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Treadmill locomotion and aversive effects induced by electrical stimulation of the mesencephalic locomotor region in the rat
Brain Research
Bullrt~n.
Vol. 25, pp.
0 Pergamon Press plc, 1990.
723-721.
0361.9?30/90
Prmted in the U.S.A.
$3 00 +
00
Treadmill Locomotion and Aversive Effects Induced by Electrical Stimulation of the Mesencephalic Locomotor Region in the Rat RONAN D.N.B.C.,
Centre
DEPOORTERE,
de Neurochimie
GEORGES
du C.N.R.S.,
DI SCALA
5, rue Blaise
AND GUY SANDNER
Pascal,
F-67084
Strashourg-Cedex,
France
Received 21 May 1990
DEPOORTERE, R., G. DI SCALA tion of the mesencephalic locomotor
AND G. SANDNER. Treadmill locomotion and aversive effects induced by electrical region in the rat. BRAIN RES BULL 25(5) 723-727, 1990. -The effects of electrical
srimula-
stimulation of the “mesencephalic locomotor region” and adjacent dorsolateral tegmentum were assessed and compared in the same rats in freely moving conditions or when lightly anesthetized and suspended over a moving treadmill. In freely moving conditions, electrical brain stimulation (EBS) of this part of the mesencephalon elicited mainly aversive effects (escape reactions: violent running and On the treadmill, EBS induced flexions of hindlimbs followed by explosive jumps), but also ipsiversive circling and “gnawing.” locomotion (stepping) or flexions only. In addition, it was found that locomotion and flexions on the treadmill were almost exclusively elicited by EBS of sites positive for escape reactions in freely moving conditions. Aversive effects
Locomotion
Mesencephalic
locomotor
region
brain stimulation
Rat
Centre de Neurochimie animal breeding unit) kept on a 12- 12 h light/dark cycle (light on: 8 a.m.-8 p.m.). They were anesthetized with pentobarbital (60 mg/kg IP) and fixed in a stereotaxic frame with the bite-bar 2 mm below the earbars. They were implanted on each side with an electrode made up of 2 intertwisted 125 km nichrome enamel insulated threads, with a 0.3 to 0.6 mm dorso-ventral intertip distance. The following coordinates were used, the lambda point serving as the reference for each plane: 0.8 to 1 mm posterior, 1.2 to 1.7 mm lateral. 5.5 to 6.5 mm ventral. Threads were soldered onto a 5-pin female connector which was embedded in acrylate resin anchored to the skull by means of 4 stainless steel screws, one of which was used as the common anode.
INVESTIGATION of supraspinal neuronal substrates subserving the induction and/or control of locomotion has gained much interest since the pioneer work of Shik et al. In their original study
(17), these authors showed that locomotion could be induced by electrical stimulation of the so-called “mesencephalic locomotor region” (MLR) in mesencephalic cats (cats with a precolliculari postmamillary transverse section) suspended over a moving treadmill. On the basis of similar experiments in rats, a MLR has been described in this species, which corresponds to the cuneiform and pedunculopontine tegmental nuclei (2,20). In addition, diverse motor/locomotor patterns like locomotion and various types of stepping have also been elicited by electrical brain stimulation (EBS) of a midbrain region including the MLR in lightly anesthetized but otherwise intact rats (16, 18, 19). We have recently observed that electrical stimulation of the MLR in intact and freely moving rats produced aversive effects (3.4). In effect, such stimulations induced escape reactions, that is a frantic locomotion followed by explosive jumps. These escape reactions are in fact considered to be the overt motor expression to an aversion generated by the EBS, since a MLR stimulated rat readily learns to switch-off the EBS by pressing a bar. In the present study, we sought to investigate in the same rats the effects of EBS of the MLR and adjacent dorsolateral tegmenturn in two different conditions: in freely moving conditions or under light anesthesia and suspended over a moving treadmill. The objective was to investigate if EBS of sites inducing aversive effects (i.e., escape reactions) in freely moving conditions would induce motor/locomotor patterns on the treadmill.
