Naunyn-Schmiedeberg's
Archivesof Pharmacology
Naunyn-Schmiedeberg's Arch. Pharmacol. 294, 121-131 (1976)
9 by Springer-Verlag 1976
Effects of Two Benzodiazepines, Phenobarbitone, and Baclofen on Synaptic Transmission in the Cat Cuneate Nucleus P. POLC and W. HAEFELY Pharmaceutical Research Division, F. Hoffmann-La Roche & Co. Ltd., CH-4002 Basel, Switzerland
Summary. The effects of diazepam, flunitrazepam, phenobarbitone and baclofen on excitatory as well as on pre- and postsynaptic inhibitory processes in the cuneate nucleus were studied in decerebrate cats. Afferent presynaptic inhibition in the cuneate nucleus, evoked by volleys in the median nerve, and assessed by the size of the positive cuneate surface potential (P wave), the dorsal column reflex (DCR), and the increased excitability of primary afferent terminals of the ulnar nerve, was markedly enhanced by diazepam (0.1- 3.0 mg/kg i.v.) and flunitrazepam (0.01-0.3mg/kg i.v.), slightly enhanced by lower doses of phenobarbitone ( 3 - 2 0 mg/kg i.v.), but depressed by baclofen ( 1 - 1 0 mg/kg i.v.). Diazepam, flunitrazepam and phenobarbitone also increased postsynaptic inhibition in the cuneate nucleus which was measured by the decrease after conditioning volleys in the median nerve of the short-latency lemniscal response to cuneate stimulation. The GABA receptor blocking agent, picrotoxin, antagonized the effects of diazepam on pre- and postsynaptic inhibition in a surmountable way. After thiosemicarbazide (TSC), an inhibitor of GABA synthesis, both preand postsynaptic inhibition were greatly reduced and the augmenting effect of diazepam on both types of inhibition was nearly abolished. Aminooxyacetic acid (AOAA), an inhibitor of GABA degradation, slightly enhanced pre- and postsynaptic inhibition; the effects of diazepam were unaffected by AOAA. Diazepam, flunitrazepam and phenobarbitone did not alter the resting excitability of primary afferent endings or of cuneo-thalamic relay (CTR) cells in the cuneate nucleus. After higher doses (30 mg/kg i.v.) of phenobarbitone pre- and postsynaptic inhibition, which were enhanced by 10 mg/kg of this drug, tended to return to pre-drug values or below. Phenobarbitone, in conSend offprint requests to the authors at the above address.
trast to benzodiazepines, also depressed in a dosedependent way the N wave, which is an index of the orthodromic excitation of the CTR cells. Baclofen strongly depressed the cuneate N wave, decreased the excitability of CTR cells, reduced pre- and postsynaptic inhibition, but had no effect on the resting excitability of primary afferent endings. Our findings suggest the following modes of action of the above mentioned drugs: 1. benzodiazepines enhance selectively the GABA-mediated pre- and postsynaptic inhibition in the cuneate nucleus; 2. phenobarbitone slightly enhances pre- and postsynaptic inhibition only in a narrow dose range, and in addition reduces the excitatory processes in the cuneate nucleus; 3. baclofen seems to depress the excitation of cuneate relay cells and interneurones postsynaptically; the depression of relay cells is probably nonspecific.
Keywords: Benzodiazepines - Phenobarbitone Baclofen - GABA - Cuneate Nucleus.
