Electrical and chemical activation of the mesencephalic and subthalamic locomotor regions in freely moving rats

Brain Research, 452 (1988) 273-285

273

Elsevier BRE 13648

Electrical and chemical activation of the mesencephalic and subthalamic locomotor regions in freely moving rats Kelly L. Milner and Gordon J. Mogenson Department of Physiology, Universityof Western Ontario, London, Ont. (Canada) (Accepted 22 December 1987)

Key words: Locomotor activity; Mesencephalic locomotor region; Subthalamic locomotor region; Pedunculopontinenucleus; Zona incerta; Subthalamic nucleus; Chemical microinjection; Electrical stimulation

The locomotor activity of freely moving rats was increased by electrical stimulation of brainstem sites, including the pedunculopontine nucleus, a major component of the mesencephalic locomotor region (MLR), and sites located in the subthalamic locomotor region (SLR), which is in the area of the zona incevta (ZI) dorsomedial to the subthalamic nucleus. Injections to the MLR of glycine, an inhibitory transmitter of the spinal cord and brainstem, had no effect on locomotion, nor did strychnine sulfate, a glycine antagonist. Unilateral injections of the excitatory amino acid, N-methyl-D-aspartic acid (NMDA), and kainic acid, a glutamate analogue, into the MLR produced an increase in locomotion not seen with glutamate, an excitatory amino acid, into the same area. A still greater response, having a later onset than NMDA but also a longer duration, was produced by administration of picrotoxin and bicuculline methiodide, GABA antagonists, to the MLR. Carbachol injections into the MLR produced two types of responses: either increased or decreased locomotion. Hypermotility resulted from microinjections of glutamate, and picrotoxin and bicuculline, into the ZI. The short latency, short duration response to glutamate resulted in a greater increase in locomotion than with picrotoxin or bicuculline when each was administered into the SLR. These results provide further evidence for the functional role of the MLR and SLR in the initiation of locomotor activity in the intact, freely behaving rat.

INTRODUCTION There are a n u m b e r of reports that locomotor activity is elicited by electrical stimulation of the brainstem 15A6'39. O n e area that has received a good deal of attention is the mesencephalic locomotor region (MLR), consisting of the p e d u n c u l o p o n t i n e nucleus (PPN) and part of the adjacent cuneiform nucleus (CNF) 1°. A n o t h e r is the subthalamic locomotor region (SLR) 10,17,44. Most studies in which locomotion was elicited by electrical stimulation of the M L R and SLR were in cats with brains transected rostral to the midbrain and tested on a treadmill owing to the deficits in equilibrium and posture after the lesion. This increase in locomotor activity was more recently d e m o n s t r a t e d in rats 6,1°29,4°, some with brains intact and in freely moving animal 2'24. In addition, locomotion was elic-

ited by injecting picrotoxin and bicuculline, ),-aminobutyric acid ( G A B A ) antagonists, or glutamate which preferentially activates cell bodies 1A4, into the M L R 2A2. This suggests that a G A B A e r g i c input normally regulates the M L R at sites which contribute to the initiation of locomotor activity. The present study was u n d e r t a k e n to investigate further the role of the M L R and SLR in locomotor activity of freely moving rats. The animals were implanted with cannulae and/or electrodes which were placed stereotaxically in the area of the M L R and SLR. The effects of electrical stimulation of these sites were first investigated with particular attention to comparing electrical stimulation of the lateral and medial M L R 38 and to comparing electrical stimulation of the subthalamic nucleus (STN) and of the zona incerta (ZI), which is dorsal to the STN. T h e n , the effects of chemical stimulation of these same sites

Correspondence: G.J. Mogenson, Department of Physiology, University of Western Ontario, London, Ont., Canada N6A 5C1. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

274 were investigated. Based on earlier studies 2'12'22'31'43 it was decided to include picrotoxin, bicuculline methiodide, strychnine sulfate, glutamate, N-methyl-D-aspartic acid (NMDA), kainic acid, glycine and carbachol. Results of these experiments will provide further information about the neural components activated (cell bodies or fibers of passage) when locomotor activity is elicited by electrical stimulation and some of the putative transmitters involved. The findings will provide the bases for further studies in which rate and pattern of limb movements can be made using photography or electromyography. MATERIALS AND METHODS

