Priming of locomotion Neuroscience Vol. 99, No. 1, pp. 77±91, 200077 q 2000 IBRO. Published by Elsevier Science Ltd Pergamon Printed in Great Britain. All rights reserved PII: S0306-4522(00)00179-2 0306-4522/00 $20.00+0.00 www.elsevier.com/locate/neuroscience

BRAINSTEM REGIONS WITH NEURONAL ACTIVITY PATTERNS CORRELATED WITH PRIMING OF LOCOMOTOR STEPPING IN THE ANESTHETIZED RAT

H. M. SINNAMON,* A. K. JASSEN and L. A. VITA Neuroscience & Behavior Program, Wesleyan University, Middletown, CT 06459-0408, USA

AbstractÐLocomotor stimulation in the perifornical hypothalamus produces a transient facilitation of subsequent locomotion, a priming effect, such that stepping to a second train of stimulation occurs with a shorter latency of onset and increased amplitude. Neurons responsible for the initiation of this facilitated stepping presumably respond to locomotor stimulation with a similar priming effect, i.e. either a shorter latency or a larger change in activity rate. This study used anesthetized rats (urethane, 800 mg/ kg) to compare regions in terms of the relative rates of occurrence of single neurons that showed both speci®c responses to locomotor stimulation and also priming effects. Speci®c responses were characterized by a progressive increase in activity prior to the ®rst step (a Type I pattern). In that they co-varied in time with the increased probability of stepping onset, Type I responses were more speci®c than Type II responses, which peaked early in the stimulation train several seconds before the onset of stepping. Regions with high proportions of neurons showing Type I responses and priming effects included the anterior dorsal lateral to the central gray, the oral pontine reticular nucleus and the medial gigantocellular nucleus. Few Type I neurons showed a modulation of activity related to the step cycle. Type I primed neurons were uncommon in the cuneiform and the pedunculopontine regions, but neurons showing other patterns (decreases and antidromic responses) were relatively prevalent there. The ventral tegmental area was generally unresponsive. The results indicate that stepping elicited by perifornical stimulation in the anesthetized rat is mediated by circuits that differ at levels from the circuits implicated in other types of locomotion. Two regions, the anterior dorsal tegmentum and the oral pontine reticular nucleus, warrant further attention to determine their possible roles in the initiation of locomotion. q 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Key words: movement initiation, midbrain, , medulla, hypothalamus.

In the locomotor preparation developed by Shik et al.,71 The anesthetized intact rat in which stepping is elicited by electrical stimulation is applied to the cuneiform region of stimulation of the perifornical hypothalamus provides an midbrain-transected cats to produce controlled locomotion. alternative model for the study of midbrain locomotor circuitry. This model has important heuristic value in providing a Consistent with early evidence for the independence of the method for the study of spinal and brainstem circuitry for ªsubthalamicº and ªmidbrainº locomotor regions in the cat,58 stepping control.16,35,55,59±61,65,68,70,72,91 It has also been elicited stepping in the anesthetized rat persists after electrolytic in¯uential in the development of a conceptual framework lesions or procaine inactivation of the cuneiformis region.44,84 for studying the circuitry involved in the initiation of Electrolytic lesions and procaine inactivation of the ventral locomotion. It has led to an emphasis on the midbrain tegmental area block hypothalamic-elicited stepping,44,82 but locomotor area that includes the nucleus cuneiformis and descending ®bers of passage from the hypothalamus appear to the pedunculopontine area (here termed the peripeduncular/ be involved here because injections of GABA, which operates midbrain locomotor area, PPA/MLA). This region is thought on postsynaptic targets in this region, are not effective.78 to provide a locomotor initiation signal to the medullary Neither the PPA/MLA nor the ventral tegmental area was reticulospinal systems that in turn activate and modulate the found to have high occurrences of neuronal responses to spinal stepping generators.12,22,24,25,27±29,35,54,68,91 hypothalamic locomotor stimulation.18 By contrast, other Studies of midbrain locomotor mechanisms in the awake midbrain regions have been implicated in hypothalamic- behaving rat have primarily concerned the control of open elicited locomotion. In both the oral pontine reticular nucleus ®eld/exploratory locomotion by the limbic system.9,33,49,86 (PnO) and the anterior dorsal tegmentum (ADT), injections