INTRODUCTION It has recently been shown that diazepam enhances the GABA-mediated presynaptic inhibition in the spinal cord (Schmidt et al., 1967; Polc et al., 1974; Haefely et al., 1975). Preliminary results indicate a similar effect of diazepam in dorsal column nuclei (Haefely et al., 1975). With moderate doses (10-25 mg/kg i.v.) of pentobarbitone, similar effects have previously been noted, whereas higher, anaesthetic doses of this drug reduced presynaptic inhibition at both levels of the neuraxis (Eccles et al., 1963 ; Banna and Jabbur, 1969). In contrast to presynaptic inhibition, postsynaptic inhibition in the spinal cord does not seem to be
122 m e d i a t e d b y G A B A b u t r a t h e r b y glycine, a n d diaz e p a m was f o u n d n o t to affect spinal p o s t s y n a p t i c i n h i b i t i o n ( S c h m i d t et al., 1967; Schlosser, 1971). Since, in c o n t r a s t to the spinal cord, b o t h pre- a n d p o s t s y n a P t i c i n h i b i t i o n o f the c u n e o - t h a l a m i c relay ( C T R ) cells seem to be m e d i a t e d b y G A B A (see C u r t i s a n d J o h n s t o n , 1974), the q u e s t i o n a r o s e w h e t h e r b e n z o d i a z e p i n e s a n d b a r b i t u r a t e s affect p o s t s y n a p t i c i n h i b i t i o n in d o r s a l c o l u m n nuclei in the s a m e w a y as t h e y a l t e r e d p r e s y n a p t i c i n h i b i t i o n in the spinal cord. Baclofen [fi(4-chlorophenyl)-GABA] readily crosses the b l o o d - b r a i n barrier. A l t h o u g h its c h e m i c a l s t r u c t u r e s t r o n g l y suggests the p o s s i b i l i t y o f a n interference with G A B A , n o evidence for this is p r e s e n t l y a v a i l a b l e ( F e h r a n d Bein, 1974; C u r t i s et al., 1974; D a v i d o f f a n d Sears, 1974; Nistri, 1975). This muscle r e l a x a n t d r u g has been r e p o r t e d to d e p r e s s selectively the s y n a p t i c e x c i t a t i o n o f m o t o n e u r o n e s w i t h o u t a l t e r i n g the i n h i b i t o r y p o s t s y n a p t i c p o t e n t i a l s i n the cat spinal c o r d ( P i e r a u a n d Z i m m e r m a n n , 1973). T h e p r e s e n t i n v e s t i g a t i o n deals with the effects o f two b e n z o d i a z e p i n e s , d i a z e p a m a n d f l u n i t r a z e p a m , o f a b a r b i t u r a t e , p h e n o b a r b i t o n e , a n d o f the G A B A derivative, baclofen, on pre- a n d p o s t s y n a p t i c inhibition as well as o n e x c i t a t o r y m e c h a n i s m s in the c u n e a t e nucleus o f d e c e r e b r a t e cats. I n s o m e e x p e r i m e n t s we p e r f o r m e d d r u g i n t e r a c t i o n studies with the G A B A r e c e p t o r b l o c k i n g agents, p i c r o t o x i n a n d bicuculline, the G A B A - s y n t h e s i s i n h i b i t o r s , t h i o s e m i c a r b a z i d e a n d 3 - m e r c a p t o p r o p i o n i c acid, a n d the i n h i b i t o r o f G A B A - d e g r a d a t i o n , a m i n o o x y a c e t i c acid ( A O A A ) . P a r t o f these results were r e p o r t e d in a s h o r t c o m m u n i c a t i o n (Polc a n d Haefely, 1975).
METHODS Fourty-eight cats were mounted under ether anaesthesia in a stereotactic holder and decerebrated by transection of the brain stem at the intercollicular level by high-frequency electrocoagulation. The ether was then withdrawn. Preparation of Cuneate Nucleus and Nerves. The dorsal column nuclei were exposed by removing the laminae of the upper two cervical vertebrae, the part of the occipital bone coveting the posterior cerebellum, and the atlanto-occipital membrane. The posterior cerebellum was removed by suction. The proximal ends of the peripherally severed median and ulnar nerves in the left foreleg were mounted on bipolar platinum electrodes for stimulation and recording, respectively. Skin flaps were used to form heated paraffin pools over the exposed peripheral nerves and the dorsal column nuclei. Stimulation and Recording. The experimental set-up is schemati-
cally presented in Figure 1. Depolarization of primary afferent endings (PAD) of dorsal column fibres, which is responsible for the presynaptic inhibition of cuneate relay (CTR) cells, was assessed on the one hand by the positive cuneate surface potential (P wave), recorded from the dorsal surface of the left cuneate nucleus using
Naunyn-Schmiedeberg's Arch. Pharmacol. 294 (1976)
orthodromic lemniscal potential
~j
cuneate stimulation
I"----~
cuneate surface
]
potential
j~
dorsal column fibre
~
antidromic action potential
\ ",4
.T- ."--'-- ,, . ~ x n ulnaris orthodromic afferent / \ stimulation (',,~ \ ~ n. medlanus Fig. I. Schematic diagram illustrating the experimental set-up. Stimulating electrodes were placed in the cuneate nucleus and on the median nerve, recording electrodes in the medial lemniscus, on the ulnar nerve and on the dorsal surface of the cuneate nucleus. A simplified picture of the synaptic organisation within the nucleus shows the terminals of primary afferents exciting on the one hand the cuneothalamic relay cells (CTR cells, solid circles), on the other hand the inhibitory interneurons (open circles). The axons of the latter cells form axo-axonal synapses with primary afferent terminals (presynaptic inhibition) and/or axodendritic and axosomatic synapses with CTR neurones (postsynaptic inhibition). Descending and recurrent inhibition is omitted in this diagram
