Experimental Brain Research · 2017. 6. 7. · Exp Brain Res (1992) 91:2945 Experimental Brain Research 9 Springer-Verlag 1992 Differential action of (--)-baclofen on the primary

Exp Brain Res (1992) 91:2945

Experimental Brain Research �9 Springer-Verlag 1992

Differential action of (--)-baclofen on the primary afferent depolarization produced by segmental and descending inputs J. Quevedo 1, J.R. Eguibar 1'2, I. Jim6nez 1, and P. Rudomin 1

Department of Physiology, Biophysics and Neurosciences, Centro de Investigacidn y de Estudios Avanzados del IPN, Apartado Postal 14-740, M+xico D.F. 07000, M6xico 2 Department of Physiological Sciences Instituto de Ciencias, UAP, M~xico D.F. 07000, M+xico

Received January 23, 1992 / Accepted April 15, 1992

Summary. The purpose of the present series of experiments was to analyze, in anesthetized and paralyzed cats, the effects of (-)-baclofen and picrotoxin on the primary afferent depolarization (PAD) generated in single Ib afferent fibers by either intraspinal microstimulation or stimulation of the segmental and descending pathways. PAD was estimated by recording dorsal root potentials and by measuring the changes in the intraspinal activation threshold of single Ib muscle afferent fibers. The PAD elicited by stimulation of group I muscle or cutaneous afferents was readily depressed and often abolished 20-40 min after the intravenous injection of 1-2 mg/kg (-)-baclofen. In contrast, the same amounts of ( - ) -ba - clofen produced a relatively small depression of the PAD elicited by stimulation of the brainstem reticular formation (RF). The monosynaptic PAD produced in single Ib fibers by intraspinal microstimulation within the intermediate nucleus was depressed and sometimes abolished following the i.v. injections of 1-2 mg/kg ( - )-baclofen. Twenty to forty minutes after the i.v. injection of picrotoxin (0.5-1 mg/kg), there was a strong depression of the PAD elicited by stimulation of muscle and cutaneous afferents as well as of the PAD produced by stimulation of the RF and the PAD produced by intraspinal microstimulation. The results obtained suggest that, in addition to its action on primary afferents, ( - )-baclofen may depress impulse activity and/or transmitter release in a population of last-order GABAergic interneurons that mediate the PAD of Ib fibers. The existence of GABAb autoreceptors in last-order interneurons mediating the PAD may function as a self-limit- ing mechanism controlling the synaptic efficacy of these interneurons.

Key words: Presynaptic inhibition - Primary afferent depolarization - Baclofen - GABA - Spinal cord - Cat

Correspondence to: P. Rudomin

Introduction

In the cat, conditioning stimulation of group I muscle afferent fibers depresses the monosynaptic excitatory postsynaptic potentials (EPSPs) produced in spinal motoneurons by Ia fibers without affecting the monosynaptic EPSPs produced in the same motoneurons by stimulation of the ventromedial fasciculus (Rudomin et al. 1975, 1991). The selective depression of the Ia EPSPs has been explained by assuming that the intraspinal terminals of Ia fibers, unlike those of descending fibers, are the targets of GABAergic interneurons whose activation produces primary afferent depolarization (PAD) and presynaptic inhibition (Rudomin et al. 1975, 1991).

The PAD elicited in group Ia muscle afferents by segmental inputs is decreased by picrotoxin or bicucul- line (Eccles et al. 1963; Rudomin et al. 1983; Curtis and Lodge 1982). This has led to the suggestion that PAD and presynaptic inhibition arise from activation of GABAa receptors. However, the monosynaptic EPSPs elicited in motoneurons by stimulation of Ia fibers are also depressed after the i.v. injection of low doses of (-)-baclofen, a GABAb agonist (Curtis et al. 1981; Ed- wards et al. 1989; Jim6nez et al. 1991). Since this depression occurs without noticeable changes in motoneuron properties, it has been suggested that the intraspinal terminals of the Ia fibers also have GABAb receptors (Edwards et al. 1989; Jim~nez et al. 1991). In contrast to Ia fibers, the intraspinal terminals of descending fibers appear not to have GABAa receptors (Curtis and Malik 1984; Curtis et al. 1984) and have a relatively low density of GABAu receptors (Jim~nez et al. 1991).

There have been no experimental studies concerning the action of GABAb agonists on primary afferent depolarization and presynaptic inhibition elicited by descending inputs. The few studies available deal with the action of (-)-baclofen on PAD of segmental origin (Kato et al. 1978; Curtis et al. 1981, 1986). These studies have shown that the PAD elicited by segmental inputs is depressed following the administration of ( - )-baclofen. Since (-)-baclofen appeared not to have any effect on

30

the exci tab i l i ty o f afferent fibers (Cur t i s et al. 1981) o r on their r e sponse to i o n t o p h o r e t i c a l l y app l i ed G A B A (Cur- tis et al. 1986), i t was cons ide red tha t the r educ t ion by ( - ) - b a c l o f e n o f the P A D gene ra t ed a t a x o - a x o n i c synapses on Ia a n d Ib t e rmina l s was due to r educed G A B A release f rom l a s t -o rde r in te rneurons . The poss ib le existence o f G A B A au to r ecep to r s in the l a s t -o rde r inter- neu rons m e d i a t i n g P A D is in te res t ing because this cou ld be the basis for a se l f - l imit ing m e c h a n i s m con t ro l l ing the a m o u n t o f p r e synap t i c i nh ib i t i on in specific pa thways . In the cat , the l a s t -o rde r i n t e rneu rons m e d i a t i n g P A D can be d i rec t ly ac t iva t ed by means o f i n t r a sp ina l m i c r o s t i m u - la t ion ( J a n k o w s k a et al. 1981; R u d o m i n et al. 1981, 1983). This a l lows a m o r e d i rec t assessment o f the ac t ion o f ( - ) - b a c l o f e n on the synap t i c effectiveness o f these i n t e rneu rons a n d o f the poss ib le exis tence o f G A B A au to recep to r s .

The p u r p o s e o f the p resen t series o f expe r imen t s was to ana lyze the effects o f ( - ) - b a c l o f e n and o f p i c ro tox in on the P A D gene ra t ed in single afferent fibers by s t imula - t ion o f segmenta l and descend ing p a t h w a y s a n d by in- t r a sp ina l m ic ro s t imu la t i on . Ib fibers were selected for this s tudy because , un l ike I a fibers, they are no t on ly d e p o l a r i z e d by g r o u p I fibers bu t also by cu t aneous and descend ing fibers ( R u d o m i n et al. 1986; JimOnez et al. 1988). This a l lowed c o m p a r i s o n o f the ac t ion o f ( - ) - b a - c lofen on the P A D gene ra t ed in the same fiber by different inputs . The resul ts o b t a i n e d suggest tha t , in add i - t ion to its ac t ion on p r i m a r y afferents, ( - ) - b a c l o f e n m a y depress impulse ac t iv i ty a n d / o r t r ansmi t t e r release in l a s t -o rde r G A B A e r g i c i n t e rneu rons tha t med ia t e the P A D o f Ib fibers. A c t i v a t i o n o f G A B A b au to r ecep to r s in axon t e rmina l s m a y func t ion as a se l f - l imit ing mechanism con t ro l l ing synap t i c release in these in te rneurons . A p r e l i m i n a r y accoun t o f these resul ts has been pub l i shed in abs t r ac t f o rm (Quevedo et al. 1990).

Materials and methods

General procedures

The investigations were undertaken in 12 cats anesthetized with pentobarbital (35 mg/kg i.p.). Anesthesia was maintained through- out the experiment by giving additional doses of pentobarbital every hour (6 mg/kg). The trachea was exposed and cannulated for artificial respiration. The left carotid artery and the radial vein were dissected and prepared for blood pressure recording and for fluid injections, respectively. The lumbosacral as well as the lower thoracic spinal cord was exposed. Both dorsal columns were removed and the right hemicord sectioned at the thoracic level. The left ventral roots S1 to L6 were dissected free and sectioned. The left posterior biceps (PB) and semitendinosus (St), sural (SU), superficial pero- neus (SP), and gastrocnemius-soleus (GS) nerves were dissected, sectioned, and their central ends prepared for stimulation. When measuring changes in the intraspinal threshold of single muscle afferent fibers, the GS and PBSt nerves were segregated into several (5-7) fine filaments for recording the antidromic action potentials elicited by intraspinal microstimulation (see below).

After the dissection, the animal was transferred to a metal frame for proper fixation of the spinal cord. The brainstem was exposed by removing the occipital bone and part of the cerebellum. Reser- voirs made with the skin flaps were filled with mineral oil. The animal was paralyzed with pancuronium bromide (Pavulon, Or-

ganon) and artificially respirated. Tidal volume was adjusted to maintain expiratory CO2 at about 4%. The temperature of the animal and of the pool was automatically kept between 37 and 38 ~ C by means of sensor-controlled radiant heat. Blood pressure was usually between 100 and 120 mm Hg. When necessary, a solution of etilefrine (Effortil, Boehringer-Ingelheim) diluted with isotonic saline (1 : 10) or dextran (10%) was infused intravenously.

Recording

Cord dorsum potentials (CDPs) were recorded with a Ag/AgC1 ball electrode placed near the entry site of the dorsal roots. The indif- ferent electrode was placed in nearby back muscles. Dorsal root potentials (DRPs) were recorded bipolarly from a L7 dorsal rootlet. One electrode was placed close to the root entry zone and the other on the distal end of the rootlet. Low noise, high gain differential amplifiers (band pass filters 0.3 Hz-10 kHz) were used to amplify the potentials.