of This work indicates that the ventral tegmental area provides both GABA78 and procaine44 block hypothalamic-induced a locomotor facilitating signal to circuits involving the stepping. Both regions show a relatively high occurrence of hippocampus, accumbens and ventral pallidum.36,51 Some neurons with responses correlated with the onset of stepping evidence indicates that the basal forebrain structures in turn elicited by hypothalamic stimulation.18 In addition, GABA provide a locomotor initiation signal to the PPA/MLA injections in the region of the median raphe have been implicated by the work in the decerebrated cat.7,9,47,48,50 shown to facilitate stepping in the anesthetized rat,80 However, other evidence indicates that additional brainstem indicating that cells in this region have a locomotor regions can mediate this type of locomotion.34,57,86 suppressive function.31,92 In summary, the picture of midbrain locomotor circuits derived from the anesthetized intact rat *To whom correspondence should be addressed. Tel.: 11-860-685-2955; may differ signi®cantly from that derived from work on either fax: 11-860-685-2761. the midbrain-transected cat or the awake behaving rat. These E-mail address: [email protected] (H. M. Sinnamon). differences may relate to the type of motivational systems Abbreviations: ADT, anterior dorsal tegmental region; CG, central gray; activated in the different preparations.76 Gi, gigantocellular nucleus; PnC, caudal pontine reticular nucleus; PnO, oral pontine reticular nucleus; PPA/MLA, peripeduncular area/midbrain This study surveyed brainstem regions for single neuron locomotor area. responses related to locomotor initiation in the anesthetized

77 78 H. M. Sinnamon et al. rat. The speci®c goal was to comprehensively compare elicited stepping compared to a region with a high rate of structures implicated in stepping of the anesthetized rat to struc- occurrence. tures implicated in stepping of other preparations. The focus of this study was the Type I pattern of response to locomotor stimulation found in a previous study.18 In Type I responses, EXPERIMENTAL PROCEDURES activity progressively increases to the onset of stepping. By Subjects and surgery contrast, in Type II responses, the activity peaks early during Male Sprague±Dawley rats (n ˆ 227), obtained from the Charles the train of locomotor stimulation, several seconds before the River Company and bred at Wesleyan University, weighing a mean onset of stepping. Type I patterns, as a class, are more speci®c of 367 g (S.D. 62 g) were used. Surgical and experimental procedures than Type II patterns in that they more closely co-vary in time were designed to avoid discomfort of the animals, and were approved with the increased probability of stepping. Compared to the by the Wesleyan Institutional Animal Care and Use Committee. For previous study, the approach used here was re®ned in three initial anesthesia, the animal was placed in a small chamber, exposed to 2% halothane in oxygen for 5±10 min and given an intraperitoneal ways. First, it increased both the range of regions studied and injection of either nembutal (25 mg/kg) or urethane (500 mg/kg). After the number of neurons sampled in each region. In addition to the an additional 5±10 min in the halothane chamber, the anesthetized rat regions described above (the PPA/MLA, the ventral tegmental was placed in a stereotaxic apparatus. The combination of halothane area, the ADT and the raphe region), other brainstem regions of and nembutal or urethane at these doses maintained the rat at a surgical level of anesthesia for the 30±45 min required for the invasive proce- locomotor signi®cance were sampled. Second, neuronal activity dures. A scalp incision was made and lidocaine was injected around its was examined for modulation with the phase of the step margins. Holes were drilled in the skull for the stimulation and record- cycle. Activity related to stepping, while not an essential char- ing electrodes, and for screws which served as connections in the acteristic of a neuron related to stepping initiation, should be ground and stimulation circuits. found in neurons involved in control of stepping execution. Following surgery, the rat remained in the stereotaxic apparatus and was maintained at the appropriate level of anesthesia by intraperitoneal Third and most important, conjunctive criteria were used to injections of nembutal or urethane. Maintenance of anesthesia by identify neurons possibly related to stepping initiation. To the continuous administration of halothane appears to be incompatible Type I property was added a property related to the excitability with the elicitation of stepping in this