Electrical Brain Stimulation The electrical brain stimulation (EBS) consisted of a continuous train of monopolar, cathodal pulses (duration: 1 ms, frequency: 70 Hz) delivered by an Alvar Physiovar TR stimulator, with a radio frequency decoupled output. Stimulation parameters were continuously monitored on a differential oscilloscope across a 100 kfi resistor in series in the stimulation circuit. Protocol A one-week postoperative delay was observed before starting the experiments. Nine rats were subjected to the behavioral observation in freely moving conditions first, then tested in the treadmill experiment. The remaining 9 rats were tested in the reverse sequence. Behavioral observation in freely moving conditions. Each rat was placed in a circular Plexiglas open field (60 cm diameter, 30
METHOD
Animals and Surgery This study was performed
Electrical
on 18 male Wistar rats (350-500 g, 723
DEPOORTERE,
724
cm high) and allowed to habituate for 10 min. All 4 stimulation sites were stimulated in a randomized order, with a 2-min pause in between each site testing, to observe the behavioral responses elicited by the EBS. The intensity was manually incremented by steps of 5 or 10 p.A (depending on the site sensitivity) every 5 s, with a cut-off value of 150 FA, and a 15-s time limit for each step. Additionally, each site was also tested at a fixed intensity having previously elicited a behavioral response with the incremented intensity procedure. For both the incremented and fixed intensity procedure, the EBS was stopped immediately when the behavioral response observed was too violent or incompatible with continuing the observation, like a violent jump out of the open field. This interruption of the EBS was done for ethical reasons. as well as to prevent the rat from hurting itself or dislodging its head connector. Treadmill experiment. The electrically driven treadmill was inspired from the one described by Sinnamon (18); its speed could be adjusted from 0 to 30 cm/s (average speed used: 25 cm/s). The rat was first lightly anesthetized with an IP injection of pentobarbital (25 mg/kg) before being fixed in a stereotaxic frame. A cloth belt was placed under the chest and belly of the animal, so as to let the anterior aspect of the hindlimbs and tip of the forelimbs contact the belt, inducing a passive extension of the hindlimbs with the belt in motion. Level of anesthesia was assessed every 15 min by testing the startle response obtained by tapping a metal object against one of the earbars (18). During the course of the experiment, the light level of anesthesia was maintained by supplemental doses of pentobarbital. Sites were stimulated in a randomized order, the intensity of the EBS being manually incremented by steps of 5 or 10 p.A (depending on the site sensitivity) every 5 s with a maximum step duration of 15 s, until a motor pattern (limb flexion or stepping) was observed. The intensity cut-off point was set at 150 pA. Each site was additionally tested at a fixed intensity having previously elicited a motor pattern with the incremented intensity method. A resting period of 2 min was applied in between each site testing. Motor positive sites were tested several times to assess the consistency of the flexion or stepping response. The EBS was immediately stopped with the appearance of overt signs of discomfort or too violent a motor response consecutively to a stimulation. Histology On completion of the experiments, rats were killed with an overdose of pentobarbital and intracardially perfused with saline followed by 4% formaldehyde. The brains were embedded in paraffin and 20 p,rn serial sections were stained with cresyl violet. The stimulation sites were localized and reported on the corresponding planes of the Paxinos and Watson atlas (14). Sites whose localization could not be precisely determined were discarded. RESULTS A total of 51 sites in 15 rats were retained and used for data. Three rats died before completion of the study; 9 sites were discarded because of imprecise histological localization. Behavioral Observation in Freely Moving Conditions Electrical stimulation of the MLR and adjacent dorsolateral tegmentum in freely moving conditions elicited 3 types of behavioral responses, The hereunder described behaviors were found to be “site-specific, ” in the sense that EBS of a distinct site induced only one type of behavioral response: 1) Escape reactions: low intensity (10-60 pA) electrical stim-
DI SCAL.4 AND SANDNEK
ulation of 32 out of the 51 sites elicited escape reactions, that 1s an initial immobility phase, followed by a frantic motor activation consisting in straight or sideways (crab-like) locomotion. interposed with arrests and/or rises and/or sudden changes of direction, This motor activation ended by a violent jump out of the open field. Stimulating at a fixed intensity (30 to 60 kA) also elicited within a few seconds a vigorous jump out of the open field, preceded or not by a short and frantic run (gallop). These 32 escape positive sites appear in the left panel of the histological figure. They were found in the cuneiform nucleus. pedunculopontine tegmental nucleus, ventral part of inferior colliculus, dorsolateral edges of periaqueductal gray and deep layers of superior colliculus. 2) Circling activity: electrical stimulation of 8 sites elicited an ipsiversive circling activity consisting in rotations accompanied by a shift of the whole body and a turn of the head towards the side ipsilateral to the site of stimulation. The frequency and vigor of this circling increased with increment in the intensity of the EBS. These 8 sites were mostly located between the cuneiform nucleus and the pedunculopontine tegmental nucleus (see Fig. 1. left panel). 3) Gnawing behavior: 7 sites induced a “gnawing” behavior when stimulated. This “gnawing” could be best described as apparent biting of the animal’s own front paws or forelimbs. This “gnawing” was limited to one front paw (either ipsilateral or contralateral to the stimulated site) at low intensities and could either increase in vigor and/or be directed towards other parts of the body, like the flank, with higher intensities. “Gnawing” ceased immediately at the offset of the stimulation, with no bleeding or apparent wounding of the gnawn parts or suffering of the animal. As shown in the left panel of Fig. 1, 6 of these sites were located in the caudal part of the inferior colliculus, the remaining one was at the border of the dorsal nucleus of lateral lemniscus. In addition, the stimulation of 4 sites generated no overt behavior up to the cut-off intensity (150 p,A). All these sites were located in the caudal part of the inferior colliculus. Treadmill Experiment Electrical stimulation of 22151 sites in the MLR and adjacent dorsolateral tegmentum in the rat lightly anesthetized and suspended over a moving treadmill elicited 2 types of motor/locomotor patterns: 1) Flenion of the hindlimbs followed by locomotion (stepping): electrical stimulation of 8 sites induced a progressive hindlimbs flexion followed by stepping. This locomotion was quadrupedal in 7/8 cases, bipedal and ipsilateral in the remaining case. With the incremented intensity protocol, stimulating at subthreshold intensity elicited a progressive flexion of the hindlimbs whereas intensity just above threshold generated a locomotion, which was fairly disorganized, mostly of a gallop type and frequently accompanied by violent leaping which necessitated the immediate cessation of the stimulation to prevent the rat from hurting itself at the level of the earbars or the bite-bar. Threshold intensities for inducing such locomotion were comprised between 40 and 90 p.A, and were the same whether the incremented intensity or fixed intensity method was aDDtied. However. successive stimulations of a locomotion positive-site, at an intensity known to induce locomotion, did not systematically elicit a locomotor activity. In effect, a mere flexion or erratic limb movements were elicited instead. Locomotion could only be elicited during the late phase of anesthesia, when it was possible to observe a startle reaction consecutively to the tapping of a metal object against one of
MLR. LOCOMOTION,
AVERSIVE
725
EFFECTS
FIG. 1. Histological localization of the 51 electrical brain stimulation (EBS) sites used in the behavioral observation and treadmill experiment. For clarity sake, all stimulation sites have been reported on the same side of the drawings, adapted from the atlas of Paxinos and Watson (14). Left panel: type of behavioral activity elicited by the EBS in the behavioral observation: (0): escape reactions; (m): ipsiversive circling; (*): gnawing; (0): no overt behavior. Right panel: type of motor/locomotor activity elicited by EBS in the treadmill experiment: (0): hindlimbs flexions followed by locomotion (stepping); (Ir): hindlimbs flexions only; (0): no motor pattern. Abbreviations: CN: cuneiform nucleus; IC: inferior colliculus; MiTg: microcellular tegmental nucleus; Mo5: motor trigeminal nucleus; PAG: periaqueductal gray; PB: parabrachial nucleus; PPTg: pedunculopontine tegmental nucleus; SC: superior colliculus; SCP: superior cerebellar peduncle.