a silver-silver chloride electrode and evoked by single supramaximal stimuli of 0.05 ms duration once every 2 s, applied to the ipsilateral median nerve. On the other hand, a more direct index of PAD was obtained by testing the excitability of primary afferent endings within the cuneate nucleus by the technique of Wail (1958). Submaximal single pulses delivered through a coaxial stainless steel semimicroelectrode (0. i mm in diameter) inserted in the left cuneate nucleus, elicited action potentials in aTraction of afferent terminals, which were conducted antidromically and recorded in the ipsilateral ulnar nerve. The amplitude of this antidromic action potential, which is a function of the number of ulnar nerve terminals excited by the constant test stimulus in the cuneate, is an index of the excitability of the resting primary afferent terminals. The increase of the excitability of primary afferent terminals, which occurs during PAD elicited by volleys in the median nerve, was measured by the increase of the size of the antidromic action potential in the ulnar nerve. PAD was induced by single conditioning supramaximal volleys in the median nerve, delivered at intervals of 4, 7, 10, 20, 30, 50, 70 and 90 or 100 ms prior to the test
P. Polc and W. Haefely: Pharmacology of Synaptic Transmission in Cuneate Nucleus pulses in the cuneate nucleus. An increased excitability of primary afferent terminals was also visible from the appearance of a late antidromic response, the dorsal column reflex (DCR). The DCR consists of late antidromic action potentials recorded in the ulnar nerve in response to direct stimuli applied to the cuneate nucleus and initiated in afferent endings by the depolarizing effect at axoaxonal synapses of impulses in inhibitory interneurones. These impulses result from either direct stimulation by the electrode or synaptic excitation through the collaterals of those primary afferents that are activated by the electrode.
Postsynaptic inhibition of CRT cells was assessed in the following way: a coaxial stainless steel electrode (0.3 mm in diameter), insulated except at the tip, was introduced into the contralateral medial lemniscus until it recorded a maximal orthodromic response to cuneate stimulation. The first short-latency spike (delay 0 . 4 0.6 ms) of this response is attributed to the direct stimulation of CTR cells (Andersen et al., 1964). The depression of this shortlatency lemniscal potential by supramaximal preceding volleys in the median nerve, delivered at intervals of 4, 7, 10, 20, 30, 50, 70 and 90 or 100 ms before the test cnneate stinmlus, was taken as an index of the reduced excitability of CTR cells which results from the hyperpolarization that underlies postsynaptic inhibition. In addition to studying inhibitory phenomena in the cuneate nucleus, we assessed excitatory mechanisms in the same nuclear region by measuring the negative wave of the cuneate surface potential (N wave) in response to a single stimulus to the median nerve, which reflects postsynaptic depolarization of relay cells, as well as the size of test short-latency lemniscal response to cuneate volleys, which indicates the resting excitability of cuneate relay cells. All the above potentials were amplified (a DC amplifier was used for the cuneate surface potential) and displayed on an oscilloscope, from which polaroid photographs were taken.
Evaluation of Drug Effects. The mean amplitudes of the test antidromic potentials in the ulnar nerve, and of the test orthodromic potentials in the medial lemniscus, both evoked by isolated cuneate volleys, measured during a 30 rain control period, were taken as 100~. The amplitudes of the corresponding responses obtained at fixed intervals after identical preceding conditioning stimuli in the pre-drug and post-drug period were expressed in percent of these control test amplitudes. In the same manner the amplitudes of the N wave and the amplitudes and the durations of the P wave were measured before and after drug administration, The effects of drugs on the dorsal column reflex (DCR) were evaluated by measuring the area determined by the response and the baseline in the control period and after drug injection. In order to obtain a dose-response curve, each drug tested (diazepam, flunitrazepam, phenobarbitone, baclofen) was given in 4 increasing cumulative doses and the effects were evaluated 20 rain after each injection. The intervals between the drug applications were 30 min. In this series of experiments each animal received only one drug. The mean values of the parameters measured in different animals were calculated for each drug series, which consisted of 5 experiments. Only preparations with stable potentials during the 30 rain control period were used for evaluation. Sometimes, 8 responses were averaged (N, P waves, test antidromic and lemniscal potentials). Student's t-test was used to calculate statistical signiflcances between pre- and post-drug values of the P wave amplitude and duration, N wave amplitude, and of the DCR area. How ever, since already in the pre-drug control period the percent reduction of lemniscal responses and the percent increase of primary afferent depolarization by a preceding volley in the median nerve varied greatly from one animal to another, it was not meaningful to pool the data from different animals. A given drug effect on the latter two parameters was considered to be statistically significant
123
(Friedman-test), if it occurred without exception in all the experiments of a series irrespective of the magnitude.