preparation. Injections were of the locomotor circuitry, the priming effect. Priming of given whenever the rat showed increased respiration, increased vibrissae movement or any limb movement in the absence of brain locomotor stimulation provides a means of modulating the stimulation. Earlier experiments were performed under nembutal 77,80,81,87 excitability of locomotor initiation. In this method, a (n ˆ 70), but when it was learned that apparently identical behavioral pair of stimulation trains, Control and Test, separated by 20 s and neuronal responses were elicited under the more stable anesthesia or less, is presented on each trial. Locomotor stimulation afforded by urethane, the remainder of the experiments (n ˆ 157) used produces a residual excitation, a priming effect, such that the this. With nembutal, injections of 3.5±7.0 mg/kg were given at approximately 25±30-min intervals, or more frequently if needed stepping occurs at a shorter latency or its amplitude is larger on throughout the course of the experiment. With urethane, the appropriate the Test compared to the Control. A neuron with a response to anesthetic level was achieved by additional injections of 50±100 mg/kg locomotor stimulation that is earlier or larger on the Test than on urethane over approximately the ®rst hour of the experiment. The the Control shows a priming effect. If a Type I response pattern average cumulative dose of urethane administered was 800 mg/kg. When the appropriate urethane level was reached, the rat would were actually speci®cally related to stepping onset, it would also maintain an anesthetized state for several hours without a supplemental have to show a priming effect. Although the set of Type I injection. Lidocaine injections were made into the incision at 3-h primed neurons may additionally include neurons not intervals. The level of anesthesia induced was deeper than that required speci®cally related to locomotion, it must include neurons to maintain the rat in a quiescent state, but not so deep that a ¯exor that are responsible for stepping initiation. Any region response of the hindlimb to a strong pinch was abolished. Body temperature during surgery was maintained by means of a heat lamp. showing a low occurrence of Type I primed neurons would During testing, when stepping episodes were periodically elicited, the have a lower likelihood of a role in the initiation of the rat effectively autoregulated its temperature.

Abbreviations used in the ®gures CI caudal interstitial nucleus of the medial longitudinal mfb medial forebrain bundle fasciculus ml CIC central nucleus of the inferior colliculus MM medial mammillary nucleus CnF cuneiform nucleus MR median raphe nucleus DA dorsal hypothalamic area Mo5 motor trigeminal nucleus Dk nucleus of Darkschewitsch Mt mammillothalamic tract DLT dorsolateral tegmental region Mve medial vestibular nucleus DM dorsomedial hypothalamic nucleus pc posterior commissure DpGi dorsal paragigantocellular nucleus Pcom nucleus of the posterior commissure DpMe deep mesencephalic nucleus PeF perifornical nucleus DR PH posterior hypothalamic area DTg dorsal tegmental pontine nucleus PnV ventral pontine reticular nucleus EICC inferior colliculus, external central PPTg pedunculopontine tegmental nucleus f fornix PR prerubral area Gem nucleus gemini R red nucleus GiA gigantocellular nucleus, anterior Rli rostral linear nucleus GiV gigantocellular nucleus, ventral RPC red nucleus, parvocellular IMLF interstitial nucleus of the medial longitudinal scp superior cerebellar peduncle fasciculus Sol solitary tract nucleus InCo intercollicular nucleus SPTg subpeduncular tegmental nucleus LH lateral hypothalamic area SuM supramammillary nucleus LPB lateral parabrachial nucleus VM ventromedial nucleus of the hypothalamus MCPC magnocellular nucleus of the posterior commissure VTA ventral tegmental area Priming of locomotion 79

Stimulation with dental acrylic. A stimulation regimen commenced in which a trial was presented every 70 s; it was composed of a pair of stimulation Brain stimulation, provided by a constant-current stimulator, was trains. A 10-s period separated the ®rst (Control) train and the second composed of 50-Hz biphasic pulses, each phase 0.5 ms, delivered in (Test) train; a 50-s period separated the offset of the Test train and the trains of 5 s. The stimulation electrode was a 125-mm-diameter tungsten next Control train. The Control/Test stimulation sequence was main- wire insulated with Te¯on except at the tip. An uninsulated wire wrapped tained for the duration of the experiment, except for periods used for around screws in the skull completed the circuit to the stimulator. determination of baseline rates of unit activity.

