 |
INTRODUCTION |
A noteworthy degree of plasticity had been revealed under certain conditions in structures of the cerebellocerebral pathways involved in the control of voluntary movements (Allen and Tsukahara 1974
; Aroniadou and Keller 1995
; Asanuma and Keller 1991
; Ito 1984
; Tsukahara et al. 1981
). Neuronal plasticity has been evidenced in the motor cortex, where pyramidal and nonpyramidal neurons receive thalamic inputs from the ventrolateral nucleus (VL) and somatosensory inputs from the primary sensory cortex (Baranyi et al. 1993
; Jones 1987
; Kosar et al. 1985
; Porter et al. 1990
; Strick and Sterling 1974
; Uno et al. 1970
; Zarzecki 1989
, 1991
; Zarzecki et al. 1993
) and a ventral component of the spinothalamic system (Padel and Relova 1988
; Padel et al. 1986
). In the motor cortex, plasticity appeared as a long-term potentiation (LTP) of the thalamocortical excitatory postsynaptic potentials (EPSPs). This phenomenon was often observed in acute preparations and was induced by pairing the VL stimulation either with intracellular depolarizing current injection or with antidromic activation of the pyramidal cells (Baranyi and Feher 1978
, 1981a
-c
), or again, with activation of corticocortical afferent fibers (Baranyi and Szente 1987
). However, it was never obtained with tetanic stimulation of the VL only (Baranyi and Feher 1981b
; Baranyi et al. 1991
; Iriki et al. 1989
, 1991
), pointing out that this LTP occurs only under associative conditions. These conditions were generally established by pairing the VL and the primary sensory cortex stimuli (Iriki et al. 1989
, 1991
). Indeed, besides their involvement in the instantaneous regulation of ongoing movements and in LTP induction, the somatosensory inputs are known to be essential for the genesis of long-lasting cortical and motor changes (Asanuma and Arissian 1984
; Keller et al. 1992
). This was evidenced by the observation of cortical synaptic proliferation following thalamic (ventral posterolateral nucleus) high-frequency stimulation (Keller et al. 1992
) and recovery of the motor function after partial thalamic (VL) lesion (Bornschlegl and Asanuma 1987
), or again, by the participation of somatosensory information in acquisition of new motor skills (Pavlides et al. 1993
; Sakamoto et al. 1989
). Somatosensory messages are also important to achieve conditioning. Those given by the unconditioned stimulus (UCS) seem to determine the characteristics of the conditioned motor responses: the pairing of an auditory conditioned stimulus (CS) with a sensory UCS inducing eyeblink has been shown to result in an association between the CS and the eyeblink, whereas when the same CS was paired with a UCS producing nose muscle contraction, the association was established between the CS and the nose muscle activation (Woody and Engel 1972
).
Owing to the importance of somatosensory inputs in functional compensation, in motor learning, and in the induction of thalamocortical LTP, we have investigated, in awake adult cats, the effects of an associative conditioning in which the CS was paired successively with two distinct UCSs applied to different somatosensory receptive fields. In a previous work, a CS applied in the cerebellar interpositus nucleus (IN) and paired with a UCS applied on the skin of the distal part of the forelimb, both stimuli initially inducing forearm flexions (Meftah and Rispal-Padel 1994
; Rispal-Padel and Meftah 1992
), produced a persistent enhancement of the transmission through the thalamocortical link of the cerebellothalamocortical (CTC) pathway. With the facilitation of the thalamocortical responses, an increase of the amplitude and rate of occurrence of the elbow flexions induced by IN stimulation was also observed. The time courses of the motor and thalamocortical changes were well correlated, suggesting that a neurobiological link between the two kinds of events could exist.
In another component of the experiment, as in the case of the conditioned facial movements (Woody and Engel 1972
), we have shown that conditioned forearm movements produced by the intracerebellar (IN) CS also depend on the cutaneous receptive field in which the unconditioned somesthetic stimulus was applied (Meftah and Rispal-Padel 1995
). In the same cats, in addition to the CS-UCS association mentioned above (Rispal-Padel and Meftah 1992
), a second conditioning procedure was also carried out. After a long period of rest, the same CS that produced forearm flexion was paired with another electrical UCS applied on a more proximal part of the forelimb that gave rise to a backward withdrawal reflex of the forelimb (forearm extension). In contrast with the first procedure, the second one resulted in a rapid decrease of the amplitude and rate of occurrence of the forearm flexions induced by the CS and the appearance of forearm extensions (Meftah and Rispal-Padel 1995
). The fact that the CS of an individual IN site can produce two distinct conditioned motor responses raises the following question: what kind of neurobiological changes produced in the CTC pathways as a result of distinct somesthetic information could lead to different conditioned movements?
Answering this question was the aim of the present experiment, in which we studied the differential effects of the two conditioning procedures on the motor responses mediated by the CTC circuits and on synaptic transmission through these circuits. The results obtained under each of the two types of experimental conditions provide information about the extent of the plasticity of the interpositus efferent pathways in adult cats; the capacity of these networks to control movements can be reinforced or depressed during conditioning, depending on the unconditioned sensory information. The mechanisms possibly underlying this capacity to bring about different motor actions were discussed in the light of the results we obtained and other theoretical and experimental data.
 |
METHODS |
The experiments were performed on four adult cats of either sex weighing 2.5-3.5 kg. Cats were initially prepared under deep anesthesia for the implantation of electrodes in the IN of the cerebellum and motor cortex sites controlling forearm flexion movements. Two electrodes were inserted in the IN and one transcortical pair in the motor cortex. After electrode implantation and a period of ~1 wk during which motor and cortical responses induced by the IN stimulation were tested, a neurotoxic lesion of the red nucleus magnocellularis (RNm) was performed in a second step. The control of the forelimb movements by the IN being exerted via interpositothalamocorticospinal and interpositorubrospinal circuits, the lesion of RNm was performed to specifically study the motor effects mediated by the CTC component. After 1 wk, postlesion tests were performed to characterize the effects of the lesion on the cortical and motor responses induced by IN stimulation. During this period one of the two IN sites was selected for application of the CS. The stimulation of the other IN site was used as a control and was never paired with any UCS. The CS and the UCS parameters as well as those of the IN control site stimulation were also determined during this period. Then two alpha conditioning procedures were successively carried out on the chronically prepared cats. In these two procedures the CS was a subthreshold stimulation applied to the IN (five 0.2-ms square pulses delivered at 500 Hz) at the origin of the CTC circuits controlling forearm flexion movements. In the first procedure the CS was paired with a transcutaneous electrical UCS, which was applied to the dorsum of the distal part of the forearm and produced a forearm flexion reflex. The movements originally induced by the CS and the UCS were in the same direction (i.e., forearm flexions); thus we termed the CS-UCS pairing a "concordant procedure." In the second procedure the same CS was paired with another UCS applied to a more proximal cutaneous receptive field located at equal distances between the wrist and the elbow. In this case the UCS produced a backward withdrawal reflex of the forelimb (extension). This procedure was called the "discordant procedure" because the CS and the UCS produced directionally opposite movements. The two UCSs activate, on the one hand, the spinal circuits supporting the unconditioned motor reflexes, and, on the other hand, long pathways ascending to the cerebellum and the brain, i.e., dorsal column lemniscal and spinocervicothalamic in addition to spinoreticular and spinothalamic pathways. These sensory pathways activate the cerebellar and cortical sites. Because these sites were also activated by the CS, the two conditioning procedures could therefore be assumed to concern sensorimotor loops involved in the control of forelimb movement.