During the experiment, raw potentials were stored on analog tape (bandpass 0-1 kHz) and averages of 32 responses elicited at 1 Hz were taken using a digital computer. After the experiment, the data were replayed for further analysis.

Intraspinal threshold testing of single muscle afferents

A glass micropipette, filled with NaC1 3M (1 2 M~ resistance), was introduced in the intermediate nucleus to record the intraspinal field potentials produced by stimulation of group I fibers in the GS and PBSt nerves. Following identification of the sites producing the largest postsynaptic responses, the recording micropipette was con- nected to a stimulating device controlled by a digital computer. Constant current pulses were passed through the micropipette at a frequency of once per second while recording from a GS or PBSt nerve filament. The stimulating micropipette was moved within the spinal cord until antidromic responses were produced in a single afferent fiber using minimal strengths. Subsequently, the computer was used to generate current pulses of the strength necessary to produce antidromic firing of the fiber with a probability of 0.5. The test current pulses were integrated, and the resulting value was maintained until the next stimulus. This gave a continuous estimate of the intraspinal threshold of the fiber.

Changes in the intraspinal threshold of the afferent fiber produced by conditioning stimuli applied to afferent nerves or to supraspinal nuclei give reliable information on PAD produced in that fiber (Rudomin et al. 1981, 1983, 1986). We have found that expressing threshold changes in absolute terms (i.e., in gA) does not allow a valid comparison of the effects produced in different fibers because the estimated "resting" threshold of the fiber depends, among other factors, on the position of the stimulating electrode relative to the afferent fiber (Rudomin et al. 1981). On the other hand, expressing the threshold changes as a percentage change relative to the resting threshold of the fiber gave a more consistent range of values and allows comparison of data obtained from different fibers (Rudomin et al. 1981, 1983, 1986).

Towards the end of this study, we developed a method to analyze, at the same time, the intraspinal threshold changes occur- ring in pairs of single afferent fibers. The method is essentially the same as that used for a single fiber, except that two stimulating micropipettes are inserted in the intermediate nucleus and stimulating pulses are delivered in alternation through each micropipette. The antidromic responses of afferent fibers are recorded from different nerve filaments and are sampled by different A/D converters. The program allows independent adjustment of the current strength required to maintain a constant antidromic firing index in each fiber. Clearly, there will be a shift of one cycle in the estimates of the intraspinal threshold of one fiber relative to the other, but this has no significance for the present study, where the concern was the steady, not the transient, threshold changes resulting during conditioning stimulation.

1 , r ~ CDP

PBSf

2"5xT�9 t C O N T R O L

. . . . . . J t / ~ ~

A

W

C

120

100

80

60

40

20

0

Bac Bac Bac $ $ $

0 \ 0 DRP �9 A f f .

~::~"L~ -~- '~' 'A-~'- , ~ / k p W a V e

\

6 I 2 0 I 4 0 I 610 I 8l 0 I 10 0

TIME (min)

31

•j• CDP ]

60 ' B a c

3 0 0 uv

I I

25 m s

100 , v

o 95. .._1 o -1-

9 0 LLI

-i- 8 5 I - -

_ J

< 80 . Z n 75 13," }..- Z 7 0

D

Fig. l A D . Effects of ( - ) -baclofen on PAD produced by stimulation of group I muscle afferents. A, B The cord dorsum potentials (CDP) and dorsal root potentials (DRP) produced by stimulation of the PBSt nerve with four pulses of 300 Hz, 2.5 x T followed by the current pulse used to test the intraspinal threshold of a single Ib fiber (see text) before and 60 rain after two injections of 1 mg/kg (-)-baclofen (Bac; see arrows in C). Note that the P waves and the

�9 Bae /

Q t

Bac �9 PBSr 2 . 5 x T

b ' 20 40 60 a'o ' 1 d o TIME (min)

DRPs were virtually abolished after (-)-baclofen. C Time course of the effect of ( -)-baclofen on afferent spikes, CDPs and DRPs produced by PBSt stimulation. Arrows show time of (-)-baclofen injections. D Effects of ( -)-baclofen on the intraspinal threshold changes produced in a single Ib fiber following conditioning stimulation of the PBSt nerve with same parameters. Further explanation in text

For the present study, we selected muscle afferents with conduction velocities above 70 m/s and peripheral thresholds (determined by collision; see Rudomin et al. 1986) between 1.12 and 2.25 x T, i.e. in the group I range, that were depolarized by stimulation of other group I and by cutaneous fibers and also by stimulation of the reticular formation. According to available information, these would be group Ib afferents (Rudomin et al. 1983, 1986; Jim6nez et al. 1988). Some group II fibers have conduction velocities above 70 m/s, peripheral thresholds below 2 x T and are depolarized by cutaneous, joint and probably also by descending inputs (Carpenter et al. 1963; Harrison and Jankowska 1989). Therefore, it is possible that a small fraction of the analyzed fibers were group II instead of group Ib. However, this would not change the basic conclusions derived from the present series of experiments.

Central stimulation

A stimulating electrode (Parylen-insulated tungsten, 20 gm tip di- ameter) was placed in the ipsilateral reticular formation (RF), using the obex as reference. The RF electrode was usually placed 4 mm rostral and 2 mm lateral to the obex at 3-3.5 mm below the surface (see Rudomin et al. 1983, 1986).

Baclofen and picrotoxin administration

After recording a series of control responses, ( - )-baclofen (Ciba- Geigy, 1-2 mg/kg) diluted in isotonic saline was injected intravenously in a volume of 5 ml over a period of 5-10 min. This amount was sufficient to produce a significant depression of the synaptic actions mediated by Ia fibers (Curtis and Malik 1985; Edwards et al. 1989; Jim6nez et al. 1991).

In several experiments, we tested the action of picrotoxin (Sig- ma) on DRPs and on the intraspinal threshold changes produced in single Ib fibers following segmental and descending inputs. Picro- toxin was given in subconvulsive doses (0.5 mg/kg) and was diluted in saline and slowly injected. This increased the blood pressure and in several experiments antidromic responses of afferent fibers could no longer be elicited, even after repositioning of the stimulating micropipette. However, in five cases we were able to keep the antidromic responses elicited in the same afferent fiber up to about 1 h after the administration of picrotoxin.

At the end of the experiment, electrolytical lesions (50 gA anodal for 40 s) were made in the brainstem. The glass micropipette was broken at the shaft, leaving the tip inserted in the spinal cord. The animal was subsequently killed with an overdose of barbiturate and perfused with formalin (10%). After fixation, histological sec-

32

tions of the brainstem were made for confirmation of stimulating sites. The piece of spinal cord was cleared with salicylate and cut to obtain a thin section containing the tip of the micropipette. This section was used to reconstruct the microelectrode position attained during the recording of the field potentials.

Results

The present series of experiments was designed primarily to test the effects of (-)-baclofen on the primary afferent depolarization (PAD) generated by segmental as well as by descending inputs. As stated in Materials and methods, the PAD was evaluated in two ways; (1) by recording the DRPs and (2) by measuring the intraspinal threshold changes of single Ib muscle afferent fibers. In every experiment, we analyzed the effects of ( - ) -ba - clofen on the PAD produced by all of the available inputs. However, the effects of (-)-baclofen on the responses produced by each individual input will be pre- sented separately.

PAD produced by 9roup I muscle afferents

Stimulation of group I fibers from the PBSt nerve with a train of 4 pulses at 300 Hz produced a negative DRP as well as a slow positive wave (P wave) in the cord dorsum. The DRP is an electrotonic recording of the intraspinal PAD. The P wave is, by contrast, produced by the current flow associated with PAD (Eccles et al. 1962).

Figure 1 illustrates the effects of several injections of ( - )-baclofen on the DRPs and P waves produced by stimulation of group I fibers. In this experiment, we also tested the intraspinal threshold changes of a single Ib fiber produced by PBSt conditioning volleys. The traces in Fig. 1A and B show the responses produced by a train of four pulses of 300 Hz, 2.5 x T, applied to the PBSt nerve followed, 25 ms later, by the pulse used to test the intraspinal threshold of the fiber (see below). Although the conditioning DRPs and the P waves are only partly shown in these records, it is clear in Fig. 1B that these responses were practically abolished following the i.v. injection of 2 mg/kg of (-)-baclofen. Figure 1C shows the complete time course of the effects produced by the i.v. injection of (-)-baclofen. The DRPs (open circles) and the P waves (open triangles) were depressed with a similar time course; 20 min after the first injection of 1 mg/kg (-)-baclofen both responses were reduced to about 40% of control values, and both were virtually abolished after the second injection. It should be noted that the group I afferent volley produced by stimulation of the PBSt nerve was not significantly changed following the injection of ( -)-baclofen (see CDP trace in Fig. 1A, B and filled circles in Fig. 1C).