Measure of hindlimb stepping Testing brainstem units for locomotor responses Descriptions and representative records of hindlimb stepping The recording electrode was introduced into the brain ipsilateral to elicited by stimulation of the hypothalamus in this preparation have the stimulation electrode or within 0.5 mm of the midline. A standar- been given in previous reports.76±78,80 The head of the rat was secured dized test was performed whenever a single unit was isolated or if no in the stereotaxic apparatus and the body was supported by an acrylic ongoing unit activity was apparent, at increments of 100 mm. At least one platform attached to the side rails. Below the platform was a wheel stimulation trial (composed of both Control and Test trains) that with a diameter of 30 cm and a surface width of 10 cm. At rest, the rat's elicited stepping was given. If a single unit appeared to respond to either hindlimbs hung in passive extension to make contact with the wheel. of the stimulation trains, or to change its rate of activity during step- When the rat stepped, the extensor phase of the hindlimb movement ping, it was tested with additional trials. If no response was apparent, caused the digits to engage the mesh surface of the wheel and rotated it. the electrode was moved 100 mm deeper or to the next single unit. If a Moderate currents were selected to elicit stepping on the Control train response was apparent, a testing sequence requiring 10±15 min was at a latency of 3 s or more, and a priming effect on the Test train. At given. It included eight trials of stimulation, a 40-s sample of baseline these currents, alternating steps of the hindlimbs, without stepping of activity after the stimulation was off for 2 min and a sample of the the forelimbs, are elicited. In general, the two hindlimbs move to a action potential waveform. Unit discrimination was frequently lost similar extent, but it was common for the limb contralateral to the before all eight test trials were completed. stimulation site to show a larger displacement amplitude. Higher If a neuron responded to individual stimulation pulses with a currents, which elicited galloping gaits (synchronous ¯exion and consistent ®xed latency, it was tested with three high-frequency pulses extension of the two hindlimbs), were avoided in order to maximize (typically an interpulse period of 3 ms). Unit responses that followed the priming effect and reduce body movements that caused loss of the high-frequency stimulation were classi®ed as antidromic, and those single-unit isolation. Hindlimb stepping movements were transduced that did not follow or that showed variable latencies were classi®ed as by a pair of accelerometers, one attached to each metatarsus. The synaptic. Collision tests were not used because most units had 0 base ampli®ed output of the accelerometers was led to an analog/digital rates in the absence of stimulation. converter sampling at 1 kHz and displayed on a computer monitor. Histology Recording of neuronal activity A marking lesion was made at the end of the recording track by Single-unit activity was recorded differentially through a pair of passing 15±30 mA anodal current through the recording wires for 30 s. 25-mm-diameter stainless steel Te¯on-insulated wires cut to expose The rat was given a lethal dose of nembutal and perfused transcardially the cross-section of the tips. The wires protruded approximately with 10% formalin. The brain was placed in formalin and sectioned 1 mm beyond the tip of a glass pipette, which was sealed by paraf®n. transversely every 100 mm with a Vibratome. The sections were Occasional dif®culty in isolating single units with these large electrodes viewed with a microscope and the locations of the sites were projected was offset by the bene®t of their matched low impedance (,1MV)in on to drawings adapted from an atlas.64 reducing stimulation artifact. Typically, the amplitude of single units was 75±300 mV. Conventional ampli®cation, ®ltering and spike Data analysis discrimination procedures were used. The discriminator output (1.2-ms pulses) was suppressed for a variable amount of time with each Response to stimulation. Sites were tested at least every 100 mm stimulator pulse (1±3 ms) to prevent spurious discriminator pulses. along a track, and the presence of a discriminated single unit was Only well-isolated, stable single units were included in the analysis. not a prerequisite for a test. However, only isolated single units were There