In two cats, the concordant procedure was carried out before the discordant one. The two other cats underwent the same procedures but in the reverse order. In the four cats, a period of 2 mo without any experimental intervention separated the two procedures. For both procedures, the motor responses induced by the conditioned IN site stimulations were examined under two conditions: during the daily conditioning sessions, when they were induced by the CS, and during a series of tests performed after each conditioning session, when they were produced by a constant intensity of stimulation at 1.5 times the initially determined threshold. The effects of the two procedures on the CTC transmission were also tested under two conditions: during the conditioning sessions, by recording the transcortical potentials evoked by the CS, and outside the conditioning sessions, by recording the transcortical responses induced by a single stimulating pulse 0.1 ms in duration delivered to the IN conditioned site. The cortical responses induced by the stimulation of the IN control site, which had never been associated with any UCS, were also recorded every day with the use of stimulation parameters identical to those of the CS (five 0.2-ms square pulses delivered at 500 Hz). At the end of each conditioning session, the motor responses induced by the stimulation of this IN control site were also tested with the use of five 0.2-ms square pulses delivered at 500 Hz with a constant intensity of 1.5 times the initially determined threshold.
Surgery
Two surgical interventions, one for electrode implantation and the other for RNm lesion, were performed in each cat. The cats were first sedated with ketamine (10 mg/kg) and then maintained under deep anesthesia with the use of an intravenous injection of
-chloralose (60 mg/kg). Respiration was artificially assisted and physiological parameters such as temperature, heart rate, electrocardiogram, and expired CO2 were monitored. In addition to the strictly sterile techniques used, the cats underwent antibiotic treatment for 3 days immediately after surgery, and the skin wounds were treated with neomycin gel and an analgesic.
IMPLANTATION OF THE ELECTRODES.
In the first intervention, several semimicroelectrodes, made of electrolytically sharpened nickel-chrome wire insulated except for the 100-µm tips, were inserted into successive relay structures of the CTC pathways. In the primary motor cortex one pair of transcortical electrodes was inserted through a trephine hole and then fixed with dental cement to the bone of the skull and soldered to a microconnector. This pair of transcortical electrodes was guided normally to the cortical surface by a micromanipulator. The deeper tip was implanted 2 mm in the depth of the motor cortex (in layers IIIb and V) and the second tip was inserted in the cortical surface just below the dura (in superficial layers I or II). The pair of transcortical electrodes was placed in the elbow motor representation zone (Nieoullon and Rispal-Padel 1976
; Woolsey 1958
). This zone in the motor cortex was carefully determined by moving the electrode tip over the cortical surface to find the place where the largest responses were evoked by stimulating the skin over the dorsum of the contralateral forelimb. Then the electrode was moved for stimulation and its position was adjusted to the point at which microstimulation induced forelimb flexion with the lowest current intensity. The electrical activity of a small population of neurons located between the two tips was transmitted to a differential amplifier and thus was selected among all the other remote activities.
Two monopolar electrodes were implanted into the IN. To implant each of them in an IN site controlling elbow movements, electrophysiological recordings were performed along electrode trajectories guided according to stereotaxic coordinates (Berman 1968
) through the IN. We looked for clusters of units excited by receptive fields located on the ipsilateral forelimb skin. When a large response was recorded, the IN electrode was used for stimulation (single 0.1-ms square pulses), and its final position was determined as the locus where large responses in the contralateral motor cortical elbow representation zone were induced with low threshold. These responses should also have the characteristics of latency and waveform previously described (Rispal-Padel and Latreille 1974
; Sasaki and Prelevic 1972
). The two electrodes were implanted in this way 1-1.5 mm apart, and their tips were located in two distinct sites of the IN, the stimulation of which induced similar cortical responses in the elbow area of the motor cortex. When tested in awake animals, these stimuli also induced similar elbow flexions. As in previous studies (Meftah and Rispal-Padel 1994
, 1995
; Rispal-Padel and Meftah 1992
), one of the two IN sites was used for the CS application, and the other one served as a control site, because its stimulation had never been paired with any UCS.
RNm NEUROTOXIC LESION.
In the second intervention, the RNm region involved in forelimb motor control and receiving projections from the IN was subjected to a neurotoxic injection. The site was determined with the use of both stereotaxic coordinates and electrophysiological methods. The RNm site that relays the influx from the previously implanted IN sites was identified during microelectrode trajectories. We noted the coordinates of the RNm site in which the largest pre- and postsynaptic field potentials were found (Fig. 1A, Microelectrode). Then a Hamilton microsyringe needle, insulated except at the tip, was used to record and find the same field potentials as those identified with the microelectrode (Fig. 1A, Microinjector). Finally, 100 nM N-methyl-D,L-aspartic acid in solution was injected into the RNm zone (Fig. 1B) to permanently inactivate the neurons of the subcortical component of the IN efferent circuits controlling forelimb movements. After 1 wk the extent of the RNm lesion and the integrity of the CTC pathway were checked. The effects of destruction of the RNm neurons on the musculature were evidenced by evaluating the amplitude of the motor responses induced by a constant IN stimulation intensity, and the integrity of the CTC pathway was checked by testing the cortical responses also induced by a constant IN stimulation intensity.

View larger version (22K):
[in this window]
[in a new window]
| FIG. 1.
Control of the effects of red nucleus magnocellularis (RNm) lesion. A: electrophysiological identification of the RNm zone receiving inputs from the interpositus nucleus (IN) conditioned stimulus (CS) site. Left: analogical traces of the presynaptic (1st negative wave) and postsynaptic (2nd negative wave) rubral field potentials induced by the IN stimulation and recorded with a microelectrode. Right: traces of the pre- and postsynaptic field potentials recorded in the same zone with the Hamilton microsyringe needle. B: schema of a frontal section of the mesencephalic region (anterior plane 5.5). Hatched zone: rubral region where degenerate neurons were found. Straight line above hatched zone: microsyringe needle trajectory. C: averaged traces of the flexion motor responses induced by identical IN stimulations before and after the RNm lesion. Each trace corresponds to the average of 10 trials. D: graph of the motor response amplitude vs. the intensity of the IN stimulating current, tested before(   ) and after ( - - - ) the RNm lesion. T, times threshold. E: cortical responses induced by identical IN stimulations. The unchanged amplitude and time course of the analogical traces of the cortical responses induced before and after lesion indicates that cerebellothalamic fibers passing through the RNm were spared.
|
|
Pretraining sessions
A few days before surgery, the cats were brought into the training room and put in a hammock for 30-90 min/day to accustom them to the training setup. After the first surgical intervention and 2-3 days of recovery, the animals were again brought into the training room for a few daily sessions, during which the chronically implanted electrodes were connected to the stimulating and recording apparatus and a potentiometer was set up on the elbow. The fixed branch of the potentiometer was attached to the hammock and the mobile branch to the distal forearm. Displacements of this body segment changing the angle between the two potentiometer branches were measured as angular deviations. Regularly calibrated, the maximum resolution of the potentiometer was 0.2°, and any angular deviations of the elbow greater than this were considered as positive responses. The experimental setup provided the possibility of simultaneously recording, in the same awake preparation, the cerebellocortical responses and the elbow joint movements induced by the CS and the UCS during the conditioning periods. The data collected in this way also enabled parallel analysis of the evolution of the changes affecting CTC transmission and of the motor responses induced by the IN stimulation.
During the postlesion pretraining periods, the parameters of the CS and the UCSs were determined as described in previous papers (Meftah and Rispal-Padel 1994
; Rispal-Padel and Meftah 1992
). Parameters were then kept rigorously constant for the entire conditioning period. The parameters of the cortical stimulations and those of the stimulations applied to the "control" IN site were also determined during the same pretraining periods. The CS consisted of five 0.2-ms square pulses delivered to the IN at 500 Hz with a subthreshold current intensity between 50 and 65 µA (depending on the cat), i.e., 70% of the threshold value determined in each cat. It should be noted, however, that the CS produced a cortical response every time it was applied. The UCS consisted of a train of five 0.5-ms square pulses delivered at 500 Hz with an intensity of ~1 mA. When the UCS was applied to the proximal cutaneous receptive field (during the discordant procedure), it produced a backward withdrawal reflex of the arm and forearm, similar to an avoidance response. In this case the cat seemed to be slightly uncomfortable, whereas it stayed quite calm when the same stimulation was applied to the dorsum of the wrist (during the concordant procedure), inducing an elbow flexion.