Drugs Used. Aminooxyacetic acid hydrochloride (AOAA), baclofen, bicuculline, diazepam, flunitrazepam, gallamine triethiodide, 3-mercaptopropionic acid (MPA), phenobarbitone, picrotoxin, thiosemicarbazide (TSC), and the solvent for diazepam and flunitrazepam were injected into the right cannulated femoral vein and flushed in with physiological saline. All drugs except diazepam and fiunitrazepam were dissolved in saline. Preparations with a sudden or strong increase of the blood pressure in response to the convulsants, bicuculline, MPA, picrotoxin and TSC were discarded as were experiments in which the blood pressure fell below 90 mm Hg. in order not to interfere with the synaptic transmission in the cuneate, gallamine triethiodide was injected only if convulsions occurred (Galindo et al., 1968). The organic solvent used for injecting diazepam and flunitrazepam did not affect any measured parameter when given in the amount (1 ml/kg) necessary for administering the highest dose (10 mg/kg) of the two benzodiazepines.
RESULTS
Effects of Diazepam on Preand Postsynaptic Inhibition and on Orthodromic Excitation of Cuneate Relay Cells A t y p i c a l e x p e r i m e n t w i t h d i a z e p a m is d e p i c t e d in F i g u r e 2. 10 m i n a f t e r a d o s e o f 0.3 m g / k g t h e foll o w i n g c h a n g e s w e r e o b s e r v e d , a) T h e P w a v e in res p o n s e to a single v o l l e y in t h e m e d i a n n e r v e a n d b) t h e d o r s a l c o l u m n reflex ( D C R ) in r e s p o n s e to a single s t i m u l u s w i t h i n t h e c u n e a t e n u c l e u s w e r e markedly enhanced, c)The increased excitability of p r i m a r y a f f e r e n t e n d i n g s o f u l n a r n e r v e fibres t h a t o c c u r s a f t e r a p r e c e d i n g v o l l e y in t h e m e d i a n n e r v e w a s a c c e n t u a t e d a n d p r o l o n g e d as s h o w n b y t h e inc r e a s e d size o f t h e a n t i d r o m i c p o t e n t i a l in the u l n a r nerve evoked by stimuli within the cuneate nucleus. In a d d i t i o n to m o d i f y i n g t h e s e 3 p a r a m e t e r s w h i c h are phenomena connected with presynaptic inhibition, d i a z e p a m a l s o e n h a n c e d a f f e r e n t p o s t s y n a p t i c inh i b i t i o n o f c u n e a t e r e l a y cells, as s h o w n b y d) a n acc e n t u a t i o n o f t h e r e d u c t i o n o f t h e s h o r t - l a t e n c y res p o n s e s in t h e m e d i a l l e m n i s c u s (to d i r e c t s t i m u l a t i o n w i t h i n t h e c u n e a t e n u c l e u s ) w h i c h is o b s e r v e d f o r a s h o r t p e r i o d a f t e r a n a f f e r e n t v o l l e y in t h e m e d i a n nerve. I n c o n t r a s t , d i a z e p a m did n o t affect t h e N w a v e e v o k e d by v o l l e y s in t h e m e d i a n n e r v e o r t h e l e m n i s c a l r e s p o n s e to i s o l a t e d s t i m u l i w i t h i n t h e c u n e a t e n u c l e u s . N e i t h e r d i d it affect t h e size o f t h e a n t i d r o m i c r e s p o n s e in t h e u l n a r n e r v e to i s o l a t e d d i r e c t c u n e a t e stimuli. ] ' h e a b s e n c e o f a n y effect o f d i a z e p a m o n t h e s e 3 r e s p o n s e s i n d i c a t e s t h a t t h e d r u g has n o effect o n t h e e x c i t a b i l i t y o f c u n e a t e r e l a y cells o r o f p r i m a r y a f f e r e n t e n d i n g s in t h e a b s e n c e o f v o l l e y s that activate inhibitory neurones within the cuneate nucleus.