Animals and surgery The experiment was performed on 60 adult, male Wistar rats weighing between 225 and 325 g at the time of surgery. The animals were anesthetized with sodium pentobarbital (60 mg/kg b. wt., i.p.; Somnotol, MTC Pharmaceuticals, Hamilton, Ont.) and placed in a Kopf stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) with the incisor bar adjusted to 3.3 m m below the interaural plane. Burr holes 1-2 mm in diameter were drilled through the skull to allow for placement of cannulae (i.e. 24 animals), electrodes (i.e. 18 animals) or cannula-electrode units (i.e. 18 animals) into the target sites. Cannula-electrode assemblies consisted of guide cannulae made from 23-gauge hypodermic needle tubing cut and ground to 14.45 mm lengths and fixed with dental acrylic (Hygenic, Akron, OH) to bipolar electrodes (model MS303/2, Plastic Products, Roanoke, VA). Details of these cannula-electrode assemblies are presented in Fig. 1 of an article by Robertson and Mogenson 34. In animals with cannulae alone, these consisted of 14.45 mm lengths of 23-gauge tubing and some animals had only electrodes as described above for the cannula-electrode units. The implants were stereotaxically placed into the area designated as the M L R and into the STN and Z I dorsal to the STN according to the atlas of Paxinos and Watson 3°. The coordinates used for the M L R were 0.2 mm anterior to the interaural line (AP), 2.0 mm lateral to the midline (LM) and 6.2 mm below the surface of the cortex (DV). The coordinates used for the medial M L R were 0.7 mm AP, 1.3 mm LM and 5.4 mm DV,

whereas those used for the lateral M L R were 0.7 mm AP, 2.3 mm LM and 6.1 mm DV. The SLR coordinates were 5.2 mm AP, 2.0 mm LM and 7.4 mm DV. The implants in the ZI were placed 5.2 mm AP, 2.0 mm LM and 7.2 mm DV, and in the STN, 5.2 mm AP, 2.5 mm LM and 7.6 mm DV. Dental acrylic was used to secure the implants to jeweller's screws (Lomat Watch Material, Montreal, Que.) fixed to the skull. Stainless-steel insert pins were placed in the guide cannulae to ensure patency during the experimental period. To prevent infection, antibiotic (0.2 ml, i.m., Pen-Di-Strep, Rogar/STB Div. BTI Products, London, Ont.) was administered immediately after surgery. The animals were housed in individual cages in an illumination-controlled room (12:12 h light/dark cycle) with controlled room temperature (22 + 1 °C) and food and water available ad libitum. The animals were given 7-10 days for recovery and initial handling and then adapted to the test apparatus for at least 3 more days before investigating the effects of electrical and chemical stimulation of the target sites on locomotor activity.

Drugs and injection protocol The following compounds were injected: sodium glutamate prepared from L-glutamic acid (L-aaminoglutaric acid, Sigma, St. Louis, MO), N M D A (Sigma), kainic acid (Sigma), glycine (Sigma), picrotoxin (Sigma), bicuculline methiodide (Sigma), strychnine sulfate (Sigma) and carbachol (prepared from carbamyl choline chloride, Nutritional Biochemicals, Cleveland, OH). All compounds were dissolved in 0.9% pyrogen-free, sterile saline (Abbott Laboratories, Montreal, Que.) and injected into the brain with a 30-gauge stainless-steel inner cannula extending approximately 1.25 m m beyond the guide cannula. The injection cannula was connected by polyethylene PE-10 tubing (Clay Adams, Div. Becton Dickinson, Parsippany, N J) to a CR-700 20/d microsyringe (Hamilton, Reno, NV) and the drug injected mantially at a rate of 0.01 ,ul/s. Unilateral injections of glutamate (2.00 M, 0.1 #1), picrotoxin (0.806 mM, 0.2/A) and bicuculline (0.368 raM, 0.2 ktl) were made in both the M L R and SLR. N M D A (4.249 mM, 0.4/zl), glycine (0.533 M, 0.5/zl), strychnine sulfate (0.875 raM, 0.2 Izl) and carbachol (1.095 mM, 0. l/A) were injected unilaterally and kainic acid (0.469 mM, 0.2/~1) and carbachol bilaterally into the

275 MLR. An injected volume of 0.2 #1 leads to diffusion of the drug to a region 0.4-0.6 mm in diameter 3. As a control for the pharmacological effects of the drugs a sham injection (the injection cannula was inserted but no injection made) was performed or the solvent, 0.9% saline, was given in the appropriate volume as indicated in the Results section. Drug treatments were given in a randomized, counterbalanced order with half the animals receiving the drug first and the other half receiving the control injection first.

Behavioral testing procedure During the testing period the animal was placed in an open-field test chamber (71.5 × 71.5 cm) containing a light beam-photocell system. Locomotor activity was recorded by a PC-800 electronic counter (Columbus Instruments, Columbus, OH) that measured the accumulated number of interruptions of 4 independent light beams. The rat was placed in this apparatus for a 20 min adaptation period in order to establish baseline activity before any intracerebral drug injections were made. Horizontal locomotor activity was recorded in 5 min intervals during the pre-injection period, the counts being automatically summed and then printed by a Canon printing calculator P10D (Canon U.S.A., Chicago, IL). The rat was removed from the testing box for a 5 min period during which the injections were made at the doses mentioned previously. The injection cannula was left in situ for an additional 60 s to allow diffusion of the solution away from the injection cannula tip. The insert pin was then replaced. Following the injection, the animal was returned to the open-field chamber for 20 rain and locomotor activity was recorded at 1 or 5 min intervals. Due to the rapid responses to glutamate injections locomotor activity was recorded every minute, but recordings at 5 min intervals were made for the other drugs. The general behavioral changes of the animals after the drug injection were also monitored. For testing days involving electrical stimulation the animal underwent a different protocol. After the 20 min adaptation period the rat was given two intervals of 5 min each of both non-stimulation (A) and stimulation (B). Thus, the pattern on one day was A B B A and then reversed (i.e. BAAB) on the following day and the results averaged. The stimulation parameters for the testing were 60 Hz pulses of 0.2 ms dura-