Training sessions
Training sessions of the two conditioning procedures consisted of 125 paired CS-UCS trials and 25 CS-alone trials evenly distributed over the session. The interstimulus interval lasted for 100 ms. An intertrial interval of 30 s was used throughout. In both conditioning procedures, the rate of occurrence and the amplitude of the elbow movements (measured as joint angular deviations) obtained at each daily session were determined from the 25 CS-alone trials. They were also determined from 20 additional trials performed at the end of each session and in which the IN site stimulation was delivered with an intensity of 1.5 times the threshold initially determined. In parallel with the motor responses induced by the CS, the transcortical potentials also evoked by the CS were recorded and averaged for each day. These were considered as an index of CTC excitability, the latter also being tested outside of the daily conditioning sessions by recording the transcortical potentials induced by single IN stimulating pulses 0.1 ms in duration.
The extinction procedures, which consisted of 150 daily presentations of the CS alone on two cats or of 150 daily UCSs followed 900 ms later by the CS on the two other cats, provided information about the reversibility of the conditioned effects and their associative nature. The associative aspect was also checked by recording the motor and the cortical responses initiated from the IN control site.
 |
RESULTS |
The RNm lesion, after the stabilization period, was found to strongly decrease the amplitude of the forearm flexions induced by the IN stimulation; the cerebellocortical transmission was not affected, indicating CTC fiber integrity. Consequently, the subsequent conditionings can be taken as being responsible for the modifications occurring in the CTC pathway. The concordant procedure was then found to result in a persistent enhancement of the excitatory cerebellocortical response that paralleled an increase of the amplitude and the rate of occurrence of the forearm flexions induced by the CS. In contrast, the discordant procedure resulted in a depression of the cerebellocortical excitatory field potentials and in the appearance of a further important positive wave inhibitory event that followed the excitatory field potential. This positive wave was previously identified as an inhibitory event (Baranyi et al. 1993
; Meftah and Rispal-Padel 1994
; Purpura et al. 1964
; Sasaki and Prelevic 1972
; Zarzecki 1991
). Concomitantly the forearm flexion induced by the IN stimulation disappeared and was replaced by a forearm extension. In each of the two conditioning procedures the central and motor effects were found to be linked. They were also found to be fairly reproducible in all the conditioned cats. In the following sections, these effects are described on the basis of the motor and cortical responses obtained in a typical cat. However, quantitative analysis took into account the data obtained in all the conditioned cats.
Effects of the RNm lesion
Examples of the averaged traces of the elbow angular deviations induced before and after the RNm lesion with the same IN stimulation parameters are illustrated in Fig. 1C. A strong amplitude decrease of the elbow flexion movements can be seen as a result of the lesion. The amplitudes were measured and plotted versus the stimulus intensity (Fig. 1D). Before the lesion (Fig. 1D,
), the flexion amplitude increased with the enhancement of the stimulation intensity from the threshold value (50 µA) to ~2 times threshold, and then reached a plateau value of ~7° for intensities between 2 and 2.5 times threshold. After the lesion (Fig. 1D,
), the amplitude of the forearm flexions also increased and reached a plateau value, but it appeared that the plateau value was two thirds lower than its prelesion value and that the threshold intensity was enhanced (70 µA). The consistently reduced amplitude independent of stimulus intensity indicates that the RNm target neurons of the IN stimulated site had indeed been destroyed. Postmortem histological analysis revealed, as described previously (Fig. 3 in Rispal-Padel and Meftah 1992
), the nearly complete disappearance of the RNm neurons located in a region that is known to be connected to the forelimb spinal segments (Pompeiano and Brodal 1957
).

View larger version (35K):
[in this window]
[in a new window]
| FIG. 3.
Motor effects produced by the discordant conditioning procedure. A: conventions as in Fig. 2A, except that the UCS produced a backward withdrawal reflex of the forelimb with elbow extension instead of the flexion seen as a response to the UCS. The new UCS was applied to the skin of a more proximal part of the forearm (short thick line). B, left: typical example of the conditioning effects on the motor responses induced by the 25 CSs given alone during each conditioning session. B, right: typical example of the conditioning effects on the motor responses recorded outside the conditioning sessions and induced by the IN conditioned site stimulation given with an intensity of 1.5 times the initial threshold. In both columns, traces correspond to the flexion (upward deflections), extension (downward deflections), and no response trials, separately averaged every 2 days during acquisition (days 1-7) and then before (day 20) and after (day 30) extinction. The numbers of averaged trials (n) are indicated beside each trace. Note the suppression of the forearm flexion movements, on the 5th day of the acquisition period, and the appearance of a forearm extension following a small flexion on the 3rd day. C: same as Fig. 2C, but concerning the percentages of flexions (black bars), extensions (gray bars), and no response (white bars) trials determined in the 4 conditioned cats from the 20 additional trials performed during each test session. D: same as Fig. 2D, but concerning the amplitudes of the flexion (black bars) and extension (gray bars) motor responses.
|
|
The decrease of the motor response amplitude was not due to an alteration of the IN projection to the thalamic VL nucleus passing through the RNm. The cortical responses induced by constant-intensity IN stimulation before and after the lesion were compared in the awake animal (Fig. 1E). Examples of the overlapping traces derived from the cortical elbow motor representation area indicate that, after the lesion, the cortical responses induced by a constant stimulating current were identical to those recorded before, indicating that the cerebellothalamic fibers of the recruited circuits were spared by the RNm lesion. The fact that RNm lesion did not produce any changes in the cortical responses 7 days after the lesion further indicates that these lesions did not produce plastic phenomena that could interfere with the subsequent conditioning effects.
Motor effects produced by the concordant procedure
Before conditioning, the movements produced by CS and UCS were first characterized with a movement analysis setup, the Elite system (Fig. 2A). The cat being sustained in the hammock with the forelimb free, the initial movements produced by CS were discrete forearm flexions produced only in a small number of trials (Fig. 2A, CS). Motor responses induced by UCS applied above the wrist were also movements in the same direction (flexions), but with a greater amplitude (Fig. 2A, UCS), and they were produced at every UCS application. On the 1st day of conditioning, as shown on averaged potentiometer traces (Fig. 2B, left, Day 1, n = 3), only small flexions (with a mean amplitude of 0.6°) were induced in 3 of the 25 trials in which CS was given alone. The remaining "no response" trials are also illustrated (Fig. 2B, right, Day 1, n = 22). On the following days of conditioning a progressive significant increase of the amplitude and of the rate of occurrence of the forearm flexions induced by the CS was observed; the successive traces (Fig. 2B, left) are averages of the positive responses (n, number of positive responses) obtained in the 25 CS trials given alone during each daily session. During the acquisition period, the no response trials decreased steadily and disappeared after the 5th day. The latencies of the CS forearm flexions (25-30 ms) were not noticeably affected by the concordant procedure (Fig. 2B, left).

View larger version (27K):
[in this window]
[in a new window]
| FIG. 2.
Motor effects produced by the concordant conditioning procedure. A, left: schema of the right forelimb of the cat. Filled circles: joints of the forelimb and the distal part of the digits. A, middle: motor response induced by the CS. Arrow and dotted line: direction and extent of the forelimb displacements. A, right: motor responses induced by the unconditioned stimulus (UCS) applied on the skin, on the wrist (short thick line). Note that the movements induced by the CS and the UCS (arrows) are in the same direction, and that the forearm flexion reflex induced by the UCS (dotted line) was of greater amplitude than that induced by CS. B, left: typical example of the conditioning effects on the angular deviation of the elbow induced by the CS. Upward deflections: flexion movements of the forearm. Averaged traces were obtained every 2 days during the acquisition period. Only the responses recorded on the 1st day (Day 20) and the last day (Day 30) of the extinction period are illustrated. B, right: averaged traces of the "no response" recordings. n, Number of trials averaged in each trace. C: averaged percentages of the flexion positive trials (black bars) and of the no response trials (white bars) obtained in the 4 conditioned cats. These percentages were determined from the 25 CS-alone trials. D: time course of the averaged amplitudes of the forearm flexions induced by the CS in the 4 conditioned cats.
