Antagonistic effects of semicarbazide and pyridoxine on cuneate presynaptic inhibition

Brain Research, 56 (1973) 249-258 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

249

ANTAGONISTIC EFFECTS OF SEMICARBAZIDE AND PYRIDOXINE ON CUNEATE PRESYNAPTIC INHIBITION

N. R. B A N N A

Department of Pharmacodynamics and Toxicology, School of Pharmacy, American Universityof Beirut, Beirut (Lebanon) (Accepted December 8th, 1972)

SUMMARY

(1) The role of ),-aminobutyric acid (GABA) in the inhibition of synaptic transmission across the dorsal column nuclei was investigated in Nembutal-anesthetized, and unanesthetized decerebrate, cats. (2) Semicarbazide (200 mg/kg) produced a pronounced depression of the surface positive wave and the dorsal column reflex. It also reduced the prolonged (over 125 msec) increase in excitability of cuneate terminals, and the inhibition of the lemniscal response, inducedby conditioning cutaneous nerve stimulation. (3) The time course of all these effects, which developed gradually over a 3--4 h period, correlated well with that of the depletion of GABA in the dorsal column nuclei. (4) Pyridoxine hydrochloride (200 mg/kg) antagonized all the effects of semicarbazide and returned GABA levels to near control values within 1 h. (5) GABA appears to play an important role in presynaptic inhibition of cuneate transmission. The possible mediation by GABA of both presynaptic and postsynaptic inhibition, partly through 'bi-inhibitory' cells, is discussed.

INTRODUCTION

The possible participation of ),-aminobutyric acid (GABA) in inhibition of synaptic transmission across the dorsal column nuclei has been the subject of various investigations. In the cat, extracellular microiontophoretic application of GABA to cuneate neurons produced a consistent and potent depression of their spontaneous and evoked activity 21. Furthermore, perfusion of the surface of the dorsal column nuclei with a 0.1 M GABA solution suppressed synaptic transmission and greatly increased the amplitude and duration of the dorsal column reflex 20. These effects were reversed

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by intravenously administered picrotoxin, an agent which blocks cuneate presynaptic inhibition 4. In the cuneate nucleus of the rat, topically applied G A B A significantly increased the resting excitability of ulnar afferent terminals and reduced the augmentation in their excitability after conditioning stimulation of the median nerve 16. It has been suggested that in the cuneate nucleus, GABA may act on both the postsynaptic membrane and the presynaptic terminals in a manner analogous to its action on the crayfish neuromuscular junctionaS, 20. In the following study, the effects of depleting GABA on cuneate synaptic transmission are described. Antagonism of these effects by pyridoxine is demonstrated. The results obtained further support the concept of a role for GABA in transmission across the dorsal column nuclei, particularly as an agent responsible for the depolarization of afferent terminals leading to presynaptic inhibition. A preliminary report on part of these results has been publishedL

METHODS

Experiments were performed on 34 cats anesthetized with Nembutal (35 mg/kg, i.p.), one cat anesthetized with a-chloralose (50 mg/kg, i.p.), and 2 unanesthetized cats decerebrated by a midcollicular section under initial ether anesthesia. All cats were paralyzed with gallamine triethiodide (Flaxedit) and artificially respired at a rate which maintained end tidal CO2 between 3.5 and 4 ~ . A bilateral pneumothorax was performed to reduce respiratory movement artifacts. In decerebrate cats, blood pressure was monitored from one carotid artery and the other artery was ligated. In non-decerebrate anesthetized cats, blood pressure was recorded from a femoral artery. Body temperature was maintained between 37 °C and 39.5 °C by a heating pad placed under the cat. The right superficial radial and ulnar nerves were isolated, crushed peripherally, and covered with mineral oil the temperature of which was thermostatically controlled at 37 °C. The dorsal column nuclei were exposed by the conventional approach through the foramen magnum. The nerve tissue surface was kept moist by intermittent washing with warm Locke's solution. In some experiments, the cats were mounted stereotaxically and a steel monopolar electrode insulated to near its tip was placed in the contralateral medial lemniscus. The indifferent electrode was attached to exposed skin or muscle tissue. The position of this stereotaxic electrode was ascertained by previously described methods 4. The following techniques were used. (1) Cuneate surface potentials were recorded by a silver, ball-tipped electrode after stimulation (0.25 c/sec, 3-4 times threshold) of the ipsilateral superficial radial nerve. While recording these slow potentials, the time constant of the preamplifier was set at 1 sec. (2) Antidromic responses were recorded in the superficial radial nerve (bipolar platinum hook electrodes) after submaximal test stimulation (15--40 V, 0.1 msec) of the ipsilateral cuneate nucleus with a glass microelectrode (3 M NaCI) and conditioning stimulation of the ulnar nerve. (3) Contralateral lemniscal responses were recorded after test stimulation of the super-