the earbars. On the other hand, flexions could be generated during earlier stages of anesthesia. These locomotion positive sites were found within the deep layers of the superior colliculus, ventral part of inferior colliculus, periaqueductal gray and cuneiform nucleus (right panel of Fig. 1). 2) Flexion of the hindlimbs only: flexion of the hindlimbs without locomotion was elicited by electrical stimulation of 14 sites, using a range of intensities of 40-90 kA. These flexions were consistently elicited upon stimulation, even at the early
stages of anesthesia, when no startle reaction could be produced by the method used. Furthermore, the threshold intensity to induce these flexions was the same, whether the intensity was incremented, or a fixed intensity was applied. These sites were located in the cuneiform nucleus, pedunculopontine tegmental nucleus, superior cerebellar peduncle, ventral part of inferior colliculus and periaqueductal gray (see Fig. 1, right panel). Stimulation of the remaining 29 sites did not generate any motor/locomotor pattern up to the cut-off intensity of 150 )*A.
DEPOORTERE, DI SC-MA
126
TABLE 1 COMBINEDFREQUENCYTABLEFOR BEHAVIORALAND MOTOR EFFECTSINDUCEDBY ELECTRICALSTIMULATIONS OF THE MESENCEPHALIC LOCOMOTORREGIONAND ADJ.ACENT ~RSOLATERAL TEGME~M Motor Positive Locomotion
Flexions
Motor Negative
8 0 0 0
10 4 0 0
14 4 7 4
Escape Reaction Positive Escape Reaction Negative
Circling Gnawing Negative
Each cell represents the number of sites having elicited the corresponding motor pattern in the column and corresponding behavior in the row (n=Sl sites). Comparison Between Effects of Electrical Stimulation in Freely Moving Conditions and in the Treadmill Experiment In the table are summarized both behavioral observation and treadmill experiment results. It clearly appears that locomotion was exclusively obtained by EBS of sites positive for escape reactions, whereas flexion of the hindlimbs was mostly obtained from escape reaction positive sites, but also from some ipsiversive circling sites. Sites whose EBS induced no motor pattern on the treadmill were equally distributed between escape reaction positive and escape. reaction negative sites. A Pearson chi-square test (5) was used to test for independence of rows and columns, after pooling of data into 4 classes: escape reaction positive, escape reaction negative, motor positive and motor negative. This statistical analysis confiied the link between induction of escape reactions in freely moving conditions and induction of motor/locomotor patterns (locomotion and hindlimbs flexions) on the treadmill, x2( 1) = 6.02, pCO.02. DISCUSSION
The present findings show that in the freely moving rat, electrical brain stim~ation (EBS) of the “mesencephalic locomotor region” and adjacent dorsolateral tegmentum induced escape reactions. These EBS also induced locomotion (stepping) and flexions of the hindlimbs in the same rats lightly anesthetized and suspended over a moving treadmill. Moreover, it was found that in the treadmill experiment, eliciting of motor~ocomotor patterns (locomotion and flexions) was almost exclusively obtained by EBS of sites positive for escape reactions in freely moving conditions. In the freely moving rat, EBS of sites in the mesencephalic locomotor region (MLR) and adjacent dorsolateral tegmentum elicited escape reactions. These present results are in line with our previous findings (3,4), as well as those of 3 other papers repoking ‘ ‘escape activity, ” “fast run following immobility” and “galloping and leaping, sometimes accompanied by vigorous jumping” with electrical stimulation of this brain region (11, 15, 22). Such escape reactions do not appear to constitute mere motor activations, but seem instead to repsent the overt behavioral response to aversive effects generated by the EBBS.In effect, in previous experiments, we have consistently found that EBS of sites positive for escape reactions in and around tbe MLR induced aversive effects, since such an EBS prompts a stimulated rat to switch-off the EBS by pressing a bar (3,4). Additionally, when tested in intact and freely moving animals, effects of EBS of the MLR have been reported to differ strikingly from those obtained
AND
SANDNER
with mesencephalic preparations. For example, in intact cat>. MLR stimulations have been shown to induce “fast walking to running movement propulsive in nature” (jumps?) and “Observation of the cat under MLR stimulation initially gave an impression to the observer that it was trying to avoid a noxious stimulus, .” (12). Escape reactions were not the only behaviors elicited by EBS of this brain region; ipsiversive circling and “gnawing” were also observed. However, and in particular for the “gnawing,” sites from which such behaviors were elicited were fairly well segregated from escape reaction positive sites. The type of ipsiversive circling we observed has been previously described in the literature (1.5). However, the description of the “gnawing” behavior we presently report does not fit with the diverse forms of “biting,” “chewing,” “gnawing,” “grooming” or ‘*nibbling” described in several studies concerned with EBS of the mesencephalon (10, 15, 21, 22). In particular. this presently reported “gnawing” never included sequences of licking or pulling at the nails or profuse salivation, and was not restricted to the oral region, as mentioned in two of these studies (lo,21 j. This “gnawing” also differed from the “nibbling” reported by EBS of sites in the cerebellum. since we never observed bleeding of the gnawn forepaws (23). Lastly, it departs from the gnawin~biting which is encountered in self-mutilation behavior, consecutive to limb deafferentation (6), intranigral GABA agonists microinjections (1) or various pharmacological treatments [see (1) for brief reviewj. This “gnawing” behavior seems, however, interesting