Similar results were obtained in other experiments, as shown in Table 1. Five minutes after the i.v. injection of 1-2 mg/kg of baclofen the DRPs elicited by a train of pulses of strength 1.5-2.5 x T applied to the PBSt nerve were reduced to a mean value of 52% relative to the control amplitude. Twenty minutes after the injection, the mean amplitude of the DRPs was 22%. The am-

Table 1. Effects of (--)-baclofen on PAD of segmental and supraspinal origin

Time PBSt (1.5-2.5 x T)

(min) Aft DRP (%) (%)

SU (2-16 x T)

P wave IT DRP CDP IT (%) (%) (%) (%) (%)

RF (97-172 gA) gSTIM

DRP CDP IT IT (%) (%) (%) (%)

Control

M 100 100 100 76.1 100 100 85.8 SD 9.3 10.2 n 4 4 4 6 3 3 5

( - ) -Bac lo fen 1-2 mg/kg

5 M 83.6 52.5 58.4 80.0 98.7 100.0 89.2 SD 18.6 48.9 48.1 14.0 18.0 n 4 4 4 6 1 1 4

10 M 100.6 35.9 47.3 84.7 61.5 76.8 93.1 SD 5.8 33.8 39.1 15.9 15.0 25.2 11.2 n 4 4 4 5 3 3 4

20 M 105.2 21.9 29.2 92.1 75.9 79.0 93.0 SD 6.7 20.3 23.3 7.2 12.1 n 4 4 4 4 1 1 3

30 M 111.7 13.3 17.6 85.3 52.6 85.8 92.7 SD 12.3 16.1 13.1 13.8 9.8 n 4 4 4 6 1 2 3

60 M 103.5 0.4 1.1 92.6 35.2 40.3 92.0 SD 7.0 0.7 2.2 12.8 18.3 14.0 n 4 4 4 5 2 3 3

100 100 76.9 78.7 9.5 11.2

4 4 5 4

67.2 72.1 84.6 84.2 30.8 33.6 12.9 12.2

3 3 5 3

75.2 70.1 82.9 89.0 32.0 24.4 13.4 12.2

4 3 5 4

68.3 82.4 83.5 84.9 23.5 21.6 14.3 7.1

4 4 3 3

64.8 79.6 82.8 91.8 29.8 25.9 13.4 7.0

4 4 5 4

57.8 65.7 88.1 91.2 32.3 33.2 7.9 10.0

3 3 6 3

gSTIM, intraspinal microstimulation; Aft, afferent spike; DRP, dorsal root potential; P wave, positive wave recorded from cord dorsum; CDP, first negative component in the cord dorsum re-

sponse; IT, changes in intraspinal threshold of a single Ib fiber relative to resting threshold.

33

plitudes of the corresponding P waves were reduced to 58 % and 29 %, respectively. One hour after the injection, DRPs and P waves were barely evident (the means were 0.4 % and 1% of the control value, respectively). It should be noted from Table 1 that the standard deviation (SD) of the mean P waves and DRPs was rather large. This is due to the variation in the time course of the action of (-)-baclofen in individual experiments.

One of the limitations in the use of DRPs as indicators of PAD is that the identity of the depolarized afferent fibers remains undetermined. Measurement of the intraspinal threshold changes of single afferents in response to segmental and descending inputs gives additional information on the type of afferent fibers exhibiting PAD

CONTROL A /zA

I I I I 1 1 1 1 PBSt RF SU ~,St 2 .5xT 97/.~A 5xT 5 /xA

B 20' AFTER BACLOFEN

11 LJ LJ I I PBSI RF SU ,u. St

C 40' AFTER BACLOFEN

LJ TT U T 1 PBSI RF SU /.L St

I I

1 min Fig. 2A-C. Effects of ( - ) -baclofen on the intraspinal threshold changes of a single Ib fiber produced by segmental and descending stimulation. A Decrease in the intraspinal threshold following stimulation of the PBSt nerve (four shocks, 300 Hz, 2.5 x T, 25 ms before test pulse), of the SU nerve (one pulse, 5 x T, 35 ms before test pulse) and of the RF (eight pulses, 700 Hz, 97 laA, 75 ms before test pulse). Intraspinal microstimulation (laSt) was a single pulse of 5 gA applied 5 ms before the threshold testing pulse through the same micropipette. Stimulus current was below the activation threshold of the test afferent fiber. B Same but 20 rain after the injection of 1 mg/kg (-)-baclofen. C Forty minutes after the first of two injections of 1 mg/kg (-)-baclofen. The second injection was given 35 min after the first injection

(Jankowska et al. 1981; Rudomin et al. 1981, 1983, 1986). As shown in Fig. 2A, before (-)-baclofen, stimulation of the PBSt nerve reduced the intraspinal threshold of a Ib fiber from about 7.0 gA to 5.8 gA, i.e. to 81.4% relative to the resting threshold. Twenty minutes after the first injection of 1 mg/kg (-)-baclofen, the resting threshold of the fiber was still at this level (6.9 gA) but the PBSt stimulus was less effective in reducing the threshold (86.9% of control; Fig. 2B). Forty minutes after the first injection, and 5 min after a second injection of the same amount of (-)-baclofen, the effect of PBSt stimulation was significantly smaller (97%; Fig. 2C). These observations are in full agreement with those of Curtis et al. (1981, 1986) and underline the finding that (-)-baclofen may depress synaptic actions produced by stimulation of group I muscle fibers practically without changing the excitability of these primary afferents.

Figure 1D illustrates the time course of the effect of (-)-baclofen on the intraspinal threshold changes produced by PBSt stimulation in a different Ib fiber. Before the injection, stimulation of the PBSt nerve reduced the intraspinal threshold to 83% relative to the resting threshold. Thirty minutes after the first injection of (-)-baclofen, the effect produced by PBSt stimulation was reduced (to 90 % relative to resting threshold). Fur- ther reduction was seen after additional (-)-baclofen, and at 95 min after the injection the PBSt conditioning stimulation produced practically no changes in the intraspinal threshold of the fiber.

In total, we have examined the effects of (-)-baclofen in 6 Ib fibers (Fig. 7A). In all cases, this GABAb agonist reduced the PAD elicited by group I PBSt volleys, but the time course and magnitude of the effects varied within wide ranges. For one Ib fiber, the depression of the PAD (threshold reduction) was rather fast, and PAD was abolished around 5 min after the injection of ( - ) -ba - clofen. In other fibers, however, depression of PAD had a slower time course and the PAD still had an appreciable magnitude 90 min after the injection of ( - ) - baclofen.

Variability in the action of (-)-baclofen on the PAD of single Ib fibers (reduction in intraspinal threshold) elicited by group I PBSt volleys cannot only be due to differences between preparations. It reflects also varia- tions in the susceptibility of the pathways producing PAD in different sets of afferent fibers, even in the same preparation, as is illustrated in Fig. 3. In this experiment, we were able to determine the effects of (-)-baclofen on the intraspinal threshold changes produced by several conditioning paradigms simultaneously in one Ib St and one Ib GSfibers (see Materials and methods). Figure 3A shows that a PBSt train of 2.6 x T reduced, very effective- ly, the intraspinal threshold of both Ib fibers. Three minutes and 25 min after the injection of 1 mg/kg (-)-baclofen, a PBSt train of 1.6 x T still produced an appreciable reduction of the intraspinal threshold of both fibers (Fig. 3D and E). However, 55 min after the first injection (and 25 min after a second injection of 1 mg/kg (-)-baclofen), PBSt conditioning produced appreciable PAD in the St Ib fiber and practically no effect on the GS Ib fiber (Fig. 3F). Eighty-five minutes later,

34

0.9 ~ h 0.6

PBSt SU RF RF

2.6xT fl.3xT 80 120 /~A

1.2

0.6 / ~ s t i m .

6.8 8.2 10 #A

JOi6 / z s t i m . (8.2 /~A)

3 5 10 m s

C J6

3' a f t e r Bac 25' a f t e r Bac

S t 1.2

PBSt SU RF 0.6

1.6xT 8.3xT 80~tA

f i b e r ~

D J6

/ z s t i m .

5.0 6.8 7.7 /zA

E

~ ] 1.2

0.9

PBSt RF 0.6

1.6xT 80/zh

J 6

55' a fLe r Bac

PBSt

1.6 2.0 2.6 xT

1.2

0.6

RF RF

80 120 /zA 10

6

F Fig. 3A-G. Effects of (-)-baclofen on the PAD elicited in a pair of Ib fibers. In this experiment simultaneous estimates were made of the intraspinal threshold changes of one Ib St afferent fiber (upper traces) and of a Ib GS afferent fiber (lower traces). A Control changes in intraspinal threshold produced by segmental and by descending stimulation, as indicated. B Control PAD (threshold reduction) produced by intraspinal conditioning (gstim.) with one pulse at the indicated strengths. Conditioning-testing stimulus interval was 5 ms. The conditioning pulse was applied through the micropipette used to test the excitability of the GS fiber. C Same, but at various conditioning-testing stimulus intervals, as indicated. Conditioning microstimulation was 8.2 pA. Note that intraspinal microstimulation produced PAD in the GS but not in the St fiber. D Effects produced by segmental and descending inputs 3 rain after

85' a f t e r Bac

PBSt

1.6 2 .0 2.6 xT

G

1.2

0.6 RF RF

80 120 # A

i i

1 r a i n

the first of two i.v. injections of 1 mg/kg (-)-baclofen. E PAD produced by segmental and descending stimulation as well as by intraspinal microstimulation 25 rain after the first injection of (-)-baclofen. Stimulus interval between conditioning intraspinal microstimulation and excitability testing pulse was 5 ms. Note that intraspinal pulses of 6.8 and 7.7 pA produced no PAD on the GS Ib fiber. F, G Effects of segmental and descending stimulation 55 and 85 min after the first injection of (-)-baclofen (25 and 55 min after the second injection). PBSt stimulus was a train of four pulses at 300 Hz applied 25 ms before the test pulse, SU stimulation was a single pulse (8.3 x T) applied 35 ms before the test pulse and RF stimulation was a train of eight pulses at 700 Hz applied 75 ms before the excitability testing pulse

SU 2xT

CDP

o

CONTROL

~ R P

120

100

,,, 8 0

~- 6 0 _ J IX. zE 4 0 <

20

A C

B

& CDP

v/"-

3 0 ' Bac

I I

25 m s

a . d o " l - (.f)

Ld n.,' " I - I - -

J

Z

U~

r ~ t-.. Z

I:)

100

95-

9 0

85-

8 0

7 5

70

Fig. 4A-D. The PAD elicited by stimulation of cutaneous afferents is depressed by (-)-baclofen. Same format as that of Fig. 1, with data collected in the same experiment. SU stimulation was a single pulse 2 x T. A Before and B 30 min after the first of two injections,

35

Bac Bac Bac ,L 4. 4.