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251

ficial radial nerve or cuneate nucleus, as described above, and conditioning stimulation of the ulnar nerve. For the determination of G A B A levels, cats were treated in exactly the same way as those used for physiological experiments, including anesthesia, muscle relaxant administration, and surgical exposure. Frequently, G A B A levels were determined in the same cats at the termination of the neurophysiological procedures. The dorsal column nuclei were then dissected to a depth of about 1.5 m m below the surface, removed along with the overlying afferent tracts, and immediately placed in chilled 90 ~ ethanol. After weighing, the tissue was homogenized and centrifuged at 12,000 rev./min at 0 °C. The supernatant was removed quantitatively, evaporated under vacuum, dissolved in distilled water, and G A B A content determined by the chromatographic-ninhydrin method a0. Semicarbazide and pyridoxine hydrochloride were dissolved in 10 ml of saline and administered over a period of 5 min through the antebrachial vein. The blood pressure alterations induced by these agents were mild and did not appear to cause any of the observed neurophysiological changes. The areas of the N and P waves were integrated by planimetry. RESULTS

Cuneate surface potentials An afferent volley in the superficial radial nerve or in other cutaneous forelimb

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Fig. I. Cuneate surface potentials. A: the effects of semicarbazide (200 mg/kg) shown 3 h after administration followed by pyridoxine hydrochloride (200 mg/kg) shown 1 h after administration. B: time course of the effects of semicarbazide on the cuneate surface P wave and their antagonism by pyridoxine. The standard errors are indicated. Calibration: 0.1 mV and 50 msec.

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nerves produces on the surface of the cuneate nucleus an initial spike potential indicating the arrival of the primary afferent volley, followed by a brief negative wave (N wave) and a prolonged positive wave (P wave). It has been suggested e that the N wave is caused by synaptically induced depolarization of cuneate relay cells and that the P wave reflects the prolonged depolarization of cuneate tract fibers close to their synaptic terminals. Semicarbazide (200 mg/kg) produced no immediate changes in surface potentials. However, within 60 min, the P wave started to gradually decrease in depth. It was appreciably depressed in size 3-4 h after drug administration (Fig. 1A and B). Three hours after semicarbazide, the average area of the depressed P wave was 13 ± S.E. 3 ~ of control size in 7 cats anesthetized with Nembutal. In 3 of these cats, pyridoxine hydrochloride (200 mg/kg) was administered intravenously. It gradually antagonized the effects of semicarbazide and in 60 min completely reversed them (Fig. IA and B). In the remaining 4 cats not given pyridoxine, the P wave was further depressed and sometimes completely blocked. Frequently, negative deflections were observed in place of the previously positive potentials, and background activity became quite pronounced (Fig. 1A). In another 3 cats given semicarbazide, the intravenous administration of 10-20 mg/kg of pentobarbital antagonized its effects provided it was given within 2.5 h. Four hours after semicarbazide, however, pentobarbital failed to antagonize the P wave depression. The N wave was more variable with time and consistent changes in this parameter could not be ascertained. In general, semicarbazide produced an increase in its amplitude but a decrease in its duration. There was no clear antagonism of these effects in those experiments where pyridoxine was administered. Since these experiments were run for prolonged periods of time, it was deemed necessary to conduct parallel experiments where the cats were identically prepared but given an equivalent volume of physiological saline containing no semicarbazide. In 4 such cats anesthetized with Nembutal and paralyzed with Flaxedil, no changes in the size of the P wave after saline administration could be observed over a 4 h period, except for a moderate increase which was not significant (Fig. 1B). Furthermore, the effects of semicarbazide on the P wave were also observed in one cat anesthetized with a-chloralose and 2 decerebrate unanesthetized cats. GABA levels in the dorsal column nuclei The levels of GABA in the dorsal column nuclei were determined in controls, 4 h after saline, 2 and 4 h after semicarbazide, and 1 h after pyridoxine given 2-3 h after semicarbazide. Saline produced no significant changes in GABA levels. On the other hand, semicarbazide reduced GABA levels to 55~/o of control values in 2 h, and completely depleted GABA within 4 h in 2 experiments and reduced these levels to 0.15 #moles/g in one experiment. Pyridoxine raised the depleted GABA levels back to near control values. These results are depicted in Table 1. Excitability of cuneate terminals The antidromic response recorded in the superficial radial nerve after direct