were two principal indications that axon spikes did not signi®cantly tested further, and if isolation was lost before suf®cient testing was contribute to the data pool. First, in recording tracks through ®ber performed, the unit was not included in the data pool. We ®rst asked bundles where somata are rare, the low-impedance electrodes that whether the unit responded to the Control stimulation. Particular were used recorded virtually no isolated spikes. Second, the testing attention was paid to the ®rst 3 s of stimulation because it preceded protocol required maintenance of isolation for several minutes during the onset of Control stepping. Neural correlates of stepping onset stepping and associated autonomic responses. Axon spikes show would have to be manifest in this pre-stepping period. The time around radical changes in amplitude or are easily lost under these conditions. stimulation onset was divided into ®ve 1-s bins, two preceding the The effectiveness of the single-unit discrimination during data onset (pre-stimulation bins) and three following it (stimulation bins). acquisition was checked by monitoring the stability of the amplitude The average rate of activity in each bin (spikes/s) was the data element and waveform of the action potential, and the absence of action for a repeated-measures analysis of variance with two factors, time-bin potentials during the refractory period. Later experiments occasionally (®ve 1-s bins) and priming condition (Control and Test phases). used a customized, off-line, spike-sorting program to separate units that Tukey's protected t-test (P , 0.05) was used for individual comparisons. had similar amplitudes of the principal peak but different waveforms. A unit was classed as responsive to the Control stimulation if any one The algorithms differentiated waveforms on the basis of the timing of the three stimulation bins was signi®cantly different from the two and amplitudes of the peaks that preceded or followed the principal pre-stimulation bins. peak. For analysis of unit responses, the discriminator pulses were passed Type I or Type II. The second question addressed was whether the to a computer-based A/D system operating at a sampling rate of 1 kHz activity of a responsive unit correlated in time with the onset of per channel and converted to a ratemeter display with a time bin of stepping. We classi®ed neurons into one of two types based on the 20 ms. The ratemeter data were transformed to a spike density record pattern of activity before the ®rst step to the Control train. Activity was by computing running averages over 100-ms intervals. The spike averaged for each Control train that had a stepping latency of at least density record, the accelerometer signals and stimulation marker 3 s. The average was synchronized to the movement of the hindlimb were stored for later analysis. Waveforms of the action potential that displayed the earliest stepping over most trials. Because the were sampled at 25 kHz. latency to step varied between trials, only the pre-stepping period common to all trials was used. Thus, the length of the pre-stepping Locomotor stimulation average was no longer than the shortest stepping latency. Almost all averages were based on four or more pre-stepping periods of 3±4 s The stimulation electrode was targeted at the perifornical hypo- duration. Type I units showed an average response that increased up to thalamus. When bilateral hindlimb stepping was reliably elicited at the point of stepping onset; the increase was gradual in some cases and an intensity of 50 mA or lower, the electrode was ®xed to the skull abrupt in others. To make this classi®cation objectively, we used the 80 H. M. Sinnamon et al. criterion that Type I increases had to have a pre-step average with a statistically signi®cant positive priming effects. Neurons with positive slope and a linear correlation coef®cient (r) of 0.67 or greater. activity patterns modulated with the phase of the locomotor Neurons that achieved this criterion were consistent on individual trials. Type II increases did not have a signi®cant positive slope over step cycle were rare (n ˆ 47, 4% of units tested). They were time during the pre-step period. The average response pattern peaked similarly rare (5.4%) among the units with Type I increases. early and changed little (or declined) in the 2±3-s period before the The most common response pattern was the Type II increase start of stepping. (43%), of which 30% showed positive priming