124
Naunyn-Schmiedeberg's Arch. Pharmacol. 294 (1976)
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Archivesof Pharmacology
Naunyn-Schmiedeberg's Arch. Pharmacol. 294, 121-131 (1976)
9 by Springer-Verlag 1976
Effects of Two Benzodiazepines, Phenobarbitone, and Baclofen on Synaptic Transmission in the Cat Cuneate Nucleus P. POLC and W. HAEFELY Pharmaceutical Research Division, F. Hoffmann-La Roche & Co. Ltd., CH-4002 Basel, Switzerland
Summary. The effects of diazepam, flunitrazepam, phenobarbitone and baclofen on excitatory as well as on pre- and postsynaptic inhibitory processes in the cuneate nucleus were studied in decerebrate cats. Afferent presynaptic inhibition in the cuneate nucleus, evoked by volleys in the median nerve, and assessed by the size of the positive cuneate surface potential (P wave), the dorsal column reflex (DCR), and the increased excitability of primary afferent terminals of the ulnar nerve, was markedly enhanced by diazepam (0.1- 3.0 mg/kg i.v.) and flunitrazepam (0.01-0.3mg/kg i.v.), slightly enhanced by lower doses of phenobarbitone ( 3 - 2 0 mg/kg i.v.), but depressed by baclofen ( 1 - 1 0 mg/kg i.v.). Diazepam, flunitrazepam and phenobarbitone also increased postsynaptic inhibition in the cuneate nucleus which was measured by the decrease after conditioning volleys in the median nerve of the short-latency lemniscal response to cuneate stimulation. The GABA receptor blocking agent, picrotoxin, antagonized the effects of diazepam on pre- and postsynaptic inhibition in a surmountable way. After thiosemicarbazide (TSC), an inhibitor of GABA synthesis, both preand postsynaptic inhibition were greatly reduced and the augmenting effect of diazepam on both types of inhibition was nearly abolished. Aminooxyacetic acid (AOAA), an inhibitor of GABA degradation, slightly enhanced pre- and postsynaptic inhibition; the effects of diazepam were unaffected by AOAA. Diazepam, flunitrazepam and phenobarbitone did not alter the resting excitability of primary afferent endings or of cuneo-thalamic relay (CTR) cells in the cuneate nucleus. After higher doses (30 mg/kg i.v.) of phenobarbitone pre- and postsynaptic inhibition, which were enhanced by 10 mg/kg of this drug, tended to return to pre-drug values or below. Phenobarbitone, in conSend offprint requests to the authors at the above address.
trast to benzodiazepines, also depressed in a dosedependent way the N wave, which is an index of the orthodromic excitation of the CTR cells. Baclofen strongly depressed the cuneate N wave, decreased the excitability of CTR cells, reduced pre- and postsynaptic inhibition, but had no effect on the resting excitability of primary afferent endings. Our findings suggest the following modes of action of the above mentioned drugs: 1. benzodiazepines enhance selectively the GABA-mediated pre- and postsynaptic inhibition in the cuneate nucleus; 2. phenobarbitone slightly enhances pre- and postsynaptic inhibition only in a narrow dose range, and in addition reduces the excitatory processes in the cuneate nucleus; 3. baclofen seems to depress the excitation of cuneate relay cells and interneurones postsynaptically; the depression of relay cells is probably nonspecific.
Keywords: Benzodiazepines - Phenobarbitone Baclofen - GABA - Cuneate Nucleus.