253

G A B A AND CUNEATE INHIBITION TABLE I GABA

(/~moles/g) IN

LEVELS

THE DORSAL COLUMN NUCLEI OF NEMBUTAL ANESTHETIZED CATS

Control•

4 h control

2 h after semicarb. (200 mg/kg)

4 h after semicarb. (200 mg/kg)

1 h after pyridoxine (200 mg/kg)

1.13 4- 0.21 (5)

1.12 4- 0.16 (3)

0.63 4- 0.10 (3)

0.05 4- 0.05 (3)

0.95 ~ 0.16 (3)

• Mean and standard error, followed by number of experiments in parentheses.

s t i m u l a t i o n o f the cuneate nucleus consists o f two spike complexes. T h e first spike results f r o m electrical s t i m u l a t i o n o f the p r e s y n a p t i c fibers close to their terminals, a n d the later, a s y n c h r o n o u s discharge is t h o u g h t to represent the d o r s a l c o l u m n reflex ( D C R ) 8. Since it is difficult to m a i n t a i n the exact l o c a t i o n o f the m i c r o e l e c t r o d e d u r i n g the p r o l o n g e d time course o f d e v e l o p m e n t o f s e m i c a r b a z i d e ' s effects, the excitability o f cuneate terminals was tested at various d e p t h s falling within a t r a c k 0.5-1.5 m m f r o m the surface. I n 5 experiments, the D C R was depressed within 60 min o f s e m i c a r b a z i d e adm i n i s t r a t i o n a n d c o m p l e t e l y b l o c k e d in 2 h at all d e p t h s tested (Fig. 2A). I n 4 o f these experiments, the excitability o f cuneate terminals to direct electrical s t i m u l a t i o n

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Fig. 2. Excitability of cune,'tte terminals. A: antidromic response in the superficial radial nerve after direct stimulation of the cuneate nucleus, shown first as control, then 2 h after semicarbazide (200 mg/kg), then 1 h after pyridoxine hydrochloride (200 mg/kg) to show recovery of the DCR. B: time course of the increase in excitability of superficial radial cuneate terminals after conditioning stimulation to the ipsilateral ulnar nerve, showing the effects of semicarbazide at 2 h antagonized by pyridoxine 1 h after its administration. Calibration: 0.15 mV and 5 msec.

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was also depressed, as shown by the decrease in the size of the first spike. This was compensated for by increasing the strength of the applied pulse. In all these experiments, the increase in excitability of superficial radial terminals after conditioning stimulation at various intervals to the ipsilateral ulnar nerve was greatly reduced within 120 min of semicarbazide administration (Fig. 2B). At this time, pyridoxine hydrochloride was administered (200 mg/kg), and within 1 h, there was complete recovery of the DCR (Fig. 2A). The ability of conditioning stimuli to increase the excitability of afferent terminals also returned to near control values (Fig. 2B). In one control experiment where the cat was given saline only, no reproducible depression of the dorsal column reflex or of the excitability increase could be dem onstrated within a 2.5 h period.