enough to warrant further study. In the rat lightly anesthetized and suspended over a moving treadmill, electrical stimulation of sites in the MLR and adjacent dorsolateral tegmentum elicited locomotion (stepping) and flexions of the h~ndlimbs. Stepping was reported in previous studies using the same protocol (16,lS) with EBS of sites localized within the same mesencephalic structures as those in which we found the motor positive sites. The vigorous type of locomotion we observed, and which is suggestive of the aversive nature of the EBS, was also described in one of these studies (18). Oddly enough. hindlimb flexions not followed by locomotion were not reported in either two of these studies. Differences in the type of anesthesia, size of electrode or characteristics of the electrical stimulation might explain such a discrepancy. The totality of sites inducing locomotion and great majority of sites inducing flexions on the treadmill were found to be positive for escape reactions in freely moving conditions. Given that EBS of sites positive for escape reactions have been repeatedly and consistently shown to induce aversive effects (3,4), our results support the hypothesis that in the rat lightly anesthetized and suspended over a treadmill, locomotion and flexions could represent the overt motor response expressed as a consequence of the aversion induced by the EBS. The induction of flexions by EBS of 4 sites positive for circling behavior might seem out of line with the above exposed hypothesis. However, we have observed in this laboratory that EBS of sites in the deep layers of the superior colliculus, which induce ipsiversive circling, are aversive, since such EBS support the learning of switch-off behavior (unpublished). Also, EBS of sites inducing a similar type of circling behavior has been previously reported to generate aversive effects (9). It is thus not impossible that EBS of sites inducing ipsiversive circling in this study had an aversive component. The absence of stepping or hindlimbs flexions with EBS of half of the sites positive for escape reactions is most probably due to interference of the anesthetic with the expression of such motor/locomotor patterns. In fact, we found that eliciting of these patterns, and especially locomotion, was highly dependent on the level of anesthesia. This interfering effect constitutes one of the main rea-
MLR, LOCOMOTION,
AVERSIVE
EFFECTS
727
sons for the use of mesencephalic preparations, which are considered not to necessitate the use of an anesthetic. The other rationale behind the choice for a mesencephalic preparation in studies of locomotion is that such a preparation is “deprived of nociceptive perception” (13). Some data in the literature do not support such an assumption. Thus, in decerebrate rats (same level of transection as the mesencephalic preparation) noxious pinching of hindlimbs consistently elicited vocalization and limb flexion. suggestive of nociceptive perception (7). Also, detelencephalated rats (same level of transection as mesencephalit preparation but sparing of hypothalamus) have been shown to present avoidance conditioning to noxious electrical shocks applied to the tail (8). Lastly, it appears that EBS of some locomotion inducing sites in mesencephalic preparations also elicits vocalization, urination and defecation. (Perret. personal commu-
nication), which are usually considered as characteristic of aversive effects. Taken together, all these considerations raise the possibility that, like the explosive type of behavior seen in intact and freely moving animals, the locomotion observed with EBS of the MLR in animals lightly anesthetized and suspended over a moving treadmill-and possibly in mesencephalic preparationscould represent the overt motor response to aversive effects generated by the EBS. ACKNOWLEDGEMEKTS This study was supported by the CNRS, INSERM. French Ministry of Education as well as by a grant from the “Fondation pour la Recherche Medicale Francaise.” drawing the figures.
We are very grateful
to Gaby Rudolf for
REFERENCES I. Baumeister. A. A.; Frye, G. D. Self-injurious behavior in rats produced by intranigral microinjection of GABA agonists. Pharmacol. Biochem. Behav. 21:89-95; 1984. 2. Coles, S. K.; Iles. J. F.; Nicolopoulos-Stoumaras, S. The mesencephalic centre controlling locomotion in the rat. Neuroscience 28: 149-157: 1989. 3. Depoortere, R.; Sandner. G.; Di Scala, G. Aversion induced by electrical stimulation of the mesencephalic locomotor region in the intact and freely moving rat. Physiol. Behav. 47:561-567; 1990. 4. Depoortere, R.: Di Scala, G.; Angst, M. J.; Sandner, G. Differential pharmacological reactivity of aversion induced by stimulation of periaqueductal gray or mesencephalic locomotor region. Pharmacol. Biochem. Behav. 37:3ll-316: 1990. 5. Dixon, W. J. BMDP statistical software. Berkeley: University of California Press; 1988. 6. Duckrow. R. B.; Taub. A. The effect of diphenylhydantoin on selfmutilation in rats produced by unilateral multiple dorsal rhizotomy. Exp. Neurol. 54:3341: 1977. 7. Grill. H. J.; Norgren, R. Neurological tests and behavioral deficits in chronic thalamic and chronic decerebrate rats. Brain Res. 143:299312; 1978. 8. Huston, J. P.: Tomaz. C.; Fix, I. Avoidance learning in rats devoid of the telencephalon plus thalamus. Behav. Brain Res. 17:87-95; 1985. 9. Kiser, R. S.; Lebovitz, R. M.; German, D. C. Anatomic and pharmacologic differences between two types of aversive midbrain stimulation. Brain Res. 155:331-342; 1978. 10. Micco. D. J. Complex behaviors elicited by stimulation of the dorsal pontine tegmentum in rats. Brain Res. 75:172-176; 1974. Il. Mimer. K. L.; Mogenson, G. J. Electrical and chemical activation of the mesencephalic and subthalamic locomotor regions in freely moving rats. Brain Res. 452:273-285; 1988. 12. Mori, S.: Sakamoto. T.: Ohta, Y.: Takakusaki. K.; Matsuyama. K. Site-specific postural and locomotor changes evoked in awake, freely