|
|
The percentages and the amplitudes of the forearm flexion responses induced by the CS were quantified separately for each cat, and then the data of all the cats were pooled and averaged for each day. The progressive changes in the mean values of percentage and amplitude during the conditioning procedure are illustrated in Fig. 2, C and D, respectively. From a mean proportion of responses of 14 ± 6% (mean ± SE) (Fig. 2B, Day 1) and a mean amplitude of 0.5 ± 0.3° (Fig. 2D, Day 1) on the 1st day of the acquisition, the forearm flexions increased regularly and occurred in 100% of the trials with a mean amplitude of 2.5 ± 0.3° at the end of the acquisition (Day 7). The no response trials, which initially occurred in 86 ± 6% (Fig. 2C, Day 1), decreased progressively and disappeared after the 5th day of the acquisition period. The high percentage (100%) and amplitude (~2.2 ± 0.2°) of the flexions remained stable even after an interruption of the CS-UCS sessions for 12 days (Fig. 2, B-D, Day 20). This revealed the long-lasting nature of the conditioned motor effects. Their associative nature was suggested by the significant decrease of the flexion proportion (to 20 ± 5%) and amplitude (to 0.6 ± 0.3°), and the reappearance of no response trials (in 80 ± 5%) after 11 days of presentation of the CS alone (Fig. 2, B-D, Day 30). In the same way, this reversibility of the conditioned motor changes ruled out the possibility of nonspecific processes such as recovery of brain function after the possible lesion of the nervous tissues located around the implanted electrodes or after the RNm lesion. Confirming previous observations (see Fig. 7 in Rispal-Padel and Meftah 1992
), the amplitude of the flexions induced by IN stimulation delivered with an intensity of 1.5 times the initial threshold was also increased during the acquisition and recovered the initial state after extinction.

View larger version (14K):
[in this window]
[in a new window]
| FIG. 7.
Conditioning effects on the motor responses induced by stimulation of the elbow area of the motor cortex. A: diagram of the primary motor cortex (cr., cruciate sulcus; cor., coronalis sulcus; d., dimple; pr. sylv., pre-sylvian sulcus). Star and dots: all sites tested on the 4 cats in the elbow area of the motor cortex. Star: site in which all the cortical responses represented in the preceding figures were recorded. B: effects produced by the discordant conditioning procedure on the motor responses induced by stimulation of the site indicated by the star. Top and bottom traces: averaged responses recorded before and after the acquisition period, respectively(n ~ 15 trials). C: graphs illustrating the pooled data obtained on the 4 cats during the concordant and the discordant procedures. In each cat, the variation of the amplitude of the motor responses induced by cortical stimulation is expressed as a percentage of the initial value of the responses. Gray histogram: average of these variations. Black histogram: initial level (taken as 100%).
|
|
Motor effects produced by the discordant procedure
After 2 mo without any experimental intervention, the second conditioning procedure, called discordant, was carried out on the same animals. The CS applied at the same IN site and, producing a slight forearm flexion (Fig. 3A, CS), was paired with another UCS that induced a backward withdrawal reflex of the whole forelimb instead of a forearm flexion reflex (Fig. 3A, UCS). As in the case of the concordant procedure, the motor effects of discordant CS-UCS pairing were also described on the basis of the motor responses induced every day during the 25 CS-alone trials (Fig. 3B, Conditioning). In addition, 20 trials in which the conditioned IN site was stimulated outside of the conditioning sessions with an intensity of 1.5 times the initially determined threshold were also performed (Fig. 3B, Test). Typical examples of the averaged traces of the responses induced in these two situations are illustrated (Fig. 3B, left, Conditioning) for the responses induced by the CS and (Fig. 3B, right, Test) for the responses induced during the 20 additional trials.
On the first day of the acquisition period, the CS induced discrete forearm flexions (Fig. 3B, Conditioning, Day 1, top trace) of ~0.4° in amplitude in only 4 of the 25 CS-alone trials. The 21 remaining trials showed no detectable responses (Fig. 3B, Conditioning, Day 1, bottom trace). During the following daily sessions of the acquisition period, a rapid decrease in the proportion of responses and the amplitude of the forearm flexions was seen. After the 5th day of acquisition it became impossible to detect any motor response to the CS. After an interruption of the pairing sessions for 12 days (Fig. 3B, Conditioning, Day 20), and during the major part of the extinction procedure, the same observation (i.e., no response to the CS) was made. The flexion motor responses were then partially recovered on the last day of extinction in 3 of the 25 CS-alone trials (Fig. 3B, Conditioning, Day 30).
The conditioned IN site stimulation with a constant intensity of 1.5 times the initial threshold was effective for producing motor responses during the entire conditioning period (Fig. 3B, Test). At the beginning of conditioning, the 20 IN stimulations performed at the end of each daily session induced forearm flexions and then began to produce different motor effects on the following days of the acquisition (Fig. 3B, Test). On the 1st day the flexions were obtained in 14 of the 20 trials (Fig. 3B, Test, Day 1, top trace) and had an amplitude of ~2.5°. Then, the rate of occurrence and the amplitude of the forearm flexions decreased rapidly. They disappeared on the 5th day of the acquisition period (Fig. 3B, Test, Day 5). On the 3rd day of the acquisition period a complex movement that was composed of a small flexion curtailed by an extension was observed in response to IN stimulation. On the following days only the extension was observed. The amplitude of these new motor responses (extension) increased regularly during acquisition (Fig. 3B, Test, bottom traces) and at the end of this period represented 100% of the 20 trials (Fig. 3B, Test, Day 7). These extensions were still observed in 100% of the trials after 12 successive days of interruption of the pairing sessions (Fig. 3B, Test, Day 20). After the 11 days of the extinction procedure (Fig. 3B, Test, Day 30), the forearm flexions reappeared in five trials (25%), with an amplitude of ~1.4°, whereas the extensions decreased and were observed in only five trials (25%), with an amplitude of 1°. In 50% of the trials no motor responses were induced. The latencies of the extensions (45 ± 5 ms) were slightly longer than those of the initial flexions (25 ± 5 ms), and during the 20-ms delay there was a complete lack of deviation of the potentiometer recording traces as shown in Fig. 3B, indicating the suppression of any movement during this period. Note that forearm extensions had never been observed before conditioning.
Because the CS became rapidly ineffective in producing detectable motor responses during the discordant conditioning period (Fig. 3B, Conditioning), the data collected during test sessions (i.e., during the 20 additional trials performed at the end of each daily session) were used for the quantitative analysis of the motor changes induced by the discordant procedure (Fig. 3, C and D). Indeed, the percentages and amplitudes of the motor responses were determined in the four cats from the 20 additional trials. They were then averaged and plotted versus the days of conditioning (Fig. 3, C and D). In all the cats, and during nearly the same periods of time, the percentages and amplitudes of the forearm flexions decreased from 75 ± 6% and 2.5 ± 0.4°, respectively, on the 1st day to 0% after the 3rd day (Fig. 3, C and D). In contrast, the forearm extensions that appeared in 40 ± 4% on the 3rd day of the acquisition period gradually increased to 100% on the 7th day (Fig. 3C). Their amplitudes also increased and reached an average 2.5 ± 0.2° (Fig. 3D). The occurrence of such reversed motor responses was also of a long-lasting nature, because they persisted in 100% of the trials (Fig. 3C, Day 20) with a mean amplitude of 2 ± 0.3° after 12 days during which the CS-UCS pairing sessions were interrupted. After 11 daily sessions of 150 CS-alone trials, or of 150 trials in which the UCS was followed 900 ms later by the CS (extinction), a decrease of both the percentage (from 100% to 25 ± 5%) and amplitude (from2 ± 0.3° to 0.8 ± 0.3°) of the forearm extensions was observed. These extinction procedures also led to the reappearance of the forearm flexions. Their averaged percentage and amplitude were 30 ± 0.2% and 1.3 ± 0.3°, respectively, at the end of the extinction procedures (Fig. 3, C and D, Day 30). The motor changes induced by the discordant procedure seem to be more persistent than those induced under the concordant procedure (see above). After the 11th day of extinction following the concordant procedure, the mean values of the percentages and the amplitudes of the forearm flexions (Fig. 2, C and D, Day 30) did not differ significantly from those obtained on the 1st day of acquisition (Fig. 2, C and D, Day 1). After extinction following the discordant procedure, however, the extensions still happened in ~25% of the trials, and flexions in only ~30% (Fig. 3C, Day 30). After 2 mo without any experimental intervention the preconditioning level was found to be partially recovered; the extension responses had disappeared and the flexion responses had reappeared in ~70% of the trials, as on the 1st day of the acquisition period (Fig. 3C, Day 1). Such reversibility of the motor changes indicates that they are indeed due to CS-UCS pairing.