\ ............ t o DRP '",\ ' 0 . OCDP

",,,,)x 0 ............ �9 P Wave

-.,., e

6 2'0 4'0 6'0 8o 16o TIME ( r a i n )

/ ?

Bac

? Bac

�9 S U 2xT

0 20 40 60 80 1do TIME ( r a i n )

each of 1 mg (- ) -baclofen (Bac; see arrows in C). D Note that 65 min after the first injection of ( - ) -baclofen the effects of SU stimulation on the intraspinal threshold of the afferent fiber were nearly abolished. Further explanation in text

the PAD elicited in the St fiber was further depressed (Fig. 3G). It thus seems that PAD elicited in different Ib fibers by the same group I input is not equally sensitive to (-)-baclofen.

Table 1 summarizes the mean changes produced by group I PBSt conditioning on the intraspinal threshold of all single Ib fibers. Before (-)-baclofen, PBSt reduced the mean intraspinal threshold to 764-9%. Twenty minutes after (-)-baclofen the effects produced by the same PBSt stimulus were clearly smaller (924-7%). The depression of the PAD persisted for at least 1 h after the injection.

PAD produced by cutaneous afferents

Figure 4 illustrates the effects of (-)-baclofen on the PAD produced by stimulation of cutaneous nerves. The format of this figure is similar to that of Fig. 1, obtained from data collected during the same experiment. It may be seen that the DRPs and the P waves, as well as the first

negative wave recorded in the cord dorsum, were reduced significantly 30 min after the injection of 1 mg/kg (-)-baclofen (Fig. 4B) and severely depressed 100 min after the third injection (Fig. 4C). The PAD produced in single Ib fibers by SU stimulation was also depressed and eventually abolished following the administration of (-)-baclofen (Fig. 2, 3A and D, 4D and 7B). In the example in Fig. 2, before (-)-baclofen, stimulation of the SU nerve reduced the intraspinal threshold of the fiber to 85.7% of the control (Fig. 2A); 20 min after (-)-baclofen, the same SU stimulus was less effective (88.2% ; Fig. 2B), and 40 min later, SU stimulation was practically ineffective (Fig 2C). Similar effects were seen in other fibers, as illustrated in Figs. 3A, D and 4D.

The (-)-baclofen-induced depression of the PAD produced by cutaneous volleys was demonstrated in five experiments (Table 1). Sixty minutes after the injection of (-)-baclofen, the SU-induced DRPs and P waves were reduced to 35.2% and 40.3% of the control values. The PAD (threshold reduction) elicited in six single Ib fibers was also reduced by (-)-baclofen. However, the

36

RF 97 /~A ~fffEEnlllm

A

~~k.~ CDP

CONTROL

Bac Bac Bac

~, 1 0 0 I v

w 8 0 a

I-- 6 0 "

. J

o_ 40-

2 0

0

C

CDP

60' Boc

! !

B 25 ms

3 0 0

v

t~ J 0 "-F (/} i , i

" l-

J

Z

O_ V}

r~

Z

100

95

90

85

80

75

70

D

Fig. 5A-D. The PAD elicited by stimulation of the RF is (-)-baclofen resistant. Same format and experiment as that of Figs. 1 and 4. The RF was stimulated with 18 pulses, 700 Hz, 97 pA, applied

120-

[] DRP

�9 CDP

[) 2'0 4'0 6'0 8'0 160 TIME (rain)

Bac Bac

Bac

�9 RF 97 /~A

() 2 '0 4 Io 60 8'0 1 (] 0 T I M E ( r a i n )

75 ms before the test pulse. Note virtual lack of changes in DRPs and P waves produced up to 60 min after the first of 2 injections of 1 mg/kg of (-)-baclofen (arrows in C). Further explanation in text

magnitude and time course of the depressive action of ( - ) -bac lo fen varied between the different fibers (Fig. 7B). Although the number of fibers examined was relatively small, it appeared that the larger the initial threshold reduction produced by the conditioning input (SU in this case), the slower and less effective the depressive action of ( - ) -bac lofen . Table 1 shows that, before the injection, SU stimulation reduced the intraspinal threshold of all the sample of Ib fibers to a mean value of 86 + 10%. Thirty minutes after ( - ) -bac lo fen , the effect was reduced to a mean value of 934- 10%.

PAD produced by reticulospinal fiber s

In contrast to the high sensitivity to ( - ) - bac lo f en of the PAD produced by stimulation of sensory nerves, we found that the PAD produced by stimulation of the RF was, in general, more resistant to this GABAb agonist. Figure 5 illustrates the effects of ( - ) -bac lo fen on the CDPs and DRPs produced by a train of 18 pulses at 700 Hz applied to the gigantocellular reticular formation

in the brainstem. These data were derived from the same experiment as that illustrated in Figs. 1 and 4. It can be seen that, in confirmation of early observations (Lund- berg, 1964), stimulation of the RF produced a negative DRP and two positive waves in the cord dorsum. The first positive wave in the CDP probably represents the synaptic activation of neurons deep in the spinal cord and the second positive wave appears to be generated during PAD (see Fig. 9A). As shown in Fig. 5A, B, in contrast with the depression of CDPs and DRPs produced in the same experiment by stimulation of muscle and cutaneous nerves, the CDPs and the DRPs produced by stimulation of the RF were not significantly affected during the first 60 rain following the first of two injections of ( - ) -bac lofen . After the third injection, there was some depression of the responses, to 60% and 80% of the control amplitude of the DRPs and CDPs, respectively (Fig. 5C).

A similar resistance to the action of ( - ) -bac lo fen was found when testing the changes produced by RF stimulation on the intraspinal threshold of single Ib fibers, as illustrated in Figs. 2, 3, 5D and 7C. In the experiment

illustrated in Fig. 2, before the injection of ( - ) -bac lo fen , stimulation of the RF reduced the intraspinal threshold of the fiber to about 85% of control (Fig. 3A). Twenty minutes after the first injection of ( - ) -bac lo fen , the PAD produced by RF stimulation of this single Ib fiber was still appreciable (about 85.7%; Fig. 2B). For ty minutes after ( - ) -bac lo fen , the threshold reduction produced by RF stimulation was practically the same (84.2%), even though the effects produced by PBSt and SU stimulation were practically abolished (Fig. 2C). The time course of the effects of ( - )-baclofen on the PAD (threshold reduction) produced by RF stimulation in 6 single Ib fibers is shown in Fig. 7C. Only in one fiber did ( - ) - bac lo f en readily reduce and finally abolish the PAD produced by RF stimulation. In the other five fibers, the depression of the PAD was less severe. In fact, 60-90 min after the first injection of ( - )-baclofen, RF stimulation still produced an appreciable PAD.

The different sensitivity to ( - ) - bac lo f en of the PAD of segmental and descending origin is further documented in Fig. 3. This is a particularly interesting observation because we were able to examine the intraspinal threshold changes produced by ( - ) -bac lo fen on two different Ib fibers simultaneously. Before (--)- baclofen, stimulation of the RF with trains of pulses of 80 and 120 gA produced a significant PAD (threshold reduction) in

37

both fibers. The effect of RF stimulation was clearly smaller than the threshold reduction produced by PBSt stimulation (Fig. 3A). Three minutes and 25 min after ( - ) -bac lo fen , RF volleys still produced an appreciable PAD in both fibers, but was stronger for the St Ib fiber than for the GS Ib fiber (Fig. 3D, E). After the second injection of ( - ) -bac lo fen , PBSt stimulation produced no PAD in the GS fiber but RF stimulation still produced a clear PAD in both Ib fibers (Fig. 3F, G).

Table 1 summarizes the results obtained from all experiments. In general, these data confirm the results illustrated in Figs. 2, 3, 5 and 7C; namely, the PAD produced by RF stimulation appears to be more resistant to ( - ) - b ac lo f en than the PAD produced following stimulation of muscle and cutaneous afferents.