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moving intact cats by stimulating the brainstem. Brain Res. 505:6674; 1989. Orlovsky. G. N.; Shik. M. L. Control of locomotion: a neurophysiological analysis of the cat locomotor system. Int. Rev. Physiol. IO: 281-317: 1976. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. New York: Academic Press; 1986. Robinson. T. E. Electrical stimulation of the brain stem in freely moving rats: I. Effects on behavior. Physiol. Behav,. 21:223-231; 1978. Ross, G. S.; Sinnamon, H. M. Forelimb and hindlimb stepping by the anesthetized rat elicited by electrical stimulation of the pons and medulla. Physiol. Behav. 33:201-208: 1984. Shik, M. L.; Severin, F. V.; Orlovsky. G. N. Control of walking and running by means of electrical stimulation of the mid-brain. Biophysics I1:756-765: 1966 (English translation of Biofizyka Il:659-666; 1966). Sinnamon, H. M. Forelimb and hindlimb stepping by the anesthetized rat elicited by electrical stimulation of the diencephalon and mesencephalon. Physiol. Behav. 33:191-199; 1984. Sinnamon, H. M.; Ginzburg, R. N.; Kurose. G. A. Midbrain stimulation in the anesthetized rat: direct locomotor effects and modulation of locomotion produced by hypothalamic stimulation. Neuroscience 20:695-707: 1987. Skinner. R. D.: Garcia-Rill, E. The mesencephalic locomotor region (MLR) in the rat. Brain Res. 323:385-389: 1984. Van Der Kooy. D. An analysis of the behavior elicited by stimulation of the dorsal pons in rat. Physiol. Behav. 23:427--132; 1979. Waldbillig, R. J. Attack, eating. drinking and gnawing elicited by electrical stimulation of rat mesencephalon and pans. J. Comp. Physiol. Psychol. 89:200-212: 1975. Watson. P. J. Behavior maintained by electrical stimulation of the rat cerebellum. Physiol. Behav. 21:749-755: 1978.
Bullrt~n.
Vol. 25, pp.
0 Pergamon Press plc, 1990.
723-721.
0361.9?30/90
Prmted in the U.S.A.
$3 00 +
00
Treadmill Locomotion and Aversive Effects Induced by Electrical Stimulation of the Mesencephalic Locomotor Region in the Rat RONAN D.N.B.C.,
Centre
DEPOORTERE,
de Neurochimie
GEORGES
du C.N.R.S.,
DI SCALA
5, rue Blaise
AND GUY SANDNER
Pascal,
F-67084
Strashourg-Cedex,
France
Received 21 May 1990
DEPOORTERE, R., G. DI SCALA tion of the mesencephalic locomotor
AND G. SANDNER. Treadmill locomotion and aversive effects induced by electrical region in the rat. BRAIN RES BULL 25(5) 723-727, 1990. -The effects of electrical
srimula-
stimulation of the “mesencephalic locomotor region” and adjacent dorsolateral tegmentum were assessed and compared in the same rats in freely moving conditions or when lightly anesthetized and suspended over a moving treadmill. In freely moving conditions, electrical brain stimulation (EBS) of this part of the mesencephalon elicited mainly aversive effects (escape reactions: violent running and On the treadmill, EBS induced flexions of hindlimbs followed by explosive jumps), but also ipsiversive circling and “gnawing.” locomotion (stepping) or flexions only. In addition, it was found that locomotion and flexions on the treadmill were almost exclusively elicited by EBS of sites positive for escape reactions in freely moving conditions. Aversive effects
Locomotion
Mesencephalic
locomotor
region
brain stimulation
Rat
Centre de Neurochimie animal breeding unit) kept on a 12- 12 h light/dark cycle (light on: 8 a.m.-8 p.m.). They were anesthetized with pentobarbital (60 mg/kg IP) and fixed in a stereotaxic frame with the bite-bar 2 mm below the earbars. They were implanted on each side with an electrode made up of 2 intertwisted 125 km nichrome enamel insulated threads, with a 0.3 to 0.6 mm dorso-ventral intertip distance. The following coordinates were used, the lambda point serving as the reference for each plane: 0.8 to 1 mm posterior, 1.2 to 1.7 mm lateral. 5.5 to 6.5 mm ventral. Threads were soldered onto a 5-pin female connector which was embedded in acrylate resin anchored to the skull by means of 4 stainless steel screws, one of which was used as the common anode.
INVESTIGATION of supraspinal neuronal substrates subserving the induction and/or control of locomotion has gained much interest since the pioneer work of Shik et al. In their original study
(17), these authors showed that locomotion could be induced by electrical stimulation of the so-called “mesencephalic locomotor region” (MLR) in mesencephalic cats (cats with a precolliculari postmamillary transverse section) suspended over a moving treadmill. On the basis of similar experiments in rats, a MLR has been described in this species, which corresponds to the cuneiform and pedunculopontine tegmental nuclei (2,20). In addition, diverse motor/locomotor patterns like locomotion and various types of stepping have also been elicited by electrical brain stimulation (EBS) of a midbrain region including the MLR in lightly anesthetized but otherwise intact rats (16, 18, 19). We have recently observed that electrical stimulation of the MLR in intact and freely moving rats produced aversive effects (3.4). In effect, such stimulations induced escape reactions, that is a frantic locomotion followed by explosive jumps. These escape reactions are in fact considered to be the overt motor expression to an aversion generated by the EBS, since a MLR stimulated rat readily learns to switch-off the EBS by pressing a bar. In the present study, we sought to investigate in the same rats the effects of EBS of the MLR and adjacent dorsolateral tegmenturn in two different conditions: in freely moving conditions or under light anesthesia and suspended over a moving treadmill. The objective was to investigate if EBS of sites inducing aversive effects (i.e., escape reactions) in freely moving conditions would induce motor/locomotor patterns on the treadmill.