Modifications of the cerebellocortical transmission
In parallel with the motor changes, the CTC transmission of the cerebellar outputs to the cortical elbow motor representation area was also affected by the concordant and discordant conditioning procedures. As in the case of the motor responses, changes affecting the CTC transmission were also found to be fairly reproducible in all the conditioned cats. The cortical responses induced by the CS and recorded during conditioning procedures simultaneously with motor responses (taken above as typical examples) allowed us to characterize the cortical modifications occurring in relation to conditioned motor effects (Figs. 4B and 5B). In addition, a more detailed analysis of the cortical modifications was made on the responses induced by single pulse of stimulation delivered out of the conditioning sessions to the IN conditioned site (Figs. 4A and 5A).

View larger version (17K):
[in this window]
[in a new window]
| FIG. 4.
Effects produced by the concordant conditioning procedure on cerebellocortical transmission. A: averaged traces of the cortical responses induced in the elbow motor representation area by single pulses delivered to the conditioned IN site during test sessions. Each trace corresponds to the average of ~15 trials. B: averaged traces of the cortical responses evoked by the CS in the same elbow motor representation area. Each trace corresponds to the average of ~40 trials. In A and B, the averaged traces obtained before (thin line) and after (thick line) the acquisition period are represented separately in the 1st and 2nd traces. They are superimposed in the 3rd traces. The averaged traces obtained before and after the extinction procedure are superimposed in the 4th traces.
|
|

View larger version (17K):
[in this window]
[in a new window]
| FIG. 5.
Effects produced by the discordant conditioning procedure on cerebellocortical transmission. A: averaged traces of the cortical responses evoked in the elbow area by a single pulse delivered to the conditioned IN site. B: averaged traces of the cortical responses evoked by the CS in the same cortical area. Acquisition and extinction effects are illustrated as in Fig. 4. Each trace corresponds to the average of ~15 trials in A, and of ~40 trials in B.
|
|
The cortical responses to IN stimulation appeared with a latency of 2.1 ms (Fig. 4, A and B, top traces), compatible with transmission through a disynaptic pathway (Uno et al. 1970
). The averaged traces of 15 cortical responses induced by a single IN pulse (Fig. 4A, top trace) first appear as a negative wave with two components in its rising phase (Fig. 4A, top trace), the second of which is assumed to be postsynaptic and excitatory. The decreasing phase (Fig. 4A, top trace), which began ~3 ms after the onset of the negative wave, did not seem to be equivalent to the simple decay of the excitatory wave, but it could be the beginning of a positive wave resulting from an inhibitory effect produced via intracortical interneurons on the deep cells of layers III and V. It could also be a thalamic excitatory effect exerted on the neurons in the superficial layers of the motor cortex (Shinoda et al. 1993
). However, many arguments supporting the idea that the negative wave is generally followed by cortical inhibitory processes have been provided previously(Fig. 4 in Meftah and Rispal-Padel 1994
; see also Baranyi et al. 1993
; Purpura et al. 1964
; Sasaki and Prelevic 1972
).
The two components of the rising phase of the negative wave were identically affected by the high frequency of IN stimulation (Fig. 4B, top trace). Indeed, as already shown in Fig. 4A for the response evoked by the CS (Fig. 4B, top trace), the two components of the ascending phase were essentially due to the first pulse. Those normally due to the following three IN pulses were prevented and the two negative components were equally abolished. This observation suggests that the first of the two negative components was probably not an incoming volley and that, like the second, it could be postsynaptically generated. Moreover, on individual recordings obtained with very thin electrode tips, spikes were sometimes seen superimposed on the top of the negative wave. Thus the negative wave corresponds to an excitatory event occurring inside layers III or V, at the level of the deepest tip of the transcortical pair of electrodes. In agreement with these observations, the cortical efferent neurons located in these deep layers are known to be activated monosynaptically and disynaptically by the excitatory endings of the VL thalamic neurons (Jones 1987
; Strick and Sterling 1974
) and to exhibit monosynaptic EPSPs in response to VL stimulation (Baranyi et al. 1993
; Purpura et al. 1964
).
The properties of the positive wave that appeared after discordant conditioning and that followed the remainder of the reduced negative wave (Fig. 5, A and B) have previously been tested, and the positive wave was assumed to be inhibitory (Meftah and Rispal-Padel 1994
). In addition, spikes superimposed on individual recording traces during the first excitatory wave or before and after the cortical response to IN stimulation disappeared at the time of the positive wave. In agreement with the suggestion of the inhibitory character of the positive wave is also the fact that the VL stimulation produced inhibitory postsynaptic potentials (IPSPs) following the monosynaptic thalamocortical EPSPs (Baranyi et al. 1993
; Purpura et al. 1964
) and the abolition of responses induced antidromically in the pyramidal neurons during IPSPs (Sasaki and Prelevic 1972
). Moreover, it must be recalled that the negative-positive responses recorded in the present work from the deep layers III and V of the motor cortex have already been extensively studied. They correspond to the positive-negative waves evoked by thalamic or brachium conjunctivum stimulation at the cortical surface (Sasaki et al. 1970
). Comparison of the cortical evoked response recorded from the surface with the postsynaptic potentials recorded simultaneously from the deep layers shows that the surface negative (or deep positive) wave corresponds to an IPSP (Purpura et al. 1964
) or to a silent period in spike discharges (Purpura et al. 1964
; Sasaki et al. 1970
).
The effects produced on cerebellocortical transmission by the concordant procedure were evaluated by comparing the cerebellocortical responses obtained at different periods of the conditioning procedure. The averaged traces obtained on the 1st day (Fig. 4, A and B, top traces) and the last day (Fig. 4, A and B, 2nd traces) of the acquisition period are superimposed in the third traces, showing a clear increase of the first part of the negative cortical evoked potential. The amplitude of the first peak of the cortical responses induced by the CS increased on average to around one-third of its preacquisition value (Fig. 4B, 3rd trace). In the rising phase of the increased wave, two events can be distinguished: 1) the enhancement of the negative wave due to the first pulse; this is also observed on the superimposition of the responses induced by single IN pulses (Fig. 4A, 3rd trace); and 2) the partial summation of the wave induced by the second pulse of the CS (Fig. 4B, 3rd trace) with the first negative wave due to the first stimulation pulse. This could be explained by a weakening of the inhibitory effect and/or the reinforcement of the excitatory effect mediated by excitatory interneurons in the deep layers. In the same way, it was observed that the decreasing phases of the negative waves induced by single pulses after conditioning (Fig. 4A, 3rd trace, thick line) start ~1 ms later than before conditioning (Fig. 4A, 3rd trace, thin line). Thus the duration of the negative wave is slightly lengthened (Fig. 4, A and B, 3rd trace). The pairing sessions were interrupted for 12 days without resulting in any changes of the responses. The effects of the extinction period were illustrated by superimposing the averaged traces obtained at the end of the acquisition period and the last day of the extinction period (Fig. 4, A and B, 4th traces). At the end of the extinction, the characteristics (amplitude and duration) of the cortical responses tended toward their initial values. However, they did not display complete recovery (Fig. 4, A and B, 4th traces). It should be noted that the latencies of these cortical responses did not show any changes.
