA uptake and primary afferent depolarization in the frog spinal cord

SILV10 GLUSMAN Section of Neural Control, Department of Physiology, Centro de lnvestigaci6n del lnstituto Politdcnico Nacional, Mexico 14, D.F. (Mexico)

(Accepted December 24th, 1974)

In the frog spinal cord, antidromic stimulation of motor nerves depolarizes the afferent fiber terminal arborizations3,1°,1a,1% The intraspinal field potentials associated with such a primary afferent depolarization (PAD) have a characteristic spatial distribution within the spinal cord suggesting that the neural elements generating the PAD are located within the dorsal horn 13. It has been suggested that GABA is the transmitter substance involved in the generation of the primary afferent depolarization 11. It fulfills several of the criteria required for a substance to be considered as a neurotransmitter. The application of GABA depolarizes the primary afferent terminals 2, GABA antagonists such as bicuculline and picrotoxin reduce the PAD produced either by nerve stimulation or by GABA application2,a,7,15,16,24, '2v and inhibitors of GABA transaminase, a major degradative enzyme for GABA, increase PAD as well as GABA concentration within the cord s. The mechanisms involved in the termination of GABA actions in the spinal cord are unknown. However, if, as has been suggested in other systems, GABA uptake is the mechanism by which GABA action terminates 5,19, the elements participating in [3H]GABA uptake should be present in the site where the action takes place. On the other hand, in the rat cerebral cortex exogenous labeled GABA has the same subcellular distribution as endogenous GABA 21. If the same occurs in frog spinal cord, GABA uptake determined after sufficiently long incubation times could be utilized as a marker of the endogenous GABA pool. The present series of experiments was performed to determine if there is any correlation between the topographical distribution of [3H]GABA uptake and of PAD within the frog spinal cord. The experiments were performed in isolated frog spinal cords at 15 °C. The methods used for the electrophysiological observations have been described previously 3,12. For the uptake experiments, hemisected spinal cords were preincubated for 15 rain in Ringer solution (NaCI, 114 mM; KC1, 2 mM; CaCl2, 1.8 mM; NaHCO3, 2 mM; and glucose, 5.5 mM). The solution was continuously bubbled with a 95°4; 02-5 ~o CO2 mixture. Then, [2,3-3H]GABA (specific activity 10 Ci/mmole) was added to the bath (final concentration 2 × 10-7 M) and incubated for various times (10-60 rain). Total incubation volume was 5 ml. After the incubation period the tissue was

If0 washed in Ringer medium v\ithout labeled G A B A . The tissue wa,,, di,~st,b,'ed in ~ ~ mi o f N C S after determining the wet and dry weight. Then radioactivity ~as measured by liquid scintillation counting after the addition of 10ml of scintillation solution ( P O P O P 100 mg, PPO 4 g, and toluene 1 liter). In 4 experiments the hemisected cords were incubated 40 min in [aH]GABA. After this period of time the tissue was washed and homogenized in 3 ml of 75'!,, ethylic alcohol at 4 C . The homogenate was maintained for at least 30 min at this temperature and then centrifuged. The supernatant was evaporated and redissolvcd in water (0.1 ml). Aliquots were taken for thin-layer c h r o m a t o g : a p h y in silica gel using butanol-acetic acid-water ( 4 : 1 : 1 ) a s the solvent system; 95",; of the total radioactivity was recovered as G A B A . The tissue protein remaining after the extraction procedure was dissolved in NCS and the radioactivity was measured. Less lhan 1 °/~I of the [ a H ] G A B A was incorporated into protein. As a first step to disclose the possible involvement o f G A B A in the P A D generation, it seemed necessary to characterize the uptake mechanism for this amino acid in the frog spinal cord, As shown in Fig. IA, the [ a H ] G A B A uptake by the hemisected spinal cord is a function of the incubation time. The uptake increases until attaining a maximum between 40-60 min o f incubation time. Tissue/medium relationship (mole/g tissue: mole/ml medium) was 20:1 at 20 min o f incubation time. Fig. I B shows the effects of replacing sodium by choline, on the [ a H ] G A B A uptake o f the hemisected spinal cord. One-half of the cord was incubated in a medium with different sodium concentrations and the other half o f the cord, kept in normal Ringer medium, was used as a control. The [ a H ] G A B A uptake was nearly zero when