tion delivered by a Grass SD9 stimulator (Grass Medical Instruments, Quincy, MA) with 20 s trains and then offfor 10 s. The timing was automated using interval timers (Model 100-C, Hunter Mfg., Iowa City, IA). The current applied was calibrated by an oscilloscope (Model $51 B, Telequipment, Div. Tektronix Canada, Downsview, Ont.) such that the applied current was 15% above that which produced the behavioral threshold. The effects of electrical stimulation were always tested before the chemical injections. The electrical stimulation was then repeated to examine the reproducibility of the results.

Histological verification At the end of the experiment a 25% India ink solution was administered in order to localize the injection sites. The rats were sacrificed by an overdose of urethane (40% (w/v, i.p.) and then perfused transcardially with 50 ml of saline, followed by 50-100 ml of 10% buffered formalin. The brains were removed, fixed in formalin for at least 24 h and then 80 p m coronal sections of the brain were cut with a cryostat (Bright Instrument, Huntingdon, U.K.). The sections were mounted on gelatinized glass slides and stained with thionin for histological determination of the injection sites. All data are presented as the mean + S.E.M. Statistical analyses involved using a t-test for paired comparisons. RESULTS The effects of electrical and chemical stimulation of sites in the area of the PPN and Z I were studied in 60 freely moving rats.

Effects of electrical stimulation of brainstem sites The effects of electrical stimulation (107 + 9 p A ) of sites located in the PPN and adjacent areas (n = 28) are shown in Fig. 1 (top). During the adaptation period, represented as PRE in Fig. 1, locomotor activity was approximately 50 responses/10 min. A comparison was made between the non-stimulation period (A) and stimulation period (B). PPN stimulation resulted in an increase in locomotion, from 21 _+ 3 to 141 _+ 14 responses/10 min (t27 = 7.82, P < 0.00001). The typical response was forward progression (walking or running) along the walls of the open-

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,ooI Fig. 1. Locomotor activity (responses/10 min) resulting from electrical stimulation of brainstem sites in the rat tested in the open-field apparatus. The rat was adapted to the apparatus for 20 min (averaged to 10 min and shown as PRE) and then underwent two 5 min intervals of non-stimulation (A) and two 5 min intervals of stimulation (B; 60 Hz, 0.2 ms duration). Top: the first period of stimulation (hatched bars) of sites in the area of the PPN of the M L R (n = 28) produced a marked 6- to 7-fold increase in locomotion. A repeated period of stimulation (stippled bars, n = 25), after all chemical testing had been completed, resulted in a similar 7-fold increase in locomotor activity. Bottom: stimulation of the lateral M L R (cross-hatched bars, n = 16) produced a similar increase in locomotion to that elicited from electrical stimulation of the medial M L R (solid bars, n = 15). The current intensities 0tA) representing 15% above that which produced a behavioral threshold are shown on the right side of each figure. The current intensities were similar to those used to elicit locomotor activity in a recent study by Mogenson and Wu in which, as shown in Fig. 1, locomotor activity increased as a function of current intensity (as reported also by Skinner and Garcia-Ril141). Mogenson and Wu estimated that current spread did not exceed 0.50 mm. The bars represent means + S.E.M.

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PRE A B Fig. 2. A comparison of the locomotor activity (responses/10 rain) resulting from electrical stimulation of brainstem sites in the freely moving rat. P R E is the 10 rain adaptation period; A, two 5 min intervals of non-stimulation and B, two 5 rain intervals of stimulation (60 Hz, 0.2 ms duration). Top: a 6- to 7-fold increase in locomotion was seen with the first period of electrical stimulation (hatched bars, n = 13) of sites in the region of the ZI. When tested a second time (stippled bars, n = 12) a 5fold increase in locomotor activity was observed. Bottom: a greater increase in locomotor activity was seen with electrical stimulation of sites in the ZI (cross-hatched bars, n = 14) than those in the area of the STN (solid bars, n = 7). The current intensities ~ A ) representing 15% above that which produced a behavioral threshold are shown on the right side of each figure. The bars represent the means + S.E.M.