INTRODUCTION It has recently been shown that diazepam enhances the GABA-mediated presynaptic inhibition in the spinal cord (Schmidt et al., 1967; Polc et al., 1974; Haefely et al., 1975). Preliminary results indicate a similar effect of diazepam in dorsal column nuclei (Haefely et al., 1975). With moderate doses (10-25 mg/kg i.v.) of pentobarbitone, similar effects have previously been noted, whereas higher, anaesthetic doses of this drug reduced presynaptic inhibition at both levels of the neuraxis (Eccles et al., 1963 ; Banna and Jabbur, 1969). In contrast to presynaptic inhibition, postsynaptic inhibition in the spinal cord does not seem to be
122 m e d i a t e d b y G A B A b u t r a t h e r b y glycine, a n d diaz e p a m was f o u n d n o t to affect spinal p o s t s y n a p t i c i n h i b i t i o n ( S c h m i d t et al., 1967; Schlosser, 1971). Since, in c o n t r a s t to the spinal cord, b o t h pre- a n d p o s t s y n a P t i c i n h i b i t i o n o f the c u n e o - t h a l a m i c relay ( C T R ) cells seem to be m e d i a t e d b y G A B A (see C u r t i s a n d J o h n s t o n , 1974), the q u e s t i o n a r o s e w h e t h e r b e n z o d i a z e p i n e s a n d b a r b i t u r a t e s affect p o s t s y n a p t i c i n h i b i t i o n in d o r s a l c o l u m n nuclei in the s a m e w a y as t h e y a l t e r e d p r e s y n a p t i c i n h i b i t i o n in the spinal cord. Baclofen [fi(4-chlorophenyl)-GABA] readily crosses the b l o o d - b r a i n barrier. A l t h o u g h its c h e m i c a l s t r u c t u r e s t r o n g l y suggests the p o s s i b i l i t y o f a n interference with G A B A , n o evidence for this is p r e s e n t l y a v a i l a b l e ( F e h r a n d Bein, 1974; C u r t i s et al., 1974; D a v i d o f f a n d Sears, 1974; Nistri, 1975). This muscle r e l a x a n t d r u g has been r e p o r t e d to d e p r e s s selectively the s y n a p t i c e x c i t a t i o n o f m o t o n e u r o n e s w i t h o u t a l t e r i n g the i n h i b i t o r y p o s t s y n a p t i c p o t e n t i a l s i n the cat spinal c o r d ( P i e r a u a n d Z i m m e r m a n n , 1973). T h e p r e s e n t i n v e s t i g a t i o n deals with the effects o f two b e n z o d i a z e p i n e s , d i a z e p a m a n d f l u n i t r a z e p a m , o f a b a r b i t u r a t e , p h e n o b a r b i t o n e , a n d o f the G A B A derivative, baclofen, on pre- a n d p o s t s y n a p t i c inhibition as well as o n e x c i t a t o r y m e c h a n i s m s in the c u n e a t e nucleus o f d e c e r e b r a t e cats. I n s o m e e x p e r i m e n t s we p e r f o r m e d d r u g i n t e r a c t i o n studies with the G A B A r e c e p t o r b l o c k i n g agents, p i c r o t o x i n a n d bicuculline, the G A B A - s y n t h e s i s i n h i b i t o r s , t h i o s e m i c a r b a z i d e a n d 3 - m e r c a p t o p r o p i o n i c acid, a n d the i n h i b i t o r o f G A B A - d e g r a d a t i o n , a m i n o o x y a c e t i c acid ( A O A A ) . P a r t o f these results were r e p o r t e d in a s h o r t c o m m u n i c a t i o n (Polc a n d Haefely, 1975).
METHODS Fourty-eight cats were mounted under ether anaesthesia in a stereotactic holder and decerebrated by transection of the brain stem at the intercollicular level by high-frequency electrocoagulation. The ether was then withdrawn. Preparation of Cuneate Nucleus and Nerves. The dorsal column nuclei were exposed by removing the laminae of the upper two cervical vertebrae, the part of the occipital bone coveting the posterior cerebellum, and the atlanto-occipital membrane. The posterior cerebellum was removed by suction. The proximal ends of the peripherally severed median and ulnar nerves in the left foreleg were mounted on bipolar platinum electrodes for stimulation and recording, respectively. Skin flaps were used to form heated paraffin pools over the exposed peripheral nerves and the dorsal column nuclei. Stimulation and Recording. The experimental set-up is schemati-
cally presented in Figure 1. Depolarization of primary afferent endings (PAD) of dorsal column fibres, which is responsible for the presynaptic inhibition of cuneate relay (CTR) cells, was assessed on the one hand by the positive cuneate surface potential (P wave), recorded from the dorsal surface of the left cuneate nucleus using
Naunyn-Schmiedeberg's Arch. Pharmacol. 294 (1976)
orthodromic lemniscal potential
~j
cuneate stimulation
I"----~
cuneate surface
]
potential
j~
dorsal column fibre
~
antidromic action potential
\ ",4
.T- ."--'-- ,, . ~ x n ulnaris orthodromic afferent / \ stimulation (',,~ \ ~ n. medlanus Fig. I. Schematic diagram illustrating the experimental set-up. Stimulating electrodes were placed in the cuneate nucleus and on the median nerve, recording electrodes in the medial lemniscus, on the ulnar nerve and on the dorsal surface of the cuneate nucleus. A simplified picture of the synaptic organisation within the nucleus shows the terminals of primary afferents exciting on the one hand the cuneothalamic relay cells (CTR cells, solid circles), on the other hand the inhibitory interneurons (open circles). The axons of the latter cells form axo-axonal synapses with primary afferent terminals (presynaptic inhibition) and/or axodendritic and axosomatic synapses with CTR neurones (postsynaptic inhibition). Descending and recurrent inhibition is omitted in this diagram