The lemniscal discharge The mass discharge in the medial lemniscal tract was recorded after direct contralateral cuneate or cutaneous nerve stimulation. The positive fl-spike complex can be taken as a record of synaptic excitation of cuneate neurons by the presynaptic volley 1. Semicarbazide produced no changes in the test lemniscal response. However, a test stimulus produced repetitive lemniscal firing, and spontaneous discharges became quite pronounced 3-4 h after drug administration. The effects ofsemicarbazide on inhibition of the lemniscal response were studied in 4 experiments and followed for 2 h only, since the instability and spontaneous depolarizations produced by this convulsant when its effects had fully developed precluded meaningful interpretation of the interaction between conditioning and test discharges beyond 2-2.5 h after its administration. Even within 2 h, however, semicarbazide reduced the prolonged inhibition of the test lemniscal response by conditioning stimuli applied at intervals of 12-200 msec preceding the test stimulus. In one experiment, administration of pyridoxine hydrochloride resulted in partial recovery within 1 h (Fig. 3).

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Fig. 3. Time course of the inhibition of the lemniscal response evoked by stimulation of the superficial radial nerve after conditioning ulnar stimulation, showing the effects of semicarbazide at 2 h, and those of pyridoxine 1 h after its administration.

GABA

AND CUNEATE INHIBITION

255

DISCUSSION

Two types of inhibition modify transmission across the dorsal column nuclei: presynaptic and postsynapticL Cuneate inhibition responds to certain pharmacological agents in a manner analogous to spinal inhibition4, and it is probable that essentially the same types of neurohumoral substances are involved. In this study, an attempt was made to clarify the role of GABA as a possible inhibitory transmitter substance in the cuneate nucleus. Presynaptic inhibition This type of inhibition, which exerts a strong influence on afferent cutaneous transmission, can be displayed by surface recordings, excitability testing, and partly as a component of inhibition of the lemniscal discharge1-a. The changes induced in these parameters by semicarbazide all indicate a probable role for GABA in presynaptic inhibition, provided it is assumed that the levels of other suspected neurotransmitters are not affected by this agent. The slowly developing effects of semicarbazide corresponded well with the depletion of GABA from the dorsal column nuclei, while the absence of early effects indicates a lack of direct action on neuronal elements. It has been shown that, of the major systems requiring pyridoxal phosphate, glutamic acid decarboxylase (GAD), an enzyme with a loosely bound cofactor involved in the synthesis of GABA ~1, is the most sensitive to in vivo inhibition by semicarbazideza. Since the activity of GABA-a-ketoglutaric transaminase, a key enzyme in the metabolism of GABA, is not altered8, the result of GAD inhibition is a decrease in the levels of this inhibitory amino acid. Of major concern here is the level of GABA present at the synaptic cleft37,41, since GAD is present in the axoplasm of nerve endingsa3. Nevertheless, the possibility should be considered that the effect of convulsant hydrazides on GABA levels may be a methodological artifact due to blockade of the rapid post-mortem increase in GABA levels by these chemicals s, or may result from an action on non-neural elementsa6. However, the antagonism of semicarbazide's effects by pyridoxine reported here as well as by topical application of GABA solutions to the cuneate nucleus 14, and the similarity of these effects to those of picrotoxin4 and bicuculline6,15, support the synaptic GABA-depletion hypothesis. Pyridoxine administration, which replenished GABA concentrations in the dorsal column nuclei, re-established presynaptic inhibition. The ability of pyridoxine to antagonize convulsions induced by semicarbazide is well known 26. Presumably, pyridoxine hydrochloride would increase the availability of pyridoxal phosphate which is a required coenzyme in the biosynthesis of GABA al. A direct action of large doses of B6 vitamers on nerve membrane surfacesa2 may account for part of this antagonism, particularly during the earlier stages. On the other hand, the ability of pentobarbital to antagonize the early effects of semicarbazide is expected, since this depressant is known to potentiate cuneate presynaptic inhibition4. It is interesting to note, however, that once GABA was almost completely depleted, pentobarbital failed to antagonize P wave depression. Apparently, the presence of sufficient amounts of GABA is essential for the action of pentobarbital on primary afferent depolarization.