277 tion, sometimes associated with rearing), were seen with electrical stimulation of some sites, particularly in and a r o u n d the central gray. In order to elucidate further the areas responsible for the initiation of locomotion with electrical stimulation, a comparison was made of the effects of stimulation of the lateral and medial M L R (see Fig. 1, bottom). W h e n 15 sites in the area lateral to the central gray (medial M L R , solid bars) were stimulated (103 + 11/~A) locomotion increased from 31 + 5 to 91 + 13 responses/10 min (t14 = 4.99, P < 0.0002). Sites in the area of the PPN (lateral MLR, cross-hatched bars; n = 16), when stimulated (122 + 11/~A) led to increased locomotion, from 22 + 5 to 104 + 27 responses/10 min (q5 = 3.12, P < 0.007). The increased 100

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sponses/10 rain, similar to that shown in Fig. 1 (top) for PPN stimulation. Electrical stimulation of the Z I resulted in an increase from 25 _+ 5 to 162 + 30 responses/10 min (t]3 = 5.07, P < 0.0003). As can be seen from Fig. 2 (bottom), stimulation of the Z I sites (cross-hatched bars, n = 14) produced a greater locomotor response than the stimulation of the 7 sites in the STN (solid bars). D u r i n g the n o n stimulation period the locomotor activity was 38 _+ 4

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Fig. 3. Locomotor activity (responses/5 min) resulting from unilateral injections (0.01 falls) of different compounds into the PPN and adjacent areas of the rat. The number of responses per 5 min interval were recorded before and after unilateral injections of: A: glutamate (L-glutamicacid, 2.00 M, 0.1 ,ul); B: glycine (0.533 M, 0.5/d); C: NMDA (4.249 mM, 0.4/d), and D: picrotoxin (0.806 raM, 0.2 M)- In each case saline was given in the appropriate volume as a control. Glutamate (n = 14) and glycine (n = 10) had little effect on locomotion. However, there was a greater than 2-fold increase in locomotor activity with NMDA (n = 16) and 5-fold increase with picrotoxin (n = 8) when compared to the saline controls. The vertical lines represent the S.E.M. and the times of injection are indicated by the arrows.

278 and 32 _+ 2 responses/10 min for the ZI and STN tests, respectively (t19 = 0 . 8 5 , P > 0.40). However, when stimulated with similar current intensities (ZI, 135 + 10/~A and STN, 134 +_ 20ktA; t19 = 0.03, P > 0.90) induced locomotion was greater from stimulation of the ZI sites (157 + 19 responses/10 min) than stimulation of the STN sites (86 + 17 responses/10 min; t19 = 2.42, P < 0.03). The effects of electrical stimulation of the PPN and ZI were repeated after the effects of chemical stimulation had been investigated. The results can be seen by comparing the stippled bars (repeated stimulation) to the hatched bars (initial stimulation) in Figs. 1 (top) and 2 (top), for the PPN and ZI, respectively. A n increase in locomotion, from 26 + 4 to 188 _+ 21 responses/10 min (t24 = 6.80, P < 0.00001) resulted when the same PPN sites (n = 25) were stimulated (146 _+ 18/~A). L o c o m o t o r activity increased from 37 _+ 11 to 189 + 31 responses/10 min (t u -- 3.96, P < 0.003) when electrical stimulation (188 _+ 17 ktA) was repeated on the ZI sites (n = 12). These observations indicated that locomotor activity elicited by electrical stimulation was reproducible. Effects o f chemical stimulation o f brainstem sites on locomotion

The effects of unilateral injection (0.01 pl/s) of different compounds into the PPN and adjacent areas on locomotor activity are summarized in Figs. 3 - 5 and Table I. There was a gradual decrease in locomotor activity during the pre-injection period. As shown in Fig. 3A, unilateral injections (n = 14) of glutamate (L-glutamic acid; 2.00 M, in 0.1 ~1 saline) did not produce a significant increase in locomotor activity during the first min (glutamate: 13 _+ 3 responses/min compared to the control of 17 _ 2 responses/min; t13 -- 1.05, P > 0.20). However, in some animals hypermotility was produced during the injection period and then subsided quickly before they were returned to the test chamber. Unilateral injections of glycine (0.533 M, 0.5 ~1) into 10 sites in the PPN did not change the locomotor activity of the animals (Fig. 3B). The total number of photocell-beam interruptions over the 20 min test period was 79 _ 15 responses after glycine administration as compared to 89 _ 21 for the saline control (t 9 = 0.41, P > 0.60) with a similar time course for the two compounds (see Fig. 3B). For the same 10 injec-

TABLE I Locomotor activity following injections of picrotoxin and strychnine sulfate into the pedunculopontine nucleus as compared to control sham injections

Locomotor activity resulting from unilateral injections (0.01 ~l/s) of picrotoxin (0.806 mM, 0.2/A; n = 10) or strychnine sulfate (0.875 mM, 0.2B1; n = 10) into the area of the PPN as compared to control sham injections. The post-injection/pre-injection (Post/Pre) value gives the ratio of activity after and before the treatment and compares it to the sham control (assigned the value 1.00). Data represent the means + S.E.M. Asterisk identifies statistically significant (P < 0.01) responses to drug injections. Treatment