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Fig. 1. A: time course of [aH]GABA uptake in hemisected frog spinal cord incubated at 15 ~C with [ZH]GABA (2 × 10 7 M). Average from 4 experiments. Vertical bars indicate S.E.M. Ordinates expressed in disint./min/mg of dry weight. B: effect of sodium concentration on [aH]GABA uptake in media in which various proportions of sodium content were replaced with choline chloride. Each point is the mean i S.E.M. of 4 experiments. In the abscissa, the sodium concentration is expressed as percentage of the sodium concentration in normal Ringer (116 mM). In the ordinates, [aH]GABA uptake is expressed as percentage of the uptake in the control hemicord of the same preparation. C: kinetic analysis of effects of GABA concentration on rate of [aH]GABA uptake. V -- rate of GABA uptake (10--~ mole/min/g cord); S .......GABA concentration (10"5 M). Each value is the mean of 4 experiments.

111 the sodium concentration in the bath was of 2 mM/liter and increased with increasing sodium concentrations. Ouabain added to a normal Ringer medium (10 -4 M final concentration) reduced [3H]GABA uptake to 25 ~ ± 4 of the uptake in the control half of the cord. To study the effect of GABA concentration on [3H]GABA uptake, hemisected spinal cords were incubated at 15 °C in a Ringer medium with a fixed [3H]GABA concentration (2 × 10-7 M) while unlabeled GABA concentration was varied between 10 4 and 10-6 M (final concentration). The [:~H]GABA uptake rate was determined at 20 rain of incubation time for different substrate concentrations. As shown in Fig. 1C when plotted in a reciprocal form using the Michaelis-Menten equation, the experimental values fall on a straight line, indicating that [3H]GABA uptake is mediated by a saturable process. The apparent K,~ for [3H]GABA uptake in these conditions is of 27 × 10-5 M and Vm~x is 6.6 × 10 .5 M/min-g dry weight. All the above findings suggest that in the frog spinal cord, like in the mammalian cerebral cortex 19, cerebellar cortex iv, retina 14 and sensory ganglia 25, the [3H]GABA uptake is an active process with a saturation kinetics, with a high affinity transport system depending on the sodium ion concentration in the medium, and inhibited by ouabain (see also ref. 4). Primary afferent depolarization is produced in the frog cord after stimulation of the motor nerves~2,15,16. Fig. 2A shows sample records of the field potentials produced by stimulation of the motor fibers in the tibial nerve recorded at various depths within the cord (see diagram in Fig. 2C). When recording from the dorsal surface, there is a delayed positive wave (VR-SFP) with a latency similar to that of the simultaneously recorded dorsal root potential (see also ref. 12). As shown in Fig. 2A and 2B, the sign of this slow field potential reverses at 100-150 #m depth. The negative VR-SFP attains maximum values between 300-500/~m depth and then decays with increasing depths towards the motor nucleus region. The region of maximal negative VR-SFP (i.e., the current sink) can be considered as the site where the mechanisms generating the PAD are located'~,12, 2s. It is also the site where most of the afferent fibers end13, 2z,23 and where the axo-axonic appositions between interneurons and afferent fiber terminals are most frequent ~3. When investigating the spatial distribution of [3H]GABA uptake, the hemisected cords were incubated in the Ringer-[3H]GABA medium for 40 min, washed for another 40 rain, fixed in cold glutaraldehyde for 1 h and sectioned every 100/~m longitudinally, perpendicular to the dorsoventral axis of the cord (see diagram in Fig. 2C). Fig. 2D shows the intraspinal distribution of [3H]GABA uptake in 100/~m thick slices obtained from a hemisected spinal cord. It can be seen that [3H]GABA uptake was not uniformly distributed within the cord. There was a maximum between 300 and 500 #m from the dorsal surface which was at least two times greater than the uptake in neighboring regions. Comparison of Fig. 2B and 2D shows that the PAD and the [3H]GABA uptake have a remarkably similar pattern of distribution within the spinal cord. The average of 5 experiments shows the same pattern of spatial distribution as indicated in Fig. 3. The finding of a close parallelism between the intraspinal distribution of [3H]-