After 2 mo at rest, the discordant conditioning procedure was carried out on the same cat. It resulted in changes of the cortical responses to IN stimulation (Fig. 5) that were completely different from those described above (Fig. 4). This was clearly shown by superimposed averaged traces (Fig. 5A, 3rd trace) of the cortical responses induced by single IN pulses before (Fig. 5A, 1st trace) and after (Fig. 5A, 2nd trace) the discordant acquisition period. The amplitude and the duration of the negative wave were dramatically reduced. Only the initial component of the rising phase of the negative wave persisted. The second component was completely abolished, and a secondary positive wave appeared 0.75 ms after the end of the negative wave (i.e., 3 ms after the onset of the initial negative event) and then increased conspicuously (Fig. 5A, 3rd trace). The cortical responses induced by the CS also presented (Fig. 5B, 3rd trace) these two distinct effects of conditioning (i.e., the depression of the initial negative wave and the appearance of the secondary positive wave). As in the case of the effects of the concordant conditioning, these changes were persistent, because the characteristics of the newly obtained responses were not affected by the interruption of the conditioning sessions over the 12 days following acquisition. However, after 11 daily sessions of repetitive CS-alone presentation (Fig. 5, A and B, 4th traces, EXTINCTION), the amplitude and the duration of the negative wave were significantly increased with respect to the values obtained at the end of the acquisition, whereas the positive wave remained important after the extinction period.
Associative nature and topographic specificity of the conditioning effects
The associative characteristics of the two conditioning procedures were tested by recording the cortical responses (Fig. 6A) induced in the same cortical location by stimulation of the IN control site that was never associated with the UCSs (see METHODS). The stimulation intensity of the IN control site was equal to that of the CS. The cortical responses had a negative waveform and a latency of 3.3 ms (Fig. 6A), compatible with disynaptic transmission (Uno et al. 1970
). The amplitude and duration were comparable with those of the responses induced by the CS. Other similarities between the conditioned and the control site stimulations concerned the characteristics of the motor responses. Indeed, the stimulation of the IN control site, delivered with a constant current intensity (130 µA) of 1.5 times the initially determined threshold, produced forearm flexions (Fig. 6B) similar to those induced by stimulation of the conditioned site. The latencies of the motor responses induced by the stimulation of the control and the conditioned sites were also found to be identical (25-30 ms). The main differences between the two stimulations were their locations in the IN nucleus (they were 1.5 mm apart in the IN) and the fact that the control stimulation site had never been associated with either of the two UCSs. These characteristics make it possible to use the second IN site as a control of both the associative nature and the topographic specificity of the conditioned central and motor effects.

View larger version (15K):
[in this window]
[in a new window]
| FIG. 6.
Effects produced by the discordant conditioning procedure on the cortical (A) and motor (B) responses, induced by stimulation of the IN control site (never paired with the UCS). A: averaged traces of the cortical responses (n ~ 40 trials) evoked in the elbow area by stimulation of the IN control site. The parameters of stimulation were identical to those of the CS. B: averaged traces of the motor responses induced in the same trials by stimulation of the IN control site with a constant intensity (~130 µA) of 1.5 times the initially determined threshold. Acquisition and extinction effects are illustrated as in Fig. 4.
|
|
Under neither of the two conditioning procedures did the stimulation of the IN control site produce changes of the evoked transcortical potentials or of the motor responses. The superimposition of the averaged traces of the cerebellocortical (Fig. 6A) and motor (Fig. 6B) responses induced by the stimulation of the IN control site before and after the acquisition (3rd traces) and the extinction (4th traces) periods of the discordant procedure revealed only slight but nonsignificant fluctuations of both cerebellocortical and motor responses. Similarly, the concordant procedure did not involve any significant changes of the cortical or motor responses. These observations provide an additional argument in favor of the associative nature of the conditioned changes (see above and Meftah and Rispal-Padel 1994
, 1995
; Rispal-Padel and Meftah 1992
), because they indicate that the conditioned changes were limited to the cerebellar efferent circuits originating in the IN conditioned site.
Analysis of the corticospinal transmission
Four sites (Fig. 7A, dots and star) located in the elbow area of the motor cortex (Nieoullon and Rispal-Padel 1976
; Woolsey 1958
) were tested separately on the four conditioned cats. The one illustrated by a star corresponds to the site where the cortical responses described in the previous sections as typical illustrative examples were recorded. The stimulation of this site produced flexions of the forearm, as illustrated by their averaged recording traces in Fig. 7B. The mean traces, obtained before and after the discordant acquisition period, did not display any significant changes of the motor responses induced by rigorously constant stimulations (Fig. 7B). Similar observations were made during the concordant procedure, confirming previous data (Rispal-Padel and Meftah 1992
). Such an absence of significant conditioning effects on the motor responses induced by cortical stimulation (Fig. 7C) was observed in all the conditioned cats under both concordant and discordant conditioning procedures. It appears from the histograms (Fig. 7C) of the evolution of the relative mean amplitude (expressed in % of the initial value) that the variations observed after each of the conditioning procedures were between 94 and 114% (104 ± 10%). Similarly, constant motor responses were also observed when stimulation was applied to other (shoulder or wrist) cortical body motor representation zones (Rispal-Padel and Meftah 1992
).
Evolution of the motor and the cerebellocortical changes in all the cats
As for the motor effects, we attempted to quantify the CTC changes on the basis of the data collected from the four conditioned cats. Because the changes affect the amplitude as well as the duration of the cerebellocortical responses, they were quantified by measuring the integrated responses corresponding to the area bounded by the response curves, with the use of a computer program. By this program the mean curve of each wave was integrated in relation to the mean level of the activity recorded for 5 ms before the stimulus. This activity preceding the stimulus was considered as the background activity and was taken as the baseline level. The integrated responses measured in this way were then expressed as a percentage of the integrated response recorded on the 1st day of the acquisition period (relative percentage) and taken as a reference (100%). This calculation of the relative integrated response makes it possible to compare and average the data obtained in the four conditioned cats. The averaged variations of the integrated responses during the acquisition period of the concordant and the discordant procedures and during the control procedure are reported in Fig. 8A. In the three conditions (Fig. 8A, Concordant, Discordant, and Control), the reference value (100%) corresponding to the initial integrated negative responses is illustrated by the black bars. The relative integrated negative waves recorded at the end of the acquisition periods are represented by the 2nd gray bar. In the discordant condition (Fig. 8A, Discordant), the 3rd bar corresponds to the positive wave (because it appeared at a later period, its area is expressed as a percentage of the the initial negative integrated response). These variations may be compared with changes of motor response amplitudes under the same three conditions (Fig. 8B, Concordant, Discordant, and Control). It appears clearly that cerebellocortical (Fig. 8A) and motor (Fig. 8B) responses evolved in the same way (i.e., concomitantly increased, decreased, or remained constant).

View larger version (24K):
[in this window]
[in a new window]
| FIG. 8.
Quantification of the changes affecting the cortical and the motor responses induced by the IN stimulation, during concordant and discordant procedures. A, left: mean variation of the surface areas of the cortical negative wave, during the concordant procedure. A, middle: mean variations of the surface areas of the cortical wave during the discordant procedure. Second bar: depression of the initial negative wave. Third bar: appearance and great increase of the positive wave. A, right: during both kinds of conditioning procedures, averaged variation of the cortical negative wave induced by the IN control site stimulation. All the variations were expressed as % of the preacquisition value of the negative integrated response. B: same as A, but concerning the amplitude of the motor responses induced by the IN stimulation. In A and B the pre- and the postacquisition values are represented by black and gray bars, respectively.
|
|
As described above, the negative integrated waves of the cerebellocortical responses were enhanced during the concordant conditioning procedure. They reached a value of ~220 ± 30% (i.e., 2.2 times their initial value, which was fixed at 100%) (Fig. 8A, Concordant). Concomitantly, the forearm flexion amplitudes significantly increased (Fig. 8B, Concordant), going from 0.5 ± 0.3° initially to ~2.5 ± 0.3° after the acquisition period (Fig. 8B, Concordant).