Electrical Brain Stimulation The electrical brain stimulation (EBS) consisted of a continuous train of monopolar, cathodal pulses (duration: 1 ms, frequency: 70 Hz) delivered by an Alvar Physiovar TR stimulator, with a radio frequency decoupled output. Stimulation parameters were continuously monitored on a differential oscilloscope across a 100 kfi resistor in series in the stimulation circuit. Protocol A one-week postoperative delay was observed before starting the experiments. Nine rats were subjected to the behavioral observation in freely moving conditions first, then tested in the treadmill experiment. The remaining 9 rats were tested in the reverse sequence. Behavioral observation in freely moving conditions. Each rat was placed in a circular Plexiglas open field (60 cm diameter, 30
METHOD
Animals and Surgery This study was performed
Electrical
on 18 male Wistar rats (350-500 g, 723
DEPOORTERE,
724
cm high) and allowed to habituate for 10 min. All 4 stimulation sites were stimulated in a randomized order, with a 2-min pause in between each site testing, to observe the behavioral responses elicited by the EBS. The intensity was manually incremented by steps of 5 or 10 p.A (depending on the site sensitivity) every 5 s, with a cut-off value of 150 FA, and a 15-s time limit for each step. Additionally, each site was also tested at a fixed intensity having previously elicited a behavioral response with the incremented intensity procedure. For both the incremented and fixed intensity procedure, the EBS was stopped immediately when the behavioral response observed was too violent or incompatible with continuing the observation, like a violent jump out of the open field. This interruption of the EBS was done for ethical reasons. as well as to prevent the rat from hurting itself or dislodging its head connector. Treadmill experiment. The electrically driven treadmill was inspired from the one described by Sinnamon (18); its speed could be adjusted from 0 to 30 cm/s (average speed used: 25 cm/s). The rat was first lightly anesthetized with an IP injection of pentobarbital (25 mg/kg) before being fixed in a stereotaxic frame. A cloth belt was placed under the chest and belly of the animal, so as to let the anterior aspect of the hindlimbs and tip of the forelimbs contact the belt, inducing a passive extension of the hindlimbs with the belt in motion. Level of anesthesia was assessed every 15 min by testing the startle response obtained by tapping a metal object against one of the earbars (18). During the course of the experiment, the light level of anesthesia was maintained by supplemental doses of pentobarbital. Sites were stimulated in a randomized order, the intensity of the EBS being manually incremented by steps of 5 or 10 p.A (depending on the site sensitivity) every 5 s with a maximum step duration of 15 s, until a motor pattern (limb flexion or stepping) was observed. The intensity cut-off point was set at 150 pA. Each site was additionally tested at a fixed intensity having previously elicited a motor pattern with the incremented intensity method. A resting period of 2 min was applied in between each site testing. Motor positive sites were tested several times to assess the consistency of the flexion or stepping response. The EBS was immediately stopped with the appearance of overt signs of discomfort or too violent a motor response consecutively to a stimulation. Histology On completion of the experiments, rats were killed with an overdose of pentobarbital and intracardially perfused with saline followed by 4% formaldehyde. The brains were embedded in paraffin and 20 p,rn serial sections were stained with cresyl violet. The stimulation sites were localized and reported on the corresponding planes of the Paxinos and Watson atlas (14). Sites whose localization could not be precisely determined were discarded. RESULTS A total of 51 sites in 15 rats were retained and used for data. Three rats died before completion of the study; 9 sites were discarded because of imprecise histological localization. Behavioral Observation in Freely Moving Conditions Electrical stimulation of the MLR and adjacent dorsolateral tegmentum in freely moving conditions elicited 3 types of behavioral responses, The hereunder described behaviors were found to be “site-specific, ” in the sense that EBS of a distinct site induced only one type of behavioral response: 1) Escape reactions: low intensity (10-60 pA) electrical stim-
DI SCAL.4 AND SANDNEK
ulation of 32 out of the 51 sites elicited escape reactions, that 1s an initial immobility phase, followed by a frantic motor activation consisting in straight or sideways (crab-like) locomotion. interposed with arrests and/or rises and/or sudden changes of direction, This motor activation ended by a violent jump out of the open field. Stimulating at a fixed intensity (30 to 60 kA) also elicited within a few seconds a vigorous jump out of the open field, preceded or not by a short and frantic run (gallop). These 32 escape positive sites appear in the left panel of the histological figure. They were found in the cuneiform nucleus. pedunculopontine tegmental nucleus, ventral part of inferior colliculus, dorsolateral edges of periaqueductal gray and deep layers of superior colliculus. 2) Circling activity: electrical stimulation of 8 sites elicited an ipsiversive circling activity consisting in rotations accompanied by a shift of the whole body and a turn of the head towards the side ipsilateral to the site of stimulation. The frequency and vigor of this circling increased with increment in the intensity of the EBS. These 8 sites were mostly located between the cuneiform nucleus and the pedunculopontine tegmental nucleus (see Fig. 1. left panel). 