PAD produced by intraspinal microstimulation

We have shown previously that microstimulation within the intermediate nucleus, with strengths below those required to activate the test afferent fiber, may produce PAD with a rather short onset (0.7-1.0 ms), consistent with a monosynaptic delay (Jankowska et al. 1981). It has been assumed that PAD elicited under these con- ditions is due to direct activation, by the intraspinal

100 ........................................................................................................................................................................................................................... �9 ~" 100-

~ I r / " 90 d o o 90

80 w

, z 80 j 70 J 0 0

/ t / 7< <

E 60J Ck:) .0 "0 z 70 I "o 0 CONTROL ~n~ ] � 9 offer Bac <

50

- 0 2'0 4'0 6'0 8'0 z

A INTERVAL (ms) B

IOO-

~ 95-

m~ 90 W

_~ 85 �84 b-

d < 80 Z

B.. u~ 75 < rw

~- 70 Z

C

t Bac

2'0 4'0 6'0

TIME (ms)

1" Bac

8'o ' ioo

60

\ \ ^

Oo-~ o

0 CONTROL �9 15' after Bac A 45' after Bac

6 �89 4 6 8 1[] 1'2 1~4

INTENSITY (#A)

Fig. 6A-C. The monosynaptic PAD of single Ib fibers that is produced by intraspinal microstimulation is (-)-baclofen sensitive. A The time course of the reduction of the intraspinal threshold of a single Ib fiber produced by a single conditioning pulse (4.5-6 laA) applied through the same micropipette used for the intraspinal threshold measurements. Open circles before, filled circles 80 rain after a first injection of 1 mg/kg (-)-baclofen and 35 rain after a second injection of the same amount. In all cases the intensity of the intraspinal conditioning pulse was below the activation threshold of the fiber. B Changes in intraspinal threshold of another Ib fiber produced by graded intraspinal conditioning stimulation. Conditioning-testing stimulus interval 5 ms. Open circles, control; filled circles and open triangles, 15 and 45 min after the i.v. injection of 2 mg/kg ( - )-baclofen. C Time course of the ( - )-baclofen-induced depression of the PAD produced by intraspinal microstimulation (4.7 ~tA) in a third single Ib fiber (same as that in Figs. 1D, 4D and 5D). Con- ditioning-testing stimulus interval 5 ms. Arrows, times of (-)-baclofen injections (1 mg/kg each). Further explanation in text

38

100-

90-

80-

E-~

70- z

m 60-

z 50

A

PBSt �9 o - . �9 �9 �9

�9 o / ~

�9

V D rn [] V 1.5 / �9 2.5

[] [] 1.6 �9 1.8

, , , , , , , , , ,

0 20 40 60 80

0

E-.

z E r / l

z

TIME (rain) B

RF

tO0'

90. 0

,~ 80.

70.

m 60'

Z - 50

........ ~ - - v ....................... v .......................... v ~ 100.

o,.~ �9 0 - - 0 - - ~ ~ o ,a 90'

~-I B O .

v lOO ~ 60. �9 17o [] 172 �9 172 Z 511

, , , , , , , , , ,

0 20 40 60 80

C TIME (min) D

Fig. 7. Time course of the effects of ( - ) -bac lofen on the PAD (threshold reduction) of individual Ib fibers produced by segmental and descending inputs and by intraspinal microstimulation. A Ef- fects on PAD produced by stimulation of group I fibers in the PBSt nerve. Conditioning stimulus was a train of four pulses at 300 Hz preceding the threshold testing pulse by 25 ms. Stimulus strength is indicated in the graph. B Same format as in A. PAD was produced by stimulation of the SU nerve (one shock preceding test stimulus by 35 ms at the indicated strengths, except in one case where conditioning stimulus was a train of seven pulses at 300 Hz (filled

t l 0 �9

1 0 0 .

V

00. 4

80-

70.

6O

5O

SURAL

,/ �9 0 2xT

V3 �9 l 0 [] 16 �9 16

I0 2'0 3'0 4'0 50 6'0

TIME (min)

MICROSTI MULATION

V ,-~-~--~ ~ V , : _ , ~ O

e/ \c~~ / - o / / - v

O 5.0 ~,A �9 5.5 V 5.56 �9 5.5 n 8.16

. . , , . , . , , ,

0 20 40 60 8 0

TIME (rain)

inverted triangles). C Effects on PAD produced by stimulation of the bulbar RF (eight pulses at 700 Hz applied 75 ms before the test stimulus at the indicated strengths). D Effects of ( - ) -bac lofen on PAD produced by intraspinal microstimulation (one pulse applied 3-5 ms before the test pulse through the same micropipette at the indicated strengths). In all graphs each symbol corresponds to a different Ib fiber. Fibers labeled with open and filled squares are from the same experiment (see Fig. 3). Values at zero time were control attained before the injection

microstimulation, of last-order GABAergic interneurons synapsing with afferent fibers (Rudomin et al. 1983). The PAD produced by intraspinal microstimulation attains maximal values at around 20 ms and lasts up to 100 ms. Suppression of sodium-dependent neuronal activity by i.v. tetrodotoxin does not appear to shorten the duration of the PAD produced by intraspinal microstimulation, a finding suggesting that the slow time course of the PAD is not due to repetitive activity of interneurons, but rather represents the slow dynamics of the GABA release, or action (Rudomin and Mufioz- Martinez, 1969). The PAD produced by intraspinal microstimulation can be detected not only by intrafiber recording of the PAD, as was done by Jankowska et al. (1981), but also by testing the effects on the intraspinal threshold of single afferent fibers. The threshold reduc-

tion produced when the intraspinal microstimulation precedes the excitability testing stimulus by 0.7-1.2 ms is probably due to direct activation of the final-order interneurons mediating the PAD (Rudomin et al. 1983). At longer conditioning-testing stimulus intervals (3-5 ms), it is possible that the final-order interneurons are not only activated directly, but also transynaptically. That is, in addition to a monosynaptic PAD, there could be some polysynaptic PAD elicited by concurrent activation of afferent fibers and/or first-order interneurons (Rudomin et al. 1983).

As indicated by the open circles in Fig. 6A, in the present series of experiments, intraspinal microstimulation was able to reduce the threshold of the single Ib fiber for a wide range of conditioning-testing stimulus intervals. This threshold reduction had a brief onset (1 ms),

attained maximal values at about 20 ms and lasted more than 80 ms. Eighty minutes after the first injection of 1 mg/kg ( - )-baclofen and 35 rain after a second injection of the same amount, there was a marked reduction in the effects produced by the intraspinal microstimulation at all conditioning-testing time intervals, including the shortest intervals ( < 1.5 ms, filled circles).

In the experiment illustrated in Fig. 6A, ( - ) -baclofen depressed the effects of intraspinal microstimulation to half. However, in other experiments, such as that in Fig. 2 (see also Figs. 3, 6C and 7D), the effects produced at a short conditioning-testing stimulus interval (3-5 ms) were completely abolished by (-)-baclofen. This needs to be stressed because it indicates that even though at these relatively short intervals the monosynaptic PAD were contaminated with a polysynaptic component, both of them were suppressed.

Further evidence supporting the view that during intraspinal microstimulation there is little coactivation of afferent fibers that may produce a significant polysynaptic PAD is provided by the experiment shown in Fig. 3. In this experiment, we made simultaneous measurements of the intraspinal threshold of two different Ib fibers. The micropipettes were inserted to converge at the intermediate nucleus and calculations indicated that the tips were separated by about 1.5 mm. Intraspinal microstim-

39

ulation, applied via the micropipette used to determine the threshold of the GS fiber, produced PAD (threshold reduction) in the GS fiber but not in the St fiber (Fig. 3B). A pulse of 6.8 gA applied 5 ms before the intraspinal threshold testing pulse produced a clear PAD. The PAD increased when stimulus strength was raised to 8.2 gA, still below the firing threshold of the GS fiber. Pulses of 10 gA activated the GS afferent fiber and also produced a stronger PAD. Figure 3C shows, in addition, the threshold reduction produced at three conditioning-testing stimulus intervals (3, 5 and 10 ms). It can be seen that PAD was elicited when the conditioning-testing stimulus interval was as short as 3 ms, and that at 5 and 10 ms there was also a clear PAD. As shown in Fig. 3E, the PAD of the GS fiber produced by intraspinal microstimulation with pulses of 6.8 and 7.7 gA was virtually abolished 25 min after (-)-baclofen and there were no significant changes in the resting threshold of the GS fiber. Before the injection of this GABA b agonist, intraspinal microstimulation with pulses of 6.8 gA produced a clear PAD (Fig. 3B).

What is remarkable in this experiment is that before (-)-baclofen, intraspinal stimulation, even with the highest intensities (10 gA), failed to produce PAD in the St fiber. This suggests that PAD produced in the GS fiber by intraspinal microstimulation is probably not due to

Table 2. Effects of picrotoxin on P A D of segmental and supraspinal origin

Time PBSt (1.5-3 x T) PBSt (4~5 x T)

(min) Aft DRP P wave IT Aft D R P (%) (%) (%) (%) (%) (%)

P wave IT (%) (%)

R F (50-130 gA)

D R P CDP1 CDP 2 IT (%) (%) (%) (%)

Control

M 100 100 100 91.8 100 100 100 86.6 SD - - - 8.2 2.9 n 3 3 2 7 2 2 2 4

Picrotoxin 0.5 mg/kg

3 M 91.5 87.6 92.6 97.2 97.6 77.4 66.0 93.5 SD 3.5 9.4 11.1 2.7 3.3 10.9 9.5 4.5 n 3 3 2 7 2 2 2 4

10 M 90.1 60.0 57.8 96.8 100 63.3 62.9 SD 3.2 17.9 8.0 2.7 0 15.6 1.1 - n 3 3 2 3 2 2 2 -

15 M 83.9 63.2 64.0 95.7 80.0 63.7 62.7 92.1 SD 6.2 20.7 10.7 7.5 0 16.2 17.8 5.8 n 3 3 2 7 2 2 2 4

Picrotoxin 0.5 mg/kg

30 M 90.8 58.7 44.7 92.6 92.8 54.6 44.3 91.8 SD 8.0 16.5 7.9 4.6 23.5 15.6 11.3 1.4 n 3 3 2 7 2 2 2 4

60 M 74.3 27.9 31.2 97.9 92.8 53.9 54.2 89.5 SD 11.5 27.2 4.9 8.8 23.5 14.7 12.3 9.0 n 3 3 2 7 2 2 2 4

75 M 62.5 16.6 15.6 97.7 - - 95.8 SD - - 6.0 - - 4.5 n 1 1 1 7 - 4

100 100 100 87.7 - 11.2

3 3 3 5

94.7 104.8 72.9 - 6.5 12.2 24.1 - 3 3 3 -

78.8 102.4 74.0 92.4 5.8 9.1 20.2 8.6 3 3 3 5

74.0 86.7 68.9 98.0 19.0 9.5 22.4 3 3 3 1

72.7 105.3 71.4 92.1 11.3 5.0 33.5 6.9 3 3 3 4

68.7 102.9 59.9 94.5 3.5 2.9 39.9 4.0 3 3 3 5

37.4 110.8 58.6 95.8 15.1 18.4 42.3 6.6 3 3 3 5

Aft, afferent spike. DRP, dorsal root potential. P wave, positive wave recorded from cord dorsum following PBSt stimulation. CDP I and CDP2, first and second positive component in the cord

dorsum response produced by RF stimulation. IT, changes in intraspinal threshold of a single Ib fiber relative to resting threshold ; M, mean; SD, standard deviation; n, number of observations.