In contrast, during the discordant procedures the negative integrated cortical responses decreased considerably and at the end of the acquisition period reached ~20 ±15% of their initial values (Fig. 8A, Discordant). Following this negative wave, a positive one appeared, the mean surface area of which increased conspicuously. If compared with the negative integrated wave recorded on the 1st day of acquisition, the positive integrated wave was increased at the end of the acquisition period to 180 ± 45% of the initial negative integrated wave. In parallel with these central changes, the forearm flexions induced by the IN stimulation disappeared in favor of forearm extensions (Fig. 8B, Discordant). The amplitude of the extensions increased and reached ~2.5 ± 0.2° at the end of the acquisition period.
The stimulation of the IN control sites, during each of the two conditioning procedures, produced only slight nonsignificant fluctuations of both the integrated surface area of the negative waves of the cortical responses (Fig. 8A, Control) and the amplitude of the forearm flexions (Fig. 8B, Control). Overall, the data indicate that the changes affecting the CTC transmission and the motor responses were reproducible in all the conditioned cats. They also seemed to be linked, because the CTC transmission and the motor responses were simultaneously reinforced, depressed, or unchanged.
Effects produced by the concordant and the discordant procedures on the cortical responses induced by the UCS
The two UCSs were applied at distinct locations on the forearm and consequently differed in the motor reflexes they produced. They also differed in their effects on the motor cortex. The transcortical evoked potentials induced in the elbow area of the motor cortex by the UCS applied to the distal part (wrist) of the dorsal forearm skin appeared with a latency of ~9 ms (Fig. 9A). This response was characterized by the succession of two negative events. In the same cortical site, the transcortical responses induced by the other UCS, which was applied on a more proximal part of the dorsal forearm skin, also appeared with a latency of ~9 ms (Fig. 9B). In this case, however, only one negative wave followed by a large positive one was seen (Fig. 9B). In addition, the amplitude and duration of the negative wave were less than those of the first negative wave induced by the distal UCS (Fig. 9A). For equivalent current intensities of stimulation, the negative wave induced by the proximal UCS was about one third the amplitude and one half the duration of the first negative wave induced by the distal UCS. No changes in these cortical responses were found to occur under either of the conditioning procedures. This is illustrated by the superimposition of the averaged traces obtained before and after each of the concordant (Fig. 9A, 3rd trace) and discordant (Fig. 9B, 3rd trace) acquisition periods. It must be noticed that these findings are also good controls supporting the argument that the modifications in response properties mentioned above are not due to physical changes at the recording site.

View larger version (11K):
[in this window]
[in a new window]
| FIG. 9.
Cortical responses induced by the 2 distinct somesthetic UCSs. A: effects of concordant conditioning procedure on cortical responses to the cutaneous UCS applied to the wrist. B: effects of discordant conditioning procedure on cortical responses to the cutaneous UCS applied to a proximal part of the forearm. Each trace corresponds to the average of ~40 responses.
|
|
 |
DISCUSSION |
The results described here indicate that during conditioning the transmission efficiency of a particular CTC circuit may undergo long-lasting modulation in awake cats when the activation of the circuit (CS) is repetitively paired with somesthetic stimulation (UCS). The modulations may be of two opposite types depending on the topographic location of the forelimb receptive fields stimulated by UCS. They can be 1) long-lasting reinforcement of the cerebellar excitatory effects on the motor cortex occurring in parallel with an increase in the amplitude and the percentage of the forearm flexions induced by the CS during the concordant procedure, and 2) long-lasting depression of the cerebellocortical excitatory effects accompanied by the development of a large inhibitory event. Both were produced under the discordant procedure that also induced directionally opposite motor responses (forearm extensions). Because the difference between the two conditioning procedures essentially concerns the unconditioned somesthetic stimulation, the difference in conditioned effects may be due to the different somesthetic information generated by each of the two UCSs that therefore seem to be the factors most probably responsible for the switch from reinforcement to depression of cerebellocortical transmission. Such bidirectional modulations that fit well with the covariance hypothesis (Bienenstock et al. 1982
; Frégnac and Shulz 1994
; Sejnowski 1977a
,b
) have been reported to occur in the hippocampus (Barr et al. 1995
; Dudek and Bear 1993
; Thiels et al. 1994
), the visual cortex (Artola et al. 1990
; Frégnac et al. 1994
; Kirkwood and Bear 1994a
,b
), and the motor cortex (Castro-Alamancos et al. 1995
) in acute or in vitro preparations. These modulations are widely believed to be involved in learning processes. However, the studies in awake animals are very rare, and until now there has been very little direct evidence supporting this suggestion. A gap therefore remains to be filled, but the present results could contribute to progress toward the demonstration of the behavioral correlate of these synaptic effects.
To check whether similar mechanisms are involved in the conditioning of motor effects in awake cats, we used alpha conditioning procedures combined with electrophysiological and behavioral approaches. The use of IN stimulation as a CS provides a way of focusing the effects of the conditioned event on specific structures involved in motor control. Indeed, it directly activates the CTC circuits controlling the ipsilateral forelimb movement, and therefore bypasses certain structures of the auditory or visual system normally involved in the perception and the integration of these usual teleceptive CSs. Moreover, the conditioning procedures we carried out can be said to concern precisely delimited sensorimotor loops involved in the control of forelimb movements, because the cortical and cerebellar sites activated by the CS were also activated via sensory pathways by the forelimb somesthetic UCS. In the cortical part of this loop, possible contamination of the responses by remote activities can be ruled out because the transcortical recording method is known to select only the activity of the cortical dipole defined by the two tips of the pair of electrodes (Hashimoto et al. 1979
; Rispal-Padel et al. 1981
; Sasaki et al. 1981
). The effects of conditioning on the cortical responses were examined during each daily session on the basis of the cortical responses to the CS, and also during tests performed outside the conditioning sessions (i.e., independently of any predictive or other cognitive aspects that may have been acquired by the CS).
Relationships between the conditioned cerebellocortical and motor changes
The motor response parameters (amplitude and percentage) and the surface area of the waves of the cortical responses to IN stimulation were found to evolve in parallel during the acquisition and the extinction periods of the two conditioning procedures. It was also shown that the CS repetitively administered alone to naive animals did not produce any detectable effects on the motor responses or on transmission in the CTC pathway (Meftah and Rispal-Padel 1994
; Rispal-Padel and Meftah 1992
). These data, which fit well with the criteria proposed by Rose (1981)
, strongly argue in favor of causal relationships between CTC and motor changes.
On repetitive presentation of the CS alone after the acquisition period, the cortical and motor response characteristics tended toward their initial levels, exactly as the CS and the UCS did when the UCS preceded the CS with a very long interstimulus interval (900 ms). In addition, the conditioned effects were found to be specific to the circuits originating in the conditioned IN sites and did not appear with the cortical and motor responses induced from the neighboring unpaired IN control loci. These observations were made in the same awake preparation and revealed the selectivity and the associative nature of the concomitantly acquired motor and central conditioned effects. It should be noted that the associative properties of conditioned behavioral changes (Brons and Woody 1980
; Gormezano et al. 1962
; Marchetti-Gauthier et al. 1990
; Tsukahara et al. 1981
; Woody and Brozek 1969
) and those of neuronal effects (Baranyi and Feher 1981b
; Baranyi et al. 1991
; Iriki et al. 1989
, 1991
) were usually shown separately in awake animals and in acute or in vitro preparations, respectively.
Location of the relay site(s) in which the plasticity occurs
In this experiment the conditioning effects seem to be essentially exerted on the interpositothalamocorticospinal pathway, because the cerebellorubral pathway, where plastic changes have been observed in another study (Pananceau et al. 1996
), was interrupted by a neurochemical lesion of the red nucleus. Moreover, in the interpositothalamocortical pathway, the conditioning effects were found to be restricted to the circuits activated by the CS. In our previous work, we have shown that these kinds of effects are essentially supported by synaptic changes in the thalamocortical (Meftah and Rispal-Padel 1994
; Rispal-Padel and Meftah 1992
) and probably intracortical connections. Despite the appearance of significant changes in the motor and cerebellocortical responses, no changes were detected in the motor responses induced by motor cortex stimulation, attesting that neither the membrane properties of the stimulated cortical neurons nor the transmission along the corticospinal pathway or in the spinal centers was affected by the conditioning procedures (see also Rispal-Padel and Meftah 1992
). On the other hand, it has been previously shown that cerebellothalamic transmission was not affected during this kind of associative conditioning (Meftah and Rispal-Padel 1994
). Taken together, these findings indicate that thalamocortical and/or intracortical synapses are the most probable location of the changes that may occur when the cortical neurons receive inputs from different origins under associative conditions.