Picrotoxin Strychnine Control (sham)

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tion sites the effects of picrotoxin and strychnine sulfate were also investigated. As seen in Table I, injections of picrotoxin, a G A B A antagonist (0.806 mM, 0.2 ~1), produced a significant increase in locomotor activity over 20 min as compared to strychnine sulfate (0.875 mM, 0.2~1, t9 = 4.00, P < 0.003) and the sham injected control (t 9 = 4.13, P < 0.01). The picrotoxin-induced locomotion was more than 4 times greater than the strychnine sulfate or sham values as seen by comparing the post-injection/pre-injection activity ratios (Post/Pre) with the sham (assigned a value of 1.00) (see Table I). Strychnine sulfate did not alter locomotor activity, as indicated by the similarity of the response to this c o m p o u n d (68 + 15 responses/20 min) and the sham control (65 + 12 responses/20 min; t9 = 0.62, P > 0.50). Unilateral injections of picrotoxin (0.806 mM, 0.2 ~1) were made in another 8 sites in the PPN and produced a significant increase in locomotion (269 + 92 responses/20 min) as compared to the saline control injections (50 + 14 responses/20 min; t7 = 2.63, P < 0.04). The 5-fold increase in locomotor activity had a long latency and duration reaching a peak between 5 and 10 min and slowly decreasing over the remaining 10 min of the test period (Fig. 3D). It can be seen in Fig. 3C that unilateral injections of N M D A (4.249 mM, 0.4~1) into the PPN (n = 16) resulted in a 2-fold increase in locomotor activity (103 + 15 responses/20 min) as compared to the control saline injections (50 _+ 7 responses/20 min; tl5 =

279 a n d b i c u c u l l i n e m e t h i o d i d e (0.368 m M , 0.2 ktl; n = 16) led to a 3- a n d 15-fold i n c r e a s e in l o c o m o t o r activity (t7 = 1.83, P > 0.10 a n d t15 = 6.24, P < 0.00002) respectively. T h e time profiles have n o t b e e n inc l u d e d for kainic acid, b i c u c u l l i n e a n d s t r y c h n i n e sulfate. It s h o u l d be n o t e d h o w e v e r , that these are similar to those p r e v i o u s l y described.

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SAL GILlNMDAKA GLY PTX BIC STRY Fig. 4. A comparison of the effects on locomotor activity of various compounds when injected into the PPN and adjacent areas of the rat. Relative locomotor activity for the first 5 rain is shown as a percentage of saline (SAL; shown as 100%) for each treatment. In each case saline was given in the appropriate volume as a control. The following compounds were injected unilaterally at a rate of 0.01 #l/s: glutamate (GLU; 2.00 M, 0.1/~1, n = 14), NMDA (4.249 mM, 0.4 Izl, n = 16), glycine (GLY; 0.533 M, 0.5/A, n = 10), picrotoxin (PTX; 0.806 mM, 0.2kd, n = 8), bicuculline methiodide (BIC; 0.368 mM, 0.2/~1, n = 16) and strychnine sulfate (STRY; 0.875 mM, 0.2 /A, n = 10). Kainic acid (KA; 0.469 mM, 0.2/~1, n = 8) was injected bilaterally. The bars represent the means + S.E.M. It appears from control experiments and the results of other studies2'4 that the differences cannot be attributed to variations in the extent of spread of the various compounds.

I n j e c t i o n s of c a r b a c h o l (1.095 m M , 0.1 ~tl) into the P P N a n d a d j a c e n t areas p r o d u c e d two types of locom o t o r r e s p o n s e s (see Fig. 5). U n i l a t e r a l m i c r o i n j e c tions in some sites (n = 10) p r o d u c e d an i n c r e a s e in l o c o m o t o r activity over saline control i n j e c t i o n s (180 + 31 vs 88 + 25 responses/20 m i n ; t9 = 4.62, P < 0.002). L o c o m o t i o n was f u r t h e r i n c r e a s e d by bilateral i n j e c t i o n s of c a r b a c h o l into 8 a n i m a l s (204 + 31 vs 87 + 31 responses/20 m i n ; t 7 = 4.96, P < 0.002). O t h e r sites (n = 14) w h e n i n j e c t e d u n i l a t e r a l l y with carbachol led to a d e c r e a s e or c o m p l e t e cessation of l o c o m o t i o n (49 + 8 responses/20 m i n ) w h e n c o m p a r e d to saline i n j e c t i o n s (78 + 11 responses/20 m i n ;