40

activation of a significant number of afferent fibers because, if this were the case, the St fiber should have also shown PAD, as it did following PBSt stimulation (Fig. 3A). Rather, it seems that the PAD produced by intraspinal microstimulation, particularly at short conditioning-testing stimulus intervals, is due to activation of a restricted number of last-order interneurons mediating the PAD.

Figure 6B illustrates another feature of the effects of ( - ) -baclofen on the PAD produced by intraspinal microstimulation. In this experiment, the conditioning-testing stimulus time interval was kept at 5 ms, and the intensity of the conditioning intraspinal microstimulation was varied over a relatively wide range (1-10 gA), in all cases below the strength required to antidromically activate the test afferent fiber. Before the injection of ( - ) -baclofen, the intraspinal microstimulation reduced the threshold of the Ib fiber. This effect grew with in- creasing stimulus strengths. However, the threshold reduction appeared to occur in two steps (Fig. 6B open circles). Fifteen minutes after the injection of 2 mg/kg (-)-baclofen, the input-output curve was displaced to the right and intraspinal stimuli below 5 gA became totally ineffective (Fig. 6B, filled circles). The reduction in the effectiveness of the intraspinal microstimulation was still observable 45 min after the injection of ( - )-baclofen (Fig. 6B triangles). It thus seems that in this experiment the i.v. injection of ( - ) -baclofen abolished the PAD produced by intraspinal microstimulation with

relatively low strengths (i.e., below 4 gA). With higher strengths of intraspinal microstimulation, PAD could be again elicited, but this PAD does not appear to be depressed by further injections of (-)-baclofen.

The effects of ( - ) -baclofen on the PAD produced in single Ib fibers by intraspinal microstimulation were examined in detail in five experiments (Fig 7D; see also Fig. 6C). These data were obtained with a conditioning- testing stimulus interval of 5 ms, to restrict the effects to monosynaptically generated PAD of the fiber, although, as discussed above, some polysynaptic activation cannot be excluded (see Jankowska et al. 1981 ; Rudomin et al. 1981, 1983). In all cases, the injection of ( - ) -baclofen decreased the PAD produced by intraspinal microstimulation. It may be seen that in four Ib fibers the PAD was severely reduced after ( - ) -baclofen and only in one Ib fiber was the PAD (threshold reduction) more resistant. As shown in Table 1, before the injection of ( - ) -ba - clofen, intraspinal microstimulation reduced the threshold of all the examined fibers to a mean value of 78 4- 11%. One hour after the injection of (-)-baclofen, the mean reduction in the threshold of the fibers was only to 91 4- 10%.

Drawing together all of the above results, it seems reasonable to conclude that ( - ) -baclofen, in addition to the action it may have on the synaptic effectiveness of afferent fibers, also depresses the monosynaptic PAD of Ib fibers produced by direct activation of the last-order interneurons mediating the PAD.

CONTROL 10' AFTER PTX

CDP

PBSI 4 x T ~

A B

W 123

I - -

._1

D

120 PT•

IO0

80

60

40

2O

0

PTX PTX

2'0 4'0 6'0

TIME

[]

PBSf 2xT �9 PBSf 3xT �9 PBSf 4xT [] PBSf 5xT

80 100 120 140

v

d O 212 O3

W n~

-1-

-J

Z

13-

O3

F-

Z

110 PTX

IO0

9O

80

7O

60

0

E

80' AFTER PTX

O

C ' ' 50 ms

PTX PTX

20 40 60

TIME

�9 PBSf 3xT �9 PBSt 4xT

PHS~" 5xT

80 100 120

Fig. 8A-E. Effects of picrotoxin on PAD of segmental origin. A-C DRPs and P waves produced by four pulses at 300 Hz applied to the PBSt nerve (4 x T) : A Con- trol; B 10 min after the injection of 0.5 mg/kg picrotoxin (PTX); C 80 min after the first of three injections of PTX (injection times are indicated in D). D Time course of amplitude changes of the DRPs partly illustrated in A-C and of DRPs produced by other stimulus strengths, as indicated. E Time course of the action of (-)-baclofen on the reduction of intraspinal threshold of a single 36 fiber produced by PBSt conditioning stimulation with several intensities, as indicated. Arrows, times of PTX injections (0.5 mg/kg each time). Further explanation in text

Effects of picrotoxin

The results described in the preceding sections indicate that the PAD produced by stimulation of the RF is more resistant to (-)-baclofen than the PAD produced by stimulation of muscle and cutaneous afferents. Although there is reasonably good evidence indicating that activation of GABA, receptors underlies the PAD produced by group I muscle and cutaneous afferents (Eccles et al. 1963; Rudomin et al. 1981; Curtis and Lodge 1982; Curtis et al. 1982), there is limited information on the transmitters involved in the generation of PAD of afferent fibers following stimulation of the reticular formation (Benoist et al. 1974; Jim~nez et al. 1987). In several experiments, we analyzed the effects of picrotoxin, a well known GABA, antagonist (Eccles et al. 1963). The results obtained in one experiment are illustrated in Figs. 8 and 9, but similar results were obtained in another four experiments (see Table 2). Following the first of two i.v. injections of picrotoxin (0.5 mg/kg), there was a clear depression of the DRPs produced by stimulation of muscle afferents (Fig. 8B) as well as by the RF (Fig. 9B). The depression of the PBSt DRPs was already noticeable 10-15 min after the injection and was at its greatest at 30 min, shortly after the second injection (0.5 mg/kg), when they were 50% of the control values (Fig. 8C, D). The DRPs produced by stimulation of the RF with strengths ranging from 90 up to 185 gA were also depressed (to 70% of control amplitude), but later there

41

was an additional depression, to 30% of control values (Fig. 9C, D).

Picrotoxin also depressed the PAD produced in single Ibfibers by PBSt or RF stimulation. Before the injection, a train of four pulses at 300 Hz (2-5 x T) applied to the PBSt nerve 25 ms before the test pulse produced a clear reduction in the intraspinal threshold of the Ib fiber (to 85 % of the control value; see Fig. 8E). Stimulation of the RF with a train of eight pulses at 700 Hz, with intensities ranging from 90-161 gA appeared to be more effective (75 % of control; Fig. 9E). Ten minutes after the injection of picrotoxin (0.5 mg/kg), the effects of PBSt and RF stimulation were already smaller, and 1 h later PBSt and RF stimulation reduced the intraspinal threshold of the fiber to only 98% and 92% of the control value, respectively. Depression of the PAD was seen for at least 2 h after the first injection of picrotoxin.

Table 2 summarizes the observations made in all experiments and shows that picrotoxin was, indeed, able to depress the PAD elicited by group I muscle afferents and by reticulospinal fibers. It should be noted that the PBSt afferent volley was also reduced following the injection of picrotoxin, but the depression of the DRPs and P waves appears to be larger. On the other hand, the first positive response recorded from the cord dorsum following stimulation of the RF (CDP1; see Fig. 9) appeared not to be significantly altered following the injection of picrotoxin, whereas the second wave (CDP2) was depressed to about the same extent as the DRPs. The DRPs

CONTROL 12' AFTER PTX

CDP

COP M,A

A B

r

I--

._1 O_ 2~ <

D

120 PTX

100

80

6O

40

20

0

PTX

2'0

PTX ~9, 110

r~ J 100 0 I #3

/~7~ rq w 9 0

8 0 _J

�9 RF 90 /~A -- 70 ~7 RF 137/~A (/) V RF 161 /~A ~E [] RF 185/~A rt~ 60

I.--

8'0 100 120 1;0 Z

(min) E

,b 8'0 TIME

8 2 ' AFTER PTX ]

J

>

O O cN

i i

C 5 0 ms

PTX PTX PTX

i 0 2'0 40

�9 R F 90 /~A ~7 RF 137/~A �9 RF 161 /~A

i i 60 80 100 120

TIME (min )

Fig. 9A-E. Effects of picrotoxin on PAD produced by stimulation of the RF. Same experiment and format as that in Fig. 8. RF stimulation was a train of eight pulses at 700 Hz, at the indicated strengths

42

produced by RF stimulation appeared to be somewhat more resistant to picrotoxin than the DRPs produced by PBSt stimulation, but these differences appear not to be significant.

In confirmation of previous observations (Rudomin et al. 1983), we found that in three Ib fibers, picrotoxin also reduced the monosynaptic PAD produced by intraspinal microstimulation. The time course of the depression of the monosynaptic PAD was similar to that of the depression of the PAD produced by stimulation of sensory nerves (not illustrated).