The plasticity evidenced in this study supports the idea that the long-lasting potentiation of the thalamocortical synapses detected in the motor cortex of acute cat preparations (Asanuma and Keller 1991
; Baranyi and Feher 1981b
; Iriki et al. 1991
; Keller et al. 1990
) could be involved in awake cats as a neuronal support of associatively conditioned motor effects. The restriction of conditioning effects to the circuits activated by the CS is in agreement with the input specificity of the long-lasting effects observed in the thalamocortical link (Baranyi and Feher 1981b
; Baranyi et al. 1991
). This property of the thalamocortical synaptic plasticity therefore may be a fundamental mechanism at the origin of conditioned behavioral discrimination tasks involving the motor cortex, such as the appearance of conditioned eye blink induced specifically by the auditory stimulus paired with the unconditioned stimulus (glabella tap) and not by another unpaired stimulus of the same modality (Engel and Woody 1972
).
Mechanisms underlying the thalamocortical changes
Among the neuronal mechanisms possibly involved in the effects observed, changes in the membrane properties of cortical neurons, like some of those assumed to occur during conditioned eye blink acquisition (Brons and Woody 1980
; Woody and Black-Cleworth 1973
), do not seem to be involved in changes of the cerebellocortical transmission seen in the present study, because motor responses induced by cortical stimulation remained unchanged after conditioning (Rispal-Padel and Meftah 1992
). This is in agreement with the fact that strong, long-lasting potentiation of the thalamocortical synaptic transmission has been observed without any changes of the membrane properties of the cortical neurons (Baranyi et al. 1991
). The enhancement of the amplitude of the excitatory negative wave under the concordant procedure therefore seems due to long-lasting facilitation of the synaptic transmission between the VL axonal endings and deep cortical neurons (Baranyi and Feher 1978
, 1981a
-c
; Baranyi et al. 1991
; Iriki et al. 1989
; Keller et al. 1990
). The enhancement of its duration could be explained by the increase of the VL effects exerted via excitatory cortical interneurons on the deep cortical cells (Purpura et al. 1964
). In contrast, the decrease of the amplitude and duration of the same cerebellocortical excitatory wave under the discordant procedure could be due to long-lasting depression of the transmission through the same mono- and polysynaptic excitatory thalamocortical pathways.
The enhancement and depression of the excitatory thalamocortical responses under the concordant and discordant procedures appear to be similar to the bidirectional modulations described in the hippocampus (Barr et al. 1995
; Dudek and Bear 1993
; Thiels et al. 1994
) and visual cortex (Artola et al. 1990
; Frégnac et al. 1994
; Kirkwood and Bear 1994a
,b
). In the CA1 pyramidal cells of the hippocampus, homosynaptic LTP (Barr et al. 1995
; Dudek and Bear 1993
; Kirkwood and Bear 1994a
) was induced by high-frequency (100 Hz) stimulation of the afferent fibers, whereas low-frequency (1 Hz) stimulation produced long-term depression (Barr et al. 1995
; Dudek and Bear 1993
; Kirkwood and Bear 1994b
). These results have been explained by the fact that CA1 pyramidal neurons are activated monosynaptically and inhibited polysynaptically via
-aminobutyric acid-releasing interneurons activated through feedforward and feedback pathways (Alger and Nicoll 1982
). As a consequence, high-frequency stimulation more likely produced repetitive coincidence of monosynaptic activation and a postsynaptic depolarized state that led to a homosynaptic LTP. In contrast, low-frequency stimulation more likely generates overlapping of monosynaptic activation with the delayed inhibition (hyperpolarization), a condition that is described as producing homosynaptic long-term depression (Thiels et al. 1994
). Induction of LTP or long-term depression therefore appears to depend on the membrane properties of postsynaptic neurons. This has been clearly demonstrated in the visual (Frégnac et al. 1994
) and motor cortex (Castro-Alamancos et al. 1995
). Our results suggest that similar bidirectional processes could also work in the motor cortex of awake cats, and could underlie the two opposite conditioned motor effects observed. Indeed, the two distinct UCSs produced different cortical responses that could induce two different patterns of cortical interactions between IN (CS) and sensory (UCS) inputs that may lead, according to the covariance hypothesis (Frégnac and Shulz 1994
), to potentiation of the thalamocortical transmission under the concordant associative condition and to its depression under the discordant condition.
The delayed positive wave of the cerebellocortical responses observed under the discordant procedure might be explained as an inhibitory effect induced in neurons of deep cortical layers (III or V) via inhibitory interneurons (Meftah and Rispal-Padel 1994
). This positive wave could also result from a reinforcement of excitatory effects mediated by superficial thalamocortical projections (Shinoda et al. 1993
); the excitatory current generated in the superficial cortical layers would then appear as a positive event on the recording from the deep layers. This latter proposal seems unlikely, because the enhancement of the positive wave remains restricted to the elbow motor representation area of the motor cortex and this is not compatible with the widespread distribution of the superficial thalamocortical projections. In addition, previous results (Meftah and Rispal-Padel 1994
) indicate that the initial cerebellocortical excitatory event was generally followed by a strong inhibition. Moreover, it is known that VL stimulation produces monosynaptic EPSPs followed by plurisynaptic IPSPs in motor cortical cells (Baranyi et al. 1993
; Purpura et al. 1964
; Sasaki and Prelevic 1972
; Sasaki et al. 1970
; Zarzecki 1991
). Then, intracortical inhibitory interneurons could be activated by collaterals of near or remote corticofugal neurons. Reinforcement of transmission to these inhibitory interneurons, and therefore the increase of their inhibitory action on the deep cortical cells, might account for the increase in the positive wave. The involvement of synaptic facilitation between inhibitory interneurons and the deep corticofugal neurons must also be taken into consideration. The latter mechanism has been described in the cerebellar cortex (Kano et al. 1992
; Vincent et al. 1992
) and in the visual cortex (Komatsu 1994
), but has not yet been investigated in the motor cortex. However, it must be noted that the latency of intracortical inhibitory effects is not compatible with their direct involvement in the depression of the earlier negative wave, which is due to another phenomenon, as discussed above.
The results of the present experiment provide information about the mechanisms by which the functional properties of the CTC pathway could be up- or down-modulated and therefore about the way in which this pathway could contribute to the elaboration of the adaptive motor responses required by external somesthetic disturbances and to motor learning processes in general. The cerebellar activation of a particular region of the motor cortex that controls forelimb movements can be reinforced or depressed in a long-lasting way depending on the somatosensory information. In addition, the intracortical inhibitory interactions that also occur could contribute to the modulation of the cerebellar influences exerted on the forelimb musculature. Such a possible function of the new inhibitory wave may also be suggested by the fact that it was not easily extinguished and persisted as long as the change (occurrence of extensions) in motor responses did. These data suggest that the functional organization of the CTC pathway, described to be at the basis of motor synergies or of simple movements and their associated postural adjustments (Rispal-Padel and Grangetto 1977
; Rispal-Padel and Latreille 1974
; Rispal-Padel et al. 1973
, 1982
), was at least partly the result of plasticity induced by repeated sensory experiences. Indeed, motor changes induced by conditioning were closely linked to modification of the thalamocortical transmission. The two kinds of events (behavioral and synaptic) strictly depended on the information given by the somesthetic UCS. Their persistence for a long period after interruption of conditioning suggests that stimuli coming from the immediate environment can, in certain associative conditions, durably contribute to adaptation of the central motor command or to learning of new motor reactions in response to a given external situation.