a silver-silver chloride electrode and evoked by single supramaximal stimuli of 0.05 ms duration once every 2 s, applied to the ipsilateral median nerve. On the other hand, a more direct index of PAD was obtained by testing the excitability of primary afferent endings within the cuneate nucleus by the technique of Wail (1958). Submaximal single pulses delivered through a coaxial stainless steel semimicroelectrode (0. i mm in diameter) inserted in the left cuneate nucleus, elicited action potentials in aTraction of afferent terminals, which were conducted antidromically and recorded in the ipsilateral ulnar nerve. The amplitude of this antidromic action potential, which is a function of the number of ulnar nerve terminals excited by the constant test stimulus in the cuneate, is an index of the excitability of the resting primary afferent terminals. The increase of the excitability of primary afferent terminals, which occurs during PAD elicited by volleys in the median nerve, was measured by the increase of the size of the antidromic action potential in the ulnar nerve. PAD was induced by single conditioning supramaximal volleys in the median nerve, delivered at intervals of 4, 7, 10, 20, 30, 50, 70 and 90 or 100 ms prior to the test
P. Polc and W. Haefely: Pharmacology of Synaptic Transmission in Cuneate Nucleus pulses in the cuneate nucleus. An increased excitability of primary afferent terminals was also visible from the appearance of a late antidromic response, the dorsal column reflex (DCR). The DCR consists of late antidromic action potentials recorded in the ulnar nerve in response to direct stimuli applied to the cuneate nucleus and initiated in afferent endings by the depolarizing effect at axoaxonal synapses of impulses in inhibitory interneurones. These impulses result from either direct stimulation by the electrode or synaptic excitation through the collaterals of those primary afferents that are activated by the electrode.
Postsynaptic inhibition of CRT cells was assessed in the following way: a coaxial stainless steel electrode (0.3 mm in diameter), insulated except at the tip, was introduced into the contralateral medial lemniscus until it recorded a maximal orthodromic response to cuneate stimulation. The first short-latency spike (delay 0 . 4 0.6 ms) of this response is attributed to the direct stimulation of CTR cells (Andersen et al., 1964). The depression of this shortlatency lemniscal potential by supramaximal preceding volleys in the median nerve, delivered at intervals of 4, 7, 10, 20, 30, 50, 70 and 90 or 100 ms before the test cnneate stinmlus, was taken as an index of the reduced excitability of CTR cells which results from the hyperpolarization that underlies postsynaptic inhibition. In addition to studying inhibitory phenomena in the cuneate nucleus, we assessed excitatory mechanisms in the same nuclear region by measuring the negative wave of the cuneate surface potential (N wave) in response to a single stimulus to the median nerve, which reflects postsynaptic depolarization of relay cells, as well as the size of test short-latency lemniscal response to cuneate volleys, which indicates the resting excitability of cuneate relay cells. All the above potentials were amplified (a DC amplifier was used for the cuneate surface potential) and displayed on an oscilloscope, from which polaroid photographs were taken.
Evaluation of Drug Effects. The mean amplitudes of the test antidromic potentials in the ulnar nerve, and of the test orthodromic potentials in the medial lemniscus, both evoked by isolated cuneate volleys, measured during a 30 rain control period, were taken as 100~. The amplitudes of the corresponding responses obtained at fixed intervals after identical preceding conditioning stimuli in the pre-drug and post-drug period were expressed in percent of these control test amplitudes. In the same manner the amplitudes of the N wave and the amplitudes and the durations of the P wave were measured before and after drug administration, The effects of drugs on the dorsal column reflex (DCR) were evaluated by measuring the area determined by the response and the baseline in the control period and after drug injection. In order to obtain a dose-response curve, each drug tested (diazepam, flunitrazepam, phenobarbitone, baclofen) was given in 4 increasing cumulative doses and the effects were evaluated 20 rain after each injection. The intervals between the drug applications were 30 min. In this series of experiments each animal received only one drug. The mean values of the parameters measured in different animals were calculated for each drug series, which consisted of 5 experiments. Only preparations with stable potentials during the 30 rain control period were used for evaluation. Sometimes, 8 responses were averaged (N, P waves, test antidromic and lemniscal potentials). Student's t-test was used to calculate statistical signiflcances between pre- and post-drug values of the P wave amplitude and duration, N wave amplitude, and of the DCR area. How ever, since already in the pre-drug control period the percent reduction of lemniscal responses and the percent increase of primary afferent depolarization by a preceding volley in the median nerve varied greatly from one animal to another, it was not meaningful to pool the data from different animals. A given drug effect on the latter two parameters was considered to be statistically significant
123
(Friedman-test), if it occurred without exception in all the experiments of a series irrespective of the magnitude.