3) Gnawing behavior: 7 sites induced a “gnawing” behavior when stimulated. This “gnawing” could be best described as apparent biting of the animal’s own front paws or forelimbs. This “gnawing” was limited to one front paw (either ipsilateral or contralateral to the stimulated site) at low intensities and could either increase in vigor and/or be directed towards other parts of the body, like the flank, with higher intensities. “Gnawing” ceased immediately at the offset of the stimulation, with no bleeding or apparent wounding of the gnawn parts or suffering of the animal. As shown in the left panel of Fig. 1, 6 of these sites were located in the caudal part of the inferior colliculus, the remaining one was at the border of the dorsal nucleus of lateral lemniscus. In addition, the stimulation of 4 sites generated no overt behavior up to the cut-off intensity (150 p,A). All these sites were located in the caudal part of the inferior colliculus. Treadmill Experiment Electrical stimulation of 22151 sites in the MLR and adjacent dorsolateral tegmentum in the rat lightly anesthetized and suspended over a moving treadmill elicited 2 types of motor/locomotor patterns: 1) Flenion of the hindlimbs followed by locomotion (stepping): electrical stimulation of 8 sites induced a progressive hindlimbs flexion followed by stepping. This locomotion was quadrupedal in 7/8 cases, bipedal and ipsilateral in the remaining case. With the incremented intensity protocol, stimulating at subthreshold intensity elicited a progressive flexion of the hindlimbs whereas intensity just above threshold generated a locomotion, which was fairly disorganized, mostly of a gallop type and frequently accompanied by violent leaping which necessitated the immediate cessation of the stimulation to prevent the rat from hurting itself at the level of the earbars or the bite-bar. Threshold intensities for inducing such locomotion were comprised between 40 and 90 p.A, and were the same whether the incremented intensity or fixed intensity method was aDDtied. However. successive stimulations of a locomotion positive-site, at an intensity known to induce locomotion, did not systematically elicit a locomotor activity. In effect, a mere flexion or erratic limb movements were elicited instead. Locomotion could only be elicited during the late phase of anesthesia, when it was possible to observe a startle reaction consecutively to the tapping of a metal object against one of
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FIG. 1. Histological localization of the 51 electrical brain stimulation (EBS) sites used in the behavioral observation and treadmill experiment. For clarity sake, all stimulation sites have been reported on the same side of the drawings, adapted from the atlas of Paxinos and Watson (14). Left panel: type of behavioral activity elicited by the EBS in the behavioral observation: (0): escape reactions; (m): ipsiversive circling; (*): gnawing; (0): no overt behavior. Right panel: type of motor/locomotor activity elicited by EBS in the treadmill experiment: (0): hindlimbs flexions followed by locomotion (stepping); (Ir): hindlimbs flexions only; (0): no motor pattern. Abbreviations: CN: cuneiform nucleus; IC: inferior colliculus; MiTg: microcellular tegmental nucleus; Mo5: motor trigeminal nucleus; PAG: periaqueductal gray; PB: parabrachial nucleus; PPTg: pedunculopontine tegmental nucleus; SC: superior colliculus; SCP: superior cerebellar peduncle.
the earbars. On the other hand, flexions could be generated during earlier stages of anesthesia. These locomotion positive sites were found within the deep layers of the superior colliculus, ventral part of inferior colliculus, periaqueductal gray and cuneiform nucleus (right panel of Fig. 1). 2) Flexion of the hindlimbs only: flexion of the hindlimbs without locomotion was elicited by electrical stimulation of 14 sites, using a range of intensities of 40-90 kA. These flexions were consistently elicited upon stimulation, even at the early
stages of anesthesia, when no startle reaction could be produced by the method used. Furthermore, the threshold intensity to induce these flexions was the same, whether the intensity was incremented, or a fixed intensity was applied. These sites were located in the cuneiform nucleus, pedunculopontine tegmental nucleus, superior cerebellar peduncle, ventral part of inferior colliculus and periaqueductal gray (see Fig. 1, right panel). Stimulation of the remaining 29 sites did not generate any motor/locomotor pattern up to the cut-off intensity of 150 )*A.
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TABLE 1 COMBINEDFREQUENCYTABLEFOR BEHAVIORALAND MOTOR EFFECTSINDUCEDBY ELECTRICALSTIMULATIONS OF THE MESENCEPHALIC LOCOMOTORREGIONAND ADJ.ACENT ~RSOLATERAL TEGME~M Motor Positive Locomotion
Flexions
Motor Negative
8 0 0 0
10 4 0 0
14 4 7 4
Escape Reaction Positive Escape Reaction Negative
Circling Gnawing Negative
Each cell represents the number of sites having elicited the corresponding motor pattern in the column and corresponding behavior in the row (n=Sl sites). Comparison Between Effects of Electrical Stimulation in Freely Moving Conditions and in the Treadmill Experiment In the table are summarized both behavioral observation and treadmill experiment results. It clearly appears that locomotion was exclusively obtained by EBS of sites positive for escape reactions, whereas flexion of the hindlimbs was mostly obtained from escape reaction positive sites, but also from some ipsiversive circling sites. Sites whose EBS induced no motor pattern on the treadmill were equally distributed between escape reaction positive and escape. reaction negative sites. A Pearson chi-square test (5) was used to test for independence of rows and columns, after pooling of data into 4 classes: escape reaction positive, escape reaction negative, motor positive and motor negative. This statistical analysis confiied the link between induction of escape reactions in freely moving conditions and induction of motor/locomotor patterns (locomotion and hindlimbs flexions) on the treadmill, x2( 1) = 6.02, pCO.02. DISCUSSION
The present findings show that in the freely moving rat, electrical brain stim~ation (EBS) of the “mesencephalic locomotor region” and adjacent dorsolateral tegmentum induced escape reactions. These EBS also induced locomotion (stepping) and flexions of the hindlimbs in the same rats lightly anesthetized and suspended over a moving treadmill. Moreover, it was found that in the treadmill experiment, eliciting of motor~ocomotor patterns (locomotion and flexions) was almost exclusively obtained by EBS of sites positive for escape reactions in freely moving conditions. In the freely moving rat, EBS of sites in the mesencephalic locomotor region (MLR) and adjacent dorsolateral tegmentum elicited escape reactions. These present results are in line with our previous findings (3,4), as well as those of 3 other papers repoking ‘ ‘escape activity, ” “fast run following immobility” and “galloping and leaping, sometimes accompanied by vigorous jumping” with electrical stimulation of this brain region (11, 15, 22). Such escape reactions do not appear to constitute mere motor activations, but seem instead to repsent the overt behavioral response to aversive effects generated by the EBBS.In effect, in previous experiments, we have consistently found that EBS of sites positive for escape reactions in and around tbe MLR induced aversive effects, since such an EBS prompts a stimulated rat to switch-off the EBS by pressing a bar (3,4). Additionally, when tested in intact and freely moving animals, effects of EBS of the MLR have been reported to differ strikingly from those obtained