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2.90, P < 0.02). T h e i n c r e a s e d activity was s e e n in the first 2 - 4 m i n . A f t e r the first 5 m i n activity h a d decreased to the level seen after saline i n j e c t i o n s . A s u m m a r y of effects o n l o c o m o t o r activity in the first 5 m i n of m i c r o i n j e c t i o n of different c o m p o u n d s in the area of the P P N is s h o w n in Fig. 4. Saline is s h o w n as 100% a n d all o t h e r c o m p o u n d s are given relative to this c o n t r o l value. G l u t a m a t e (2.00 M, 0.1 /,tl; n = 14) did n o t differ from the saline c o n t r o l over the first 5 m i n (tl3 = 0.63, P > 0.50) w h e r e a s injections of N M D A (4.249 m M , 0.4~1; n = 16) a n d kainic acid (0.469 m M , 0.2ktl; n = 8), a g l u t a m a t e a n a l o g u e , led to i n c r e a s e d activity (greater t h a n 3- a n d 10-fold, t15 = 4.04, P < 0.002 a n d t7 = 7.55, P < 0.0001, respectively). G l y c i n e (0.533 M, 0.5 ktl; n = 10) a n d s t r y c h n i n e sulfate (0.875 m M , 0.2/A; n = 10) did n o t increase l o c o m o t o r activity over the saline c o n t r o l (t9 = 0.026, P > 0.90 a n d t9 = 0.74, P > 0.40, respectively); h o w e v e r , p i c r o t o x i n (0.806 m M , 0.2/~1; n = 8)

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Fig. 5. Locomotor activity resulting from microinjections (0.01 ~l/s) of carbachol (1.095 mM, 0.1/A) into the PPN and adjacent areas of the rat. The number of photocell interruptions in the first 5 min after unilateral (UNI) or bilateral (BI) injections of carbachol were compared to the saline control (SAL; shown as 100%) over a similar 5 min period. Two types of responses were seen: those in which unilateral injections (n = 10) led to increased locomotion and further enhanced by bilateral injections (n = 8), or a reduction in locomotor activity after unilateral injections (n = 14) which was attenuated with bilateral injections (n = 4) of carbachol into these sites. The bars represent the means + S.E.M.

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Fig. 6. Coronal sections showing the cannula sites (n = 24) in the area of the PPN into which carbachol was injected. Closed circles, cannula sites producing an increase in locomotion; open circles, cannula sites eliciting a decrease in locomotor activity. Aq, aqueduct; CG, central gray; CNF, cuneiform nucleus; IC, inferior colliculus; ml, medial lemniscus; PPN, pedunculopontine nucleus; SC, superior colliculus; scp, superior cerebellar peduncle; xscp, decussation of superior cerebellar peduncle. A n t e r i o r - p o s t e r i o r coordinates are given with respect to interaural zero. Bar = 1 mm. (Drawings are modified, from the atlas of Paxinos and Watson 3°.)

3.67, P < 0.003); this decrease was greater with bilateral carbachol injections (n = 4) (38 + 11 responses/20 min) although this decrease was not significantly different from the saline control (42 + 7 responses/20 min; t3 = 0.73, P > 0.50). As can be seen from Fig. 6 those sites from which decreased locomotor activity was induced by carbachol injections are located predominantly within the PPN whereas increased locomotion resulted from microinjections in sites dorsal and medial to the PPN. Unilateral injections (0.01 #l/s) of glutamate (2.00 M, 0.1 #1) into 14 sites in the area of the ZI produced a short latency, short duration 3- to 4-fold increase in locomotor activity, as illustrated in Fig. 7A. This significant increase lasted about 1-2 min (as compared to saline for the first rain, t13 = 3.02, P < 0.01, with the locomotor response 1 min after glutamate of 23 + 3 responses/min compared to the control of 13 + 2 responses/min). Locomotion after chemical injections consisted of walking or running along the walls of the testing apparatus and usually appeared more natural than that seen during electrical stimulation. Unilateral injections of picrotoxin (0.806 mM, 0.2 #1) into the area of the ZI (n = 30) produced a small but sustained increase in locomotor activity developing to a peak response over the first 5-10 min after

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Fig. 7. Locomotor activity (responses/5 min) recorded during the 20 min adaptation period in the open-field apparatus and following unilateral injections (0.01 #l/s) of different compounds into the area of the diencephalon in the rat. Unilateral injection of: A: glutamate (2.00 M, 0.1/d), B: picrotoxin (0.806 mM, 0.2#1), and C: bicuculline methiodide (0.368 raM, 0.2#1). Saline was administered in the appropriate volume as a control. There was a greater effect of glutamate (n = 14) than bicuculline (n = 16) and picrotoxin (n = 30). The drugs were injected at the time indicated by the arrow. The bars represent the means + S.E.M.

281 injection (see Fig. 7B). The picrotoxin-induced locomotion over the 20 min test period was 150 _+ 22 responses as c o m p a r e d to 85 + 9 responses/20 min for the saline control (t29 = 2.54, P < 0.002). As shown in Fig. 7C unilateral injections of bicu-

culline methiodide (0.368 mM, 0.2pl) into the area of the ZI (n = 16) produced an increase in locomotor activity (101 _+ 16 responses/20 min) over the saline control (44 _+ 6 responses). This bicuculline-induced locomotor activity was approximately 2-fold that seen with injections of saline (t15 = 3.87, P < 0.002). The effect of bicuculline was similar to the locomotor activity resulting from injections of picrotoxin (t44 = 1.51, P > 0.10).