effects. Negative priming effects, smaller responses in the Test Priming effect. The next analysis compared Control and Test activity phase, occurred in 13% of responses. Negative priming to determine whether the activity of a responsive unit showed a priming effect. The principal interest was in positive priming effects. Positive effects were common (43%) for an unanticipated subtype of priming effects were declared for either of two cases. In stimulation Type II increase units that showed a decline before stepping priming, the Test response was greater (t-test, P , 0.05) than the onset (II 1 decline). They were rare (4%) for the more Control. In pre-stimulation priming, the response to Control stimulation common type of Type II increase pattern, which was termed continued into the Test period, and caused the pre-Test activity to differ constant II1. Other response types included decreases (26%) (t-test, P , 0.05) from the pre-Control activity. When responses were signi®cantly smaller on the Test phase than on the Control, the priming and antidromically activated (5%). effect was termed negative. Type I primed increases. As shown in Fig. 2, Type I Phase relations. The ®nal analysis looked for modulation of activity increases had progressive increases up to the time of the in phase with the step cycle. Each locomotor bout was examined for ®rst step in the locomotor episode. Note the priming effects indications of phase relations and, where appropriate, averages of unit on stepping as re¯ected in the shorter latencies and/or greater activity synchronized to normalized step cycles were computed. In summary, the analyses determined four independent properties amplitudes of Test stepping. Unit priming effects are re¯ected of the activity of units that responded to locomotor stimulation: (i) in the differences between the Test trace (heavy line) and the whether the activity increased or decreased; (ii) whether the response Control trace (thin line). The units in Fig. 2B±E showed both pattern correlated with the onset of stepping (Type I or Type II); (iii) pre-stimulation and stimulation priming effects. The effect of whether the response correlated with the facilitated stepping during the Test train (positive or negative priming effect or neither); and (iv) the priming stimulation was to advance or amplify the whether the activity pattern was modulated with respect to the phase of increase in activity during the Test train. Although all Type stepping (phase related or not). I responses had the characteristic positive slope in the pre-step period, there were several indications that the class was not Regional analysis. The incidence rate of the response types was homogeneous. First, in 34% of the Type I units, an increase in computed for each brainstem region. The regions varied in terms of the activity appeared at the offset of the stimulation train. The occurrence of the amount of neural activity occurring in the absence of locomotor stimulation (spontaneous activity). Most responsive units magnitude of the offset increase varied, and often it was had 0 base rates and were found at the standard 100-mm steps along small, appearing only on stimulation averages. Second, for the electrode track. Therefore, it was not appropriate to use the number most units, the increase in activity started in the ®rst second of tested units in a region as the base for the incidence rate because it of Control stimulation, but for others (16%) the rise began would penalize regions with a higher incidence of spontaneously active later. An example of a unit with both a large offset increase neurons. For this reason, the number of standard tests at 100-mm steps through a region was used as the base, expressing the incidence rate of and a late-onset pattern is shown in Fig. 2E. Third, 6% of each response type as the occurrence per 100 sites tested. This measure Type I patterns showed an initial decrease in activity that was less biased in the sense of making regions with differing amounts preceded the upward ramp. It was often undetectable on of spontaneous activity and different sampling densities comparable. single trial records and appeared on the stimulation averages. To evaluate the incidence rate for each region, it was compared to the remainder of the population by chi-square tests or, when expected This pattern can be seen in stimulation averages for the Test in frequencies were ,5, by Fisher's Exact Probability Test. Fig. 2B and for the Control in Fig. 2D.