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Fig. 2. A: intraspinal field potentials recorded at various depths within the cord alter single shock stimulation of the tibial nerve (2 :< threshold strength). Dorsal roots Lg-L7 were sectioned. Negativity upwards. Each trace is the average of 50 responses evoked at 0.4/sec. DRP, dorsal root potential; SURF, record from dorsal surface; MNN, record from motoneuron nucleus. B shows the amplitude changes of the slow potential recorded at various depths within the spinal cord. The potentials were measured at the time indicated by the vertical line in A. The dots are the average of 4 experiments. Approximate direction of electrode presentation is illustrated in C which also shows how the spinal cord was sliced for the experiment illustrated in Figs. 2D and 3. D shows the spatial distribution of [3H]GABA uptake. Radioactivity was determined in every 100 ftm thick slice of a single hemisected cord as indicated. Abscissa in B and D, depth in t+m from the dorsal surface. V.R. shows the [3H]GABA uptake measured from a ventral root. Calibration: 100 HV and [0 msec.

G A B A uptake a n d of P A D is of great interest with regard to the m e c h a n i s m s of P A D because it suggests that in the frog spinal cord the elements that take up G A B A are preferentially f o u n d in the region where the P A D itself is originated, a necessary c o n d i t i o n for the hypothesis that G A B A is a possible t r a n s m i t t e r in the P A D genera t i o n 11. These findings agree with recent observations made in the cat 20 a n d in the rat spinal cord 1 where most of the G A B A or G A D are also f o u n d in the dorsal horn, the site of g e n e r a t i o n of PADg, '~8. F o r the time being the dorsal horn elements involved in G A B A uptake have

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Fig. 3. Spatial distribution of [aH]GABA uptake derived from 5 experiments, as in Fig. 2D. For each experiment the maximum [aH]GABA uptake was considered as 100~ and the other values were expressed relative to this maximum. The bars show the mean of such values for the 5 experiments plus 1 S.E.M. Note a similar spatial distribution as in Fig. 2D.

not been identified. Important uptake by afferent fibers seems to be excluded since no significant differences in the [aH]GABA uptake were found when comparing the normal with the chronically (6 days) deafferented lumbosacral cord (see ref. 12 for Methods). In rat sensory ganglia most of [aH]GABA uptake occurs in glial cells 2a. However, autoradiographic studies in rat spinal cord homogenates 18 and tissue culture of rat cerebellum 26 show that the higher proportion of [aH]GABA is present in nerve terminals and a significantly lower proportion in glial cells. On the other hand, Miyata and Otsuka 2° showed that the G A B A content of the cat spinal cord decreased after anoxic destruction of interneurons. If the same occurs in the frog spinal cord, the spatial distribution of [aH]GABA uptake presently described probably results from uptake by some fraction of the interneurons in the dorsal horn. Whether or not the interneurons mediating the PAD belong to the set of GABA uptaking cells in the dorsal horn cannot be decided with the available evidence. Experiments are now being performed to further characterize the dorsal horn elements involved in GABA uptake. I thank Drs. P. Rudomin and D. Erlij for their useful comments and for reviewing the manuscript, and to Mrs. L. Arditti and Mr. M. Pacheco for their participation in some of the experiments. This work was partly supported by N I H G r a n t NS 09196-04 NEUB.

l BARBER, R., McLAUGHLIN, B. J., SAITO, K., AND ROBERTS, t . , Light microscopic localization glutamate decarboxylase in boutons of rat spinal cord before and after dorsal rhizotomy, Soc. Neurosci., 4 (1971) 127. 2 BARKER, J. L., AND NICOLL, R . A . , The pharmacology and ionic dependence of amino acid responses in the frog spinal cord, J. Physiol. (Lond.), 228 (1973) 259-277.

114 3 CARPENTER, D. O., AND RUI)OMiN, P., The organizatiol; of primary afferent depolarization m lhc isolated spinal cord of the frog, J. Physio/. (Lond.), 229 (1973) 471-493. 4 COLLINS, G. G. S., The sponlaneous and electrically evoked release of [:~H]GABA From the isolated hemisected fi'og spinal cord, Brah~ Research, 66 (1974) 121-137. 5 CURTIS, D. R., DUGGAN, A. W., AND JOtq,'