Sites of stimulating electrodes and cannulae Histological examination of the brains revealed the locations of 112 cannula and 124 electrode sites equally distributed between the mesencephalon and ~7 m m

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Fig. 8. Coronal sections showing the cannula and electrode sites (n = 112) in the area of the PPN, a major component of the MLR. Closed circles, cannula sites producing a locomotor response; open circles, non-responsive cannula sites; closed triangles, responsive electrode sites; open triangles, non-responsive electrode sites. Aq, aqueduct; CG, central gray; CNF, cuneiform nucleus; DpMe, deep mesencephalic nuclei; IC, inferior colliculus; ml, medial lemniscus; PPN, pedunculopontine nucleus; SC, superior colliculus; scp, superior cerebellar peduncle; SNC/SNR, substantia nigra, pars compacta/reticulata; VTA, ventral tegmental area; xscp, decussation of superior cerebellar peduncle. Anterior-posterior coordinates are given with respect to interaural zero. Bar = 1 mm. (Drawings modified from the atlas of Paxinos and Watson3°.)

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Fig. 9. Coronal sections through the rat brain showing the locations of the cannula and electrode sites (n = 124) in the region of the ZI, dorsal to the medial STN. Closed circles, cannula sites producing a locomotor response; open circles, non-responsive cannula sites; closed triangles, responsive electrode sites; open triangles, non-responsive electrode sites. 3V, third ventricle; CM, centre median nucleus; CP, caudate-putamen; EP, entopeduncular nucleus; f, fornix; GP, globus pallidus; LH, lateral hypothalamic area; MD, mediodorsal thalamus; MM, mammillary bodies; mt, mammillothalamic tract; opt, optic tract; STN, subthalamic nucleus; VT, ventral thalamus; ZI, zona incerta. Anterior-posterior coordinates are given with respect to interaural zero. Bar = 1 mm. (Drawings are modified from the atlas of Paxinos and Watson3°.)

282 diencephalon in 52 rats, as seen in Figs. 8 and 9. In the mesencephalon there was a total of 112 implant sites, of which 47 were for cannulae and 65 for electrodes. Forty sites were located on the edge of, or lateral to, the central gray and more caudally in the area of the CNF, in the area designated as the medial MLR. An area more lateral to this which includes the PPN, and called the lateral MLR, contained 50 implant sites. In addition, there were sites located more dorsal in the superior colliculus (n = 7) and lateral and dorsolateral to the central gray (n = 15). Diencephalic implants (n = 124) were located in the area of the SLR (n = 75), in the region of the STN and dorsomedially in the ZI. The remaining sites were located dorsally in the ventral thalamus (n = 43), the centre median nucleus (n = 4) and in the lateral hypothalamic area (n = 2). DISCUSSION Locomotor activity of freely moving rats was increased by electrical stimulation of locomotor regions in the mesencephalon and diencephalon confirming and extending a number of previous studies. There was at least a 3-fold increase in locomotion when these sites were stimulated with a current intensity 15% above behavioral threshold. The typical response was forward progression along the wall of the apparatus, similar to the spontaneous behavior of rats in this test chamber 24. The elicited locomotor responses were reproducible after a period of several weeks during which the effects of centrally administered chemical compounds were investigated. The classical experiments in which the so-called mesencephalic locomotor region (MLR) was demonstrated were with cats, their brains transected at the precollicular-postmammillary level, tested on a treadmill 16'39. The MLR was used as a functional term but the CNF was usually assumed to be the critical site of stimulation. However, observations from more recent experiments with both cats and rats have suggested that the PPN is also an important component of the M L R 1°. In the present study a comparison was made of the effects of electrical stimulation of the medial MLR, located in the vicinity of the CNF and lateral border of the central gray, and the lateral MLR, consisting of the PPN and adjacent areas. Locomotor activity was elicited from both areas and the