RESULTS Type II increases. Type II increases, the most common response type, had early peaks that preceded stepping by 1 Stimulation sites or more seconds. In the more common constant II 1 pattern, The locations of the stimulation sites used to elicit stepping the rate increased to an early peak and maintained a constant are shown in Fig. 1. They were in the perifornical area and high level. Examples are shown in Fig. 3A±C. In an unantici- in the medial and lateral hypothalamus adjacent to it. The pated variation that was seen in 29% of the Type II increases, locations of effective sites are consistent with previous termed declining II1, the peak was early, but activity studies.76 It was usually necessary to raise the current over declined before the onset of stepping. The decline usually the course of an experiment to maintain stepping. The mean did not reach a level lower than the pre-stimulation rate. An initial current was 37 mA(S.D.ˆ 9.6) and the mean increment example is shown in Fig. 3D. Note the negative stimulation was 10 mA (S.D. ˆ 9.2). priming effect, i.e. less increase in activity on the Test than the Control. Response types Antidromic responses. Antidromic responses appeared with A total of 1159 single units was tentatively classed as the ®rst pulse of the stimulation and continued at a virtually responsive to locomotor stimulation. Analysis showed 82% constant rate throughout the train. The latencies of 50 anti- (n ˆ 959) of them to have statistically signi®cant responses dromic responses ranged from 0.7 to 7.0 ms, with a median of during the ®rst 3 s of Control stimulation. The 52 units that 1.9 ms. There was no indication of increased latencies for failed to reach the response criterion indicate that the testing sites at greater distances from the site of stimulation. An procedure was not biased toward units with large responses. example of an antidromically activated unit showing an The response type of main interest, the Type I increase, was entrained response to the 50-Hz hypothalamic stimulation shown by 25% of the responsive units, 57% of which showed and a post-stimulation depression in rate is shown in Fig. 4. Priming of locomotion 81

locomotor stimulation are illustrated in Figs 6±8. Type I increases are indicated on the left side, and Type II increases and antidromic responses on the right side. The frequency of responses should be viewed in the context of the sampling density of the recording tracks, which are represented by grey lines. The ®lled circles represent the Type I and II units that showed positive priming effects. The grey squares indicate the II 1 decline units that showed negative priming effects. For simplicity, the locations of units that showed decreased patterns are not illustrated. Generally, they occurred proportionally among units showing increased patterns. Table 1 compares the regions of interest in terms of the rate of incidence of selected response patterns. The population value in Table 1 shows that the overall incidence rate of all responses to locomotor stimulation was 10.3 per 100 sites. The total number of recording sites in the regions of interest constituted approximately 41% of the population sites, and no one region accounted for more than 9% of the population. Signi®cant differences relative to the population values are indicated with asterisks. Three regions, the ADT, the caudal pontine reticular nucleus (PnC) and the gigantocellular nucleus (Gi), were particularly responsive (.20%) to locomotor stimulation, compared to the 10.3% incidence rate for the population. Two other regions, the PnO and the PPA/MLA, were less responsive but also had incidence rates greater than the population. The ventral tegmental area was signi®cantly less responsive than the population.

Anterior midbrain. In Fig. 6, the grey rectangles show the ADT. The boundaries are based on the ®ndings of previous studies18,78 and encompass the magnocellular nucleus of the posterior commissure, the interstitial nucleus of the medial longitudinal fasciculus and the adjacent tegmental area. As shown in Table 1, the ADT had an incidence of Type I primed and constant II 1 primed increases that was more than three times the population value. Type I primed units were localized to the ADT compared to the rest of the anterior midbrain (Fig. 6). The ADT was the only region with a high incidence of both Type I and II 1 primed units. Constant II 1 primed units were actually more common than the Type I primed units in the ADT, but they were more widely distributed. Decrease responses showed a similar pattern. Declining Type II increases were rare in the ADT region and in the anterior Fig. 1. Schematic representation of the locations of the stimulation sites used to elicit locomotion. The anterior±posterior (AP) numbers on the left midbrain generally. of each panel indicate the distance in mm posterior to bregma. A total of Responses to locomotor stimulation in general, and primed 238 sites is represented; 12 rats had two sites. One site located at AP 2.3 is responses in particular, were rare in several well-sampled not represented. Sites on both sides were used but all are plotted on the left. 64 regions of the anterior midbrain. Responses of all types, Drawings adapted from the atlas of Paxinos and Watson. except for decreases, had a low incidence in the ventral tegmental area. Other low-response regions included the Decrease responses. The typical (n ˆ 217) pattern of lateral tegmentum at AP 4.8 and 5.3, the medial central decrease in activity in response to locomotor stimulation is gray (CG), the red nucleus and the rostral linear nucleus. shown in Fig. 5A. Post-stimulation increases in the rate of Antidromic responses were found only in the nucleus of the activity were common (33%) among these units. An example posterior commissure. of a unit that decreased in rate during Control and Test stimulation with a progressive decrease in activity that Posterior midbrain. The locations of units in the posterior was inverse to the Type I increases is given in Fig. 5A. midbrain showing the various response types are represented Units of this type were rare, and few showed positive priming in Fig. 7. The dorsolateral tegmentum region includes effects. structures in the posterior midbrain dorsal to the peduncle and cuneiform nucleus, usually not considered part of the midbrain locomotor area. The region includes the lateral Regional comparison of response types CG, the intercollicular nucleus and the tegmentun lateral to The locations of units showing increases in activity to the CG. As shown in Table 1, this well-sampled region 82 H. M. Sinnamon et al.