Drugs Used. Aminooxyacetic acid hydrochloride (AOAA), baclofen, bicuculline, diazepam, flunitrazepam, gallamine triethiodide, 3-mercaptopropionic acid (MPA), phenobarbitone, picrotoxin, thiosemicarbazide (TSC), and the solvent for diazepam and flunitrazepam were injected into the right cannulated femoral vein and flushed in with physiological saline. All drugs except diazepam and fiunitrazepam were dissolved in saline. Preparations with a sudden or strong increase of the blood pressure in response to the convulsants, bicuculline, MPA, picrotoxin and TSC were discarded as were experiments in which the blood pressure fell below 90 mm Hg. in order not to interfere with the synaptic transmission in the cuneate, gallamine triethiodide was injected only if convulsions occurred (Galindo et al., 1968). The organic solvent used for injecting diazepam and flunitrazepam did not affect any measured parameter when given in the amount (1 ml/kg) necessary for administering the highest dose (10 mg/kg) of the two benzodiazepines.
RESULTS
Effects of Diazepam on Preand Postsynaptic Inhibition and on Orthodromic Excitation of Cuneate Relay Cells A t y p i c a l e x p e r i m e n t w i t h d i a z e p a m is d e p i c t e d in F i g u r e 2. 10 m i n a f t e r a d o s e o f 0.3 m g / k g t h e foll o w i n g c h a n g e s w e r e o b s e r v e d , a) T h e P w a v e in res p o n s e to a single v o l l e y in t h e m e d i a n n e r v e a n d b) t h e d o r s a l c o l u m n reflex ( D C R ) in r e s p o n s e to a single s t i m u l u s w i t h i n t h e c u n e a t e n u c l e u s w e r e markedly enhanced, c)The increased excitability of p r i m a r y a f f e r e n t e n d i n g s o f u l n a r n e r v e fibres t h a t o c c u r s a f t e r a p r e c e d i n g v o l l e y in t h e m e d i a n n e r v e w a s a c c e n t u a t e d a n d p r o l o n g e d as s h o w n b y t h e inc r e a s e d size o f t h e a n t i d r o m i c p o t e n t i a l in the u l n a r nerve evoked by stimuli within the cuneate nucleus. In a d d i t i o n to m o d i f y i n g t h e s e 3 p a r a m e t e r s w h i c h are phenomena connected with presynaptic inhibition, d i a z e p a m a l s o e n h a n c e d a f f e r e n t p o s t s y n a p t i c inh i b i t i o n o f c u n e a t e r e l a y cells, as s h o w n b y d) a n acc e n t u a t i o n o f t h e r e d u c t i o n o f t h e s h o r t - l a t e n c y res p o n s e s in t h e m e d i a l l e m n i s c u s (to d i r e c t s t i m u l a t i o n w i t h i n t h e c u n e a t e n u c l e u s ) w h i c h is o b s e r v e d f o r a s h o r t p e r i o d a f t e r a n a f f e r e n t v o l l e y in t h e m e d i a n nerve. I n c o n t r a s t , d i a z e p a m did n o t affect t h e N w a v e e v o k e d by v o l l e y s in t h e m e d i a n n e r v e o r t h e l e m n i s c a l r e s p o n s e to i s o l a t e d s t i m u l i w i t h i n t h e c u n e a t e n u c l e u s . N e i t h e r d i d it affect t h e size o f t h e a n t i d r o m i c r e s p o n s e in t h e u l n a r n e r v e to i s o l a t e d d i r e c t c u n e a t e stimuli. ] ' h e a b s e n c e o f a n y effect o f d i a z e p a m o n t h e s e 3 r e s p o n s e s i n d i c a t e s t h a t t h e d r u g has n o effect o n t h e e x c i t a b i l i t y o f c u n e a t e r e l a y cells o r o f p r i m a r y a f f e r e n t e n d i n g s in t h e a b s e n c e o f v o l l e y s that activate inhibitory neurones within the cuneate nucleus.
124
Naunyn-Schmiedeberg's Arch. Pharmacol. 294 (1976)
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