magnitudes of the increases in locomotion were similar (Fig. 1, bottom). This result is of special interest since these two regions are the origins of two descending pathways from the MLR. Neural projections from the lateral M L R appear to project via Probst's tract to the dorsal tegmental reticular formation of the pons and medulla 13,38,42. Projections from the medial M L R project via the d0rsolateral pontomedullary locomotor strip (PLS) 2~'3s. The results of the present study suggest that both of the descending MLR pathways may contribute to the initiation of locomotor behavior. However, there are also ascending neural projections of the M L R 19'25'36, to the nucleus accumbens and cortex, for example, and as suggested previously 24, they may be associated with locomotor activity. Locomotor activity was also elicited by electrical stimulation of the ZI and adjacent areas (Fig. 2, top). A number of classical studies have also demonstrated that locomotion results from the electrical stimulation of this region 8'17'20'21'27'44. However, they concluded that the critical site of stimulation was the STN and the term subthalamic locomotor region (SLR) gained wide acceptance. In the present study a greater locomotor response was elicited by electrical stimulation of the ZI than the STN (Fig. 2, bottom). These observations support the previous suggestion 1°'41 that the ZI, and not the STN, is the site of the classical SLR. Locomotor activity was elicited by injecting certain chemical compounds into the same regions from which locomotor activity had been elicited previously by electrical stimulation. Injections of picrotoxin and bicuculline, G A B A antagonists, into the PPN (Figs. 3D and 4) produced a substantial increase in locomotor activity consistent with recent observations 2. A smaller, but statistically significant, increase in locomotor activity was also observed when picrotoxin and bicuculline were administered to the Z I (Fig. 7B,C). There is a good deal of evidence that both the PPN and the ZI have GABA-mediated inputs which have been implicated in locomotor activity 2'5'11. Glycine receptors have been demonstrated in the PPN 32. However, injections of glycine or strychnine sulfate, a glycine antagonist (Fig. 4) to the PPN, unlike other areas of the mesencephalon 18, did not produce a change in locomotor activity suggesting that the density of glycine receptors is low and might not be asso-

283 ciated with locomotor responses. Glutamate was also injected into the PPN and ZI. Glutamate was used because there is some evidence that it excites cell bodies and not fibers of passage 1'a4. Glutamate injections into the PPN (Fig. 3A) made rats more active, as reported by Brudzynski et al. z but these brief effects had subsided by the time the animals were returned to the open-field apparatus. Glutamate injections into the ZI increased locomotor activity for 1-2 min and the effect was statistically significant (Fig. 7A). NMDA, an excitatory amino acid, was also injected into the PPN. There was a significant increase in locomotor activity for 2 or 3 min, as shown in Fig. 3C. The greater than 10-fold increase in locomotor activity after kainic acid injections, as seen in Fig. 4, provides further evidence for an excitatory role for glutamate in the PPN since kainic acid, a glutamate analogue, stimulates neurons directly 22. The short duration of the hyperkinetic effect of glutamate as compared to N M D A and kainic acid can be explained by the fact that there are effective uptake mechanisms for glutamate but not for NMDA and kainic acid 9. The results with glutamate, NMDA and kainic acid suggest that locomotor activity from electrical stimulation of the PPN and ZI may not be merely activation of fibers of passage but rather, excitation of neurons in these two regions. The cholinergic system has been implicated in the regulation of locomotor activity using anatomical techniques which located neurons in the area of the PPN which may contain acetylcholine as a neurotransmitter 43. Cholinergic muscarinic receptors have been mapped in this region 35,45. The direct application of carbachol into this area led to two responses: an increase or a decrease or complete cessation of locomotion (as seen in Fig. 5). Brudzynski et al. 4 also made similar observations and subsequently described the location of the sites which corresponded to these responses. Decreased locomotion was seen with carbachol injection into sites within the area of the PPN suggesting a modulatory role on locomotor activity. Although early anatomical and clinical studies led to the suggestion that the STN is the anatomical substrate for the SLR, it now seems likely, based on the results of this study and others, that the ZI forms the neural substrate for the SLR. Other studies involving

electrophysiological and behavioral techniques, have also confirmed a functional role for the ZI as the SLR 7'23. With its numerous connections, the ZI is strategically located to integrate activities between the MLR, basal ganglia and limbic system 2°'23'33'37. Orlovsky27 showed that cats with bilateral lesions in the MLR could still walk when the SLR was electrically stimulated and bilateral SLR lesions eliminated spontaneous locomotion for several weeks 44. Thus, the ZI may have a functional role as the SLR and is probably capable of initiating goal-directed locomotor behavior independently of the MLR. In the precollicular-postmammillary transected cat it has been reported that spontaneous locomotion does not o c c u r 16'28. The sub stantia nigra pars reticulata is the only known structure located caudally to this transection that supplies known inputs to the PPN l° and it has been suggested that these are tonically active GABAergic inputs 5. A premammillary transection results in spontaneous stepping 16 suggesting the importance of a structure between these two transections which acts either to directly activate the MLR and/or disinhibit the GABAergic inputs from the substantia nigra to the PPN 12. The SLR is a likely candidate for this role. In studies using rats and cats, electrical and in the present study, chemical stimulation of this area produced locomotor activity. Reciprocal projections pass through the SLR between the STN and PPN as do projections to the substantia nigra. Thus, it may be the role for the SLR to influence the MLR through direct pathways 36 or via modulation of inputs such as those from the substantia nigra. Both the PPN and ZI are strategically located and connected with their numerous projections to many motor structures and thus are likely candidates as the neural substrates for the mesencephalic and subthalamic locomotor regions.

ACKNOWLEDGEMENTS Th authors express appreciation to Becky Woodside, Michael Wu and Bruce Arppe for their assistance in the preparation of the illustrations. The research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.

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