Fig. 2. Examples of Type I primed patterns. Each panel shows a representative trial, an average of unit activity synchronized to the ®rst step, and averages of the unit activity synchronized to the start of the Control and Test trains. (A) Stimulation priming effect. (B±E) Pre-stimulation and stimulation priming effects. The left column shows the spike density and accelerometer traces from single trials consisting of a Control train followed in 10 s by a Test train. The stimulation marker at the bottom shows a break, which indicates a 5-s gap between Control and Test records. The upper trace in each panel shows the spike density record. The vertical calibration marker to the left of the trace represents ®ve spikes/100 ms; all spike density calibration markers in subsequent illustrations have this value. The bottom of the calibration marker locates the 0 value. The trace below each spike density trace represents the outputofan accelerometer attached to the metatarsus of the hindlimb that most frequently led the stepping sequence. Flexion produced an upward de¯ection; extension produced downward de¯ection. The middle column, Step Average, shows the average of the spike density and accelerometer traces synchronized to the start of stepping during the Control train. The length of the average trace to the left of the ®rst step re¯ects the trial with the shortest latency of stepping; thus, the average is of activity occurring between the stimulation onset and stepping onset on all trials. The right column, Stimulation Averages, shows the average spike density records synchronized to the start of stimulation. The Control (thin trace) and Test (heavy trace) records are overlapped to highlight unit priming effects. Locations of units: (A, B) magnocellular nucleus of the posterior commissure; (C) PnO; (D, E) Gi. showed a general responsiveness and a distribution of responsiveness moderately larger than the general population responses that paralleled the general population. The exception that was attributable to a relatively high incidence of was the high incidence of antidromic responses (1.5% of decreased (4.5%) and antidromic responses (1.3%). There sites). They were particularly common in the ventrolateral was no indication in the region of a high incidence of primed posterior CG and adjacent tegmentum at AP 7.8 (Fig. 7). Type I or Type II responses. The boundaries of the PPA/MLA were based on data for The PnO was well sampled, particularly at AP 8.3. the rat.7±9,12,22,23,45,50,63,67,78,79,85 The region includes the areas Although the region showed a general responsiveness similar adjacent to the superior cerebellar peduncle at AP 7.3, 7.8 and to the PPA/MLA, it showed a large incidence of Type I 8.3 (chie¯y the pedunculopontine tegmental nucleus, the responses with priming effects. As shown in Fig. 7, they subpeduncular tegmental nucleus and the cuneiform nucleus appeared to be present throughout the region. at AP 8.3 and 8.8). The superior cerebellar peduncle (which The median raphe region included the median raphe contains few cell bodies) occupies a signi®cant proportion of nucleus at AP 7.3±8.3 and the paramedian raphe nucleus. the region, and to ensure comparability of incidence rates, the At AP 8.3, the dorsal boundary extended up to the ventral estimated number of recording sites in the PPA/MLA was aspect of the dorsal raphe. The general responsiveness and adjusted by subtracting the number in the superior cerebellar the distribution of response types were similar to the general peduncle. The PPA/MLA was densely sampled, particularly population, with the exception of a relatively high incidence at AP 8.3. As shown in Table 1, it showed a general of declining II 1 responses with negative priming effects. Priming of locomotion 83

Fig. 3. Four units showing Type II increases, level (or declining) spike density traces for several seconds prior to the onset of stepping. (A±C) Constant II1 patterns; A shows a stimulation priming effect, B shows stimulation and pre-stimulation effects, and C shows no priming effect. (D) Declining II1 pattern showing negative priming effect. Unit locations: (A) anteromedial Gi; (B) medial to the red nucleus; (C) magnocellular nucleus of the posterior commissure; (D) ventral margin of the medial longitudinal fasciculus at AP 78. Rate calibration: ®ve spikes/100 ms.