Discussion

The PAD produced in Ib fibers by descending inputs is less sensitive to ( - )-baclofen than the P A D elicited by sensory fibers

The observations reported here refer specifically to the action of (-)-baclofen on the PAD elicited in Ib afferents. These afferents were selected for the present investigation because, unlike Ia fibers, they are depolarized by group I fibers as well as by cutaneous and reticulospinal fibers (Rudomin et al. 1983, 1986; Jim6nez et al. 1988). This has allowed comparison of the action of (-)-baclofen on the PAD elicited by different inputs on the same Ib afferent fiber. Our data indicate that the PAD of Ib fibers that is produced by stimulation of group I muscle and by cutaneous afferents is readily depressed, and often abolished, following the injection of (-)-baclofen, a GABAb agonist (Price et al. 1984). In contrast, the PAD produced by stimulation of the bulbar reticular formation appears to be more resistant to (-)-baclofen than the PAD produced by stimulation of the afferent fibers.

We have recently examined the action of (-)-baclofen on monosynaptic EPSPs produced in motoneurons by Ia and descending fibers (Jim6nez et al. 1991), and found that the synaptic actions produced by Ia afferents are clearly more susceptible to depression by (-)-baclofen than the monosynaptic responses produced by descending fibers (see also Edwards et al. 1989). This has been explained by assuming that the intraspinal terminals of the afferent fibers have a higher density of GABAb receptors than the terminals of the descending fibers (Jim6nez et al. 1991). The same explanation appears to account for the lower sensitivity to (-)-baclofen of the PAD produced by RF stimulation relative to the PAD produced by segmental inputs documented in the present series of investigations. However, as discussed below, this may not be the only factor explaining the differential sensitivity, because (-)-baclofen also appears to act on the final-order interneurons mediating the PAD of Ib fibers.

The final-order GABAergic interneurons mediating the PAD of segmental origin have GABA b autoreceptors

We have found that (-)-baclofen depresses not only the PAD elicited by segmental inputs but also the PAD

produced by intraspinal microstimulation. As discussed in Results, at short conditioning-testing stimulus intervals (< 5 ms) the PAD elicited in Ib fibers by intraspinal microstimulation appears to be mostly due to direct activation of the last-order interneurons (i.e., monosynaptic), although there could be a small polysynaptic component as well. Depression of the polysynaptic components of the PAD produced by intraspinal microstimulation can be explained by assuming that the PAD was elicited by activation of afferent fibers (and/or first order interneurons) and that (-)-baclofen reduced their synaptic efficacy. On the other hand, depression of the monosynaptic PAD could be due to prevention of GABA release from the last-order GABAergic interneurons, because (-)-baclofen appears to have no effect on the intraspinal excitability of afferent fibers (see Figs. 2 and 3 and also Curtis et al. 1981, 1986). It thus seems reasonable to suggest that the last-order interneurons mediating the PAD have GABAb receptors.

The spatial distribution of the GABA b receptors in the last-order interneurons mediating the PAD is unknown. As a consequence, it is not clear whether (-)-baclofen

Ib f i be r A

RF Gr.

~ ~ su

2Hm

RF Gr. l [. PBSt

SU

• GABA a ~st im.

�9 GABA b , ' ! �9 / / '5," / /

/

Ib fiber B

Fig. 10A, B. Diagram of the neuronal connections explaining the results. A The first-order interneurons that are activated by segmental and descending inputs end on common last-order GABAer- gic interneurons. B First-order interneurons receiving RF and segmental inputs have projection patterns on the population of last- order GABAergic interneurons that are related to the density of GABAb receptors. Thickness of axonal branches in first-order interneurons indicates density of projections of last-order interneurons. Size of triangles is proportional to density of receptors. Further explanation in text

43

acts by hyperpolarizing the cell bodies and proximal dendrites of these interneurons, thus decreasing their capability to generate and conduct action potentials (see for example Wang and Dun 1990), or by decreasing calcium currents and transmitter release at their axonal terminations (as indicated in Fig. 10; Robertson and Taylor 1986). The first possibility seems unlikely in view of the observations of Alford and Grillner (1991) who showed that (-)-baclofen may be relatively ineffective in preventing spike initiation in neurons in the lamprey spinal cord. The presence of GABAb receptors in the intraspinal terminals of last-order GABAergic interneurons mediating the PAD would act as a negative feedback system controlling transmitter release as has been described in other systems (for review see Starke et al. 1989). This appears to be one of the first available examples of GABAb autoreceptors in spinal interneurons. However, strictly speaking, an autoreceptor function of GABA b receptors at GABAergic terminals of interneurons remains to be demonstrated.

The final-order interneurons mediatin9 the PAD of segmental and descending origin are GABAergic

Although there is reasonably good evidence suggesting that the PAD elicited in Ia and Ib fibers by stimulation of segmental afferents is mediated by GABAergic interneurons (Eccles et al. 1963; Rudomin et al. 1981 ; Curtis and Lodge 1982; Curtis et al. 1986), the transmitters mediating the PAD generated in Ib fibers by supraspinal stimulation have not been fully characterized. In the barbiturate-anesthetized cat, topical application of picrotoxin on the spinal cord depresses the DRPs produced by segmental and cortical stimulation (Benoist et al. 1974), as would be expected if GABA were the transmitter involved in the generation of the PAD. More recently, Jim6nez et al. (1987) have shown that picrotoxin depresses the PAD of cutaneous fibers that is produced following stimulation of the red nucleus and of the reticular formation. The evidence provided here indicates, additionally, that the PAD elicited in Ib fibers, both by segmental and RF stimulation, is also picrotoxin sensitive (Figs. 8 and 9). Therefore, we suggest that the final-order interneurons mediating the PAD produced in Ib fibers by segmental as well as by reticulospinal inputs are GABAergic.

Segmental and descending inputs have different convergence patterns on last-order interneurons mediating the PAD of Ib fibers

As a first approximation, the differential sensitivity to (-)-baclofen of the segmental and descending PAD can be explained by assuming that afferent fibers have a higher density of GABAb receptors than descending fibers and that the last-order interneurons mediating the PAD (which also have GABAb receptors) receive conver- gent inputs from descending and segmental pathways, as indicated in Fig. 10A.

Convergence of segmental and descending pathways on common last-order interneurons mediating the PAD is suggested by the finding of non-linear interactions between the PAD of segmental and descending origin and the monosynaptic PAD elicited by intraspinal microstimulation (Rudomin et al. 1983). However, this could be due to saturation of the PAD elicited at the level of the afferent fiber. More direct evidence on convergence, at an interneuronal level, has been provided by Rudomin et al. (1987), who showed that last-order interneurons presumably mediating the PAD of Ib fibers (type C interneurons) receive inputs from muscle and cutaneous afferents, as well as from reticulospinal fibers.

However, the simple model of afferent and descending fibers with different densities of GABAb receptors con- verging onto common last-order interneurons mediating the PAD (as in Fig. 10A) may not explain the differential sensitivity to (-)-baclofen of the PAD produced by the various inputs, particularly the persistence of PAD produced by RF stimulation once the PAD produced by segmental stimulation and by intraspinal microstimulation has been abolished (Fig. 2), or the selective depression of the PAD produced by PBSt group I volleys in one Ib fiber but not in the other (Fig. 3).

It thus seems reasonable to assume that more than one last-order interneuron synapses with a single afferent fiber and that the various pathways producing the PAD of a given afferent fiber do not all converge on the same last-order interneuron mediating the PAD. That is, the last-order interneurons mediating PAD receive different proportions of segmental and descending inputs, as is the case with the Ib interneurons mediating the nonrecipro- cal postsynaptic inhibition (Harrison and Jankowska, 1985).

The diagram in Fig. 10B was constructed to explain our results and is based on the following assumptions: (a) that there is a wide variation in the density of GABAb receptors in the terminal arborizations of the last-order interneurons synapsing with Ib fibers, and (b) that segmental pahways may have preferential connections with last-order interneurons with a high density of GABAb receptors, whereas descending pathways have preferential connections with last-order interneurons with a low density of GABA b receptors. One additional assumption would be that the cell bodies of the last-order interneurons with a low density of GABA b receptors have a higher threshold to electrical stimulation than final-order interneurons with higher density of GABAb receptors. This would probably explain our inability to activate, with low stimulus strengths, the "(-)-baclofen-resistant" last-order interneurons (see below).

The first assumption seems reasonable in view of available information. However, at the present time there is no evidence supporting the second assumption, namely the existence of differential patterns of connectivity of RF and segmental pathways with last-order interneurons mediating the PAD that are related to their density of GABAb receptors. Therefore, we must regard the explanation illustrated in Fig. 10B as highly speculative. Nevertheless, we feel it is of some interest and may lead to experiments aimed to test this possibility.

44

The two-step increase in the PAD elicited by graded intraspinal microstimulation, illustrated by the open circles in Fig. 6B, also suggests that more than one last- order interneuron converges onto the same Ib afferent fiber. This is in agreement with a recent proposal made by Maxwell et al. (1990) in relation to anatomical observations made on Ia fibers. Considering the suggestion made above, that more than one set of last-order interneurons could mediate the P A D of Ib fibers of segmental and of descending origin, it seems tempting to conclude that the two steps seen in the PAD produced by intraspinal microst imulation were due to the recruitment of the interneurons mediating the P A D of afferent and descending origin, respectively, in agreement with what is illustrated in Fig. 10B. However, it should be pointed out that discrete steps in the PAD produced by intraspinal microstimulation have not been observed in every case (see Rudomin et al. 1981). This may depend on the location of the stimulating micropipette relative to the populat ion of final-order interneurons mediating the P A D of Ib fibers, and it may not be possible to decide, on the basis of the relative thresholds of the various components in the monosynapt ic PAD, which kind of interneurons were activated.