AND
SANDNER
with mesencephalic preparations. For example, in intact cat>. MLR stimulations have been shown to induce “fast walking to running movement propulsive in nature” (jumps?) and “Observation of the cat under MLR stimulation initially gave an impression to the observer that it was trying to avoid a noxious stimulus, .” (12). Escape reactions were not the only behaviors elicited by EBS of this brain region; ipsiversive circling and “gnawing” were also observed. However, and in particular for the “gnawing,” sites from which such behaviors were elicited were fairly well segregated from escape reaction positive sites. The type of ipsiversive circling we observed has been previously described in the literature (1.5). However, the description of the “gnawing” behavior we presently report does not fit with the diverse forms of “biting,” “chewing,” “gnawing,” “grooming” or ‘*nibbling” described in several studies concerned with EBS of the mesencephalon (10, 15, 21, 22). In particular. this presently reported “gnawing” never included sequences of licking or pulling at the nails or profuse salivation, and was not restricted to the oral region, as mentioned in two of these studies (lo,21 j. This “gnawing” also differed from the “nibbling” reported by EBS of sites in the cerebellum. since we never observed bleeding of the gnawn forepaws (23). Lastly, it departs from the gnawin~biting which is encountered in self-mutilation behavior, consecutive to limb deafferentation (6), intranigral GABA agonists microinjections (1) or various pharmacological treatments [see (1) for brief reviewj. This “gnawing” behavior seems, however, interesting enough to warrant further study. In the rat lightly anesthetized and suspended over a moving treadmill, electrical stimulation of sites in the MLR and adjacent dorsolateral tegmentum elicited locomotion (stepping) and flexions of the h~ndlimbs. Stepping was reported in previous studies using the same protocol (16,lS) with EBS of sites localized within the same mesencephalic structures as those in which we found the motor positive sites. The vigorous type of locomotion we observed, and which is suggestive of the aversive nature of the EBS, was also described in one of these studies (18). Oddly enough. hindlimb flexions not followed by locomotion were not reported in either two of these studies. Differences in the type of anesthesia, size of electrode or characteristics of the electrical stimulation might explain such a discrepancy. The totality of sites inducing locomotion and great majority of sites inducing flexions on the treadmill were found to be positive for escape reactions in freely moving conditions. Given that EBS of sites positive for escape reactions have been repeatedly and consistently shown to induce aversive effects (3,4), our results support the hypothesis that in the rat lightly anesthetized and suspended over a treadmill, locomotion and flexions could represent the overt motor response expressed as a consequence of the aversion induced by the EBS. The induction of flexions by EBS of 4 sites positive for circling behavior might seem out of line with the above exposed hypothesis. However, we have observed in this laboratory that EBS of sites in the deep layers of the superior colliculus, which induce ipsiversive circling, are aversive, since such EBS support the learning of switch-off behavior (unpublished). Also, EBS of sites inducing a similar type of circling behavior has been previously reported to generate aversive effects (9). It is thus not impossible that EBS of sites inducing ipsiversive circling in this study had an aversive component. The absence of stepping or hindlimbs flexions with EBS of half of the sites positive for escape reactions is most probably due to interference of the anesthetic with the expression of such motor/locomotor patterns. In fact, we found that eliciting of these patterns, and especially locomotion, was highly dependent on the level of anesthesia. This interfering effect constitutes one of the main rea-
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sons for the use of mesencephalic preparations, which are considered not to necessitate the use of an anesthetic. The other rationale behind the choice for a mesencephalic preparation in studies of locomotion is that such a preparation is “deprived of nociceptive perception” (13). Some data in the literature do not support such an assumption. Thus, in decerebrate rats (same level of transection as the mesencephalic preparation) noxious pinching of hindlimbs consistently elicited vocalization and limb flexion. suggestive of nociceptive perception (7). Also, detelencephalated rats (same level of transection as mesencephalit preparation but sparing of hypothalamus) have been shown to present avoidance conditioning to noxious electrical shocks applied to the tail (8). Lastly, it appears that EBS of some locomotion inducing sites in mesencephalic preparations also elicits vocalization, urination and defecation. (Perret. personal commu-
nication), which are usually considered as characteristic of aversive effects. Taken together, all these considerations raise the possibility that, like the explosive type of behavior seen in intact and freely moving animals, the locomotion observed with EBS of the MLR in animals lightly anesthetized and suspended over a moving treadmill-and possibly in mesencephalic preparationscould represent the overt motor response to aversive effects generated by the EBS. ACKNOWLEDGEMEKTS This study was supported by the CNRS, INSERM. French Ministry of Education as well as by a grant from the “Fondation pour la Recherche Medicale Francaise.” drawing the figures.
We are very grateful
to Gaby Rudolf for
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