256

N. R, BANNA

Postsynaptie inhibition GABA has been shown to depress the activity of cuneate cells el, an action blocked by bicuculline 27, hence may also play an important role in postsynaptic inhibition. Since the time course of inhibition of the lemniscal response by conditioning cutaneous volleys displays both postsynaptic and presynaptic components, depletion of GABA will result in reduction of both types at intervals ranging fi'om 10 to 200 msec. Complex interactions (summation, repetitive discharge) between test and conditioning lemniscal potentials prevented a study of early inhibition (below 10 msec). Since picrotoxin, a specific GABA antagonist in the dorsal column nuclei '-'°, does not increase the direct excitability of cuneothalamic relay (CTR) neurons, it is probable that the inhibitory interneurons which release GABA form remote axodendritic synapses. On the other hand, glycine has also been reported to inhibit cuneate neurons 9-1, and strychnine, which is a potent and specific anatagonist of glycine in the cuneate nucleus 2°, increases the direct excitability of CTR cells4 and blocks postsynaptic inhibition ~°. Glycine-releasing interneurons may thus form axosomatic or close axodendritic synapses with relay cells. Both types of synaptic contacts have been described in the cuneate nucleus 40. Furthermore, two types of inhibitory potentials can be recorded from CTR neurons: a brief hyperpolarization analogous to that observed in the spinal cord, and a prolonged one lasting about 200 msec 1. This may reflect different transmitter processes and/or dissimilar firing patterns. A prolonged postsynaptic inhibitory time course for GABA would, temporally, fit well with a simultaneous presynaptic action. Possible dual function of GABA From the previous discussion, it can be seen that GABA may be playing a dual role in inhibition of transmission across the dorsal column nuclei, hyperpolarizing CTR neurons while depolarizing cuneate primary afferent terminals and thus reducing the amount of transmitter released. The concept of a presynaptic action of GABA is supported by the present findings as well as those of others 7,9,1a,16,19,2°,2z, a4,38. The possibility that GABA may be acting by disinhibition, i.e. by inhibiting (hyperpolarizing) neurons which in turn inhibit interneurons that depolarize presynaptic terminals, should also be considered. It has already been proposed to explain the action of bicuculline in the spinal cord 29, and is in keeping with the finding that GABA depresses the excitability of afferent fiber terminals when applied electrophoretically 12. However, this hypothesis presupposes that the interneuron which depolarizes presynaptic terminals is itself not inhibited by GABA. In the cuneate nucleus, all cells are inhibited by this amino acid 21. On the other hand, increased background activity induced by this convulsant could contribute to the observed neurophysiological changes through occlusion, particularly when the effects of semicarbazide are fully developed 3-4 h after administration. However, a definite reduction in P waves was always observed before the onset of spontaneous discharges. Thus, at least in the early stages of semicarbazide action, occlusion is not a major factor contributing to the observed changes. Furthermore, another convulsant, strychnine, in fact potentiates the P wave and the excitability increase of presynaptic terminals 4.

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A depolarizing action of GABA on central terminals of afferent fibers is not contradictory to its hyperpolarizing action on central neurons and may be due to different ionic gradients. Thus, it has been shown that GABA depolarizes cells of the vagal sensory ganglion17. Indeed, GABA may be released by one neuron at two transsynaptic sites, simultaneously hyperpolarizing one and depolarizing the other. This has already been proposed by several investigators6,15,z°,3L The morphological substrate of such a cell, which may be called a 'bi-inhibitory' or 'Walberg' neuron, has been described in the dorsal column nuclei 4°. Functionally inhibitory interneurons in the cuneate nucleus are exposed to a variety of influences 11,22,24,2s,39 and partake in the overall integration of sensory information passing across this first relay. ACKNOWLEDGEMENTS

I thank Mrs. L. Jeha for valuable technical assistance. This work was supported by grants from the Lebanese National Research Council and the American University of Beirut.

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