Concluding remarks

The possibility that segmental and descending inputs may have different convergence patterns on last-order interneurons mediating the PAD of Ib fibers comple- ments our previous proposal that different last-order interneurons mediate the PAD of Ia, of Ib and of cutaneous fibers (Jankowska et al. 1981 ; Rudomin et al. 1983, 1986), and stresses the selectivity of the presynaptic control of the synaptic efficacy of muscle afferents (Rudomin et al. 1987; Rudomin 1991).

In humans with partial spinal cord lesions, spasticity is reduced following the intrathecal administrat ion of ( - ) - b a c l o f e n but with little effect on the voluntary control of movements (Burke et al. 1971 ; Latash et al. 1989). The present results, taken together with previous investigations (Edwards et al. 1989; Jim6nez et al. !991), suggest that the relief of spasticity is due, at least in part , to a reduction in the synaptic effectiveness of afferent fibers, together with decreased transmission of impulses in specific subsets of GABAergic interneurons. The persistence of voluntary control of muscle activity may be explained because ( - ) - b a c l o f e n affects, to a lesser degree, the synaptic efficacy of descending pathways, including some of the last-order interneurons mediating presynaptic inhibition of descending origin.

I t is tempting to suggest that the presynaptic inhibition of descending origin continues to play some role in the presence of ( - ) - b ac l o f en , by acting on those afferent fibers whose synaptic efficacy was not completely abolished by the drug. It should be noted that the last- order interneurons mediating the P A D of segmental origin appear to have direct inhibitory connections with motoneurons (Rudomin et al. 1987), and so, even in the absence of an effective afferent input, there could be an

inhibitory GABAergic descending control on motoneurons which may be relevant for motor performance (Rudomin 1991).

Acknowledgements. We would like to thank Drs. E. Jankowska and L. Mendell for their valuable comments on the manuscript, J. Gon- zfilez and C. Rodriguez for the implementation of the computer programs required for data acquisition and processing, A. Rivera and C. Ledn for technical support and Ciba-Geigy, Basel, Switzer- land, for the generous gift of (-)-baclofen~ This work was partly supported by NIH grant NS 09196, CONACyT grant PCEXCCNA 41739 and the Sistema Nacional de Investigadores, M6xico.

References

Alford S, Grillner S (1991) The involvement of GABA b receptors and coupled G-proteins in spinal GABAergic presynaptic inhibition. J Neurosci 11 : 3718-3726

Benoist JM, Besson JM, Boissier JR (1974) Modifications of presynaptic inhibition of various origins by local application of convulsant drugs on cat's spinal cord. Brain Res 71 : 172-177

Burke D, Andrews CJ, Knowles L (1971) The action of a GABA derivative in human spasticity. J Neurol Sci 14:199-208

Carpenter D, Lunberg A, Norrsell U (1963) Primary afferent depolarization evoked from the sensory motor cortex. Acta Physi- ol Scand 59:126-142

Curtis DR, Lodge D (1982) The depolarization of feline ventral horn group Ia spinal afferent terminations by GABA. Brain Res 46:215-233

Curtis DR, Malik R (1984) The effect of GABA on lumbar terminations of rubrospinal neurons in the cat spinal cord. Proc R Soc Lond B223 : 25-33

Curtis DR, Malik R (1985) The differential effects of Baclofen on segmental and descending excitation of spinal interneurons in the cat. Exp Brain Res 58 : 333-337

Curtis DR, Lodge D, Bornstein JC, Peet MJ (1981) Selective effects of baclofen on spinal synaptic transmission in the cat. Exp Brain Res 42:158-170

Curtis DR, Lodge D, Bornstein JC, Peet MJ, Leath JU (1982) The dual effects of GABA and related aminoacids on the electrical threshold of ventral horn group Ia afferent terminations in the cat. Exp Brain Res 48:387400

Curtis DR, Wilson VJ, Malik R (1984) The effect of GABA on the terminations of vestibulospinal neurons in the cat spinal cord. Brain Res 295: 372-375

Curtis DR, Gynther BD, Malik R (1986) A pharmacological study of group I muscle afferent terminals and synaptic excitation in the intermediate nucleus and Clarke's column of the cat spinal cord. Exp Brain Res 64:105-113

Eccles JC, Magni F, Willis WD (1962) Depolarization of central terminals of group I afferent fibres from muscle. J Physiol (Lond) 160: 62-93

Eccles JC, Schmidt RF, Willis WD (1963) Pharmacological studies on presynaptic inhibition. J Physiol (Lond) 168:500-530

Edwards FR, Harrison P J, Jack JB, Kullman DM (1989) Reduction by baclofen of monosynaptic EPSPs in lumbosacral motoneurones of the anaesthetized cat. J Physiol (Lond) 416:53%556

Harrison PJ, Jankowska E (1985) Organization of input to the interneurones mediating group I non-reciprocal inhibition of motoneurones in the cat. J Physiol (Lond) 361:403-418

Harrison PJ, Jankowska E (1989) Primary afferent depolarization of central terminals of group II muscle afferents in the cat spinal cord. J Physiol (Lond) 411 : 71-83

Jankowska E, McCrea D, Rudomin P, Sykova E (1981) Observa- tions on neuronal pathways subserving primary afferent depolarization. J Neurophysiol 46: 506-516

Jim~nez I, Rudomin P, Solodkin M (1987) Mechanisms involved in the depolarization of cutaneous afferents produced by segmental and descending inputs in the cat spinal cord. Exp Brain Res 69 : 195-207

45

Jim6nez I, Rudomin P, Solodkin M (1988) PAD patterns of physi- ologically identified afferent fibers from the medial gastrocnemius muscle. Exp Brain Res 71:643-657

Jim6nez I, Rudomin P, Enrlquez M (1991) Differential effects of (-)-baclofen on Ia and descending monosynaptic EPSPs. Exp Brain Res 85:103-113

Kato M, Waldman U, Murakami S (1978) Effects of baclofen on spinal neurones of cats. Neuropharmacol 17:827-833

Latash ML, Penn RD, Corcos DM, Gottlieb GL (1989) Short-term effects of intrathecal baclofen in spascticity. Exp Neurol 103:165-172

Lundberg A (1964) Supraspinal control of transmission in reflex paths to motoneurons and primary afferents. In: Eccles JC, Schade JP (eds) Progress in Brain Research, vol 12, Physiology of spinal neurons. Elsevier, Amsterdam, pp 197-219

Maxwell DJ, Christie WM, Short AD, Brown, AG (1990) Direct observations of synapses between GABA-immunoreactive boutons and muscle afferent terminals in lamina VI of the cat's spinal cord. Brain Res 530:215-222

Price GW, Wilkin GP, Turnbul MJ, Bowery NG (1984) Are baclofen-sensitive GABAb receptors present on primary afferent terminals of the spinal cord? Nature 307:71-72

Quevedo J, Eguibar JR, Jim~nez I, Rudomin P (1990) Action of (-)-baclofen on segmental pathways producing PAD. Abstr Soc Neurosci 16: 111

Robertson B, Taylor WR (1986) Effects of gamma-aminobutyric acid and (-)-baclofen on calcium and potassium currents in cat dorsal root ganglion neurones in vitro. Br J Pharmacol 89: 661-672

Rudomin P (1991) Presynaptic inhibition of muscle spindle and tendon organ afferents in mammalian spinal cord. Trends Neurosci 13(12): 499-505

Rudomin P, Mufioz-Martinez J (1969) A tetrodotoxin-resistant primary afferent depolarization. Exp Neurol 25:106 115

Rudomin P, Nufiez R, Madrid J (1975) Modulation of synaptic effectiveness of Ia and descending fibers in the cat spinal cord. J Neurophysiol 38 : 1181-1195

Rudomin P, Engberg I, Jim6nez I (1981) Mechanisms involved in presynaptic depolarization of group I and rubrospinal fibers in cat spinal cord. J Neurophysiol 46: 532-548

Rudomin P, Jim~nez I, Solodkin M, Duefias S (1983) Sites of action of segmental and descending control of transmission on pathways mediating PAD of Ia- and Ib-afferent fibers in cat spinal cord. J Neurophysiol 50:743-769

Rudomin P, Solodkin M, Jim6nez I (1986) PAD and PAH response patterns of group Ia and Ib fibers to cutaneous and descending inputs in the cat spinal cord. J Neurophysiol 56:987-1006

Rudomin P, Solodkin M, Jim6nez I (1987) Synaptic potentials of primary afferent fibers and motoneurons evoked by single intermediate nucleus interneurons in the cat spinal cord. J Neuro- physiol 57:1288-1313

Rudomin P, Jim~nez I, Enriquez M (1991) Effects of stimulation of group I afferents from flexor muscles on heterosynaptic facilita- tion of monosynaptic reflexes produced by Ia and descending inputs: a test for presynaptic inhibition. Exp Brain Res 85:93-102

Starke H, Gothert M, Kilbinger H (1989) Modulation of neuro- transmitter release by presynaptic autoreceptors. Physiol Rev 69: 864-989

Wang MY, Dun JN (1990) Phaclofen-insensitive presynaptic inhibitory action of (+/-)-baclofen in neonatal rat motoneurones in vitro. Br J Pharmacol 99: 413M21

Documents

Experimental Brain Research · 2017. 6. 7. · Exp Brain Res (1992) 91:2945 Experimental Brain Research 9 Springer-Verlag 1992 Differential action of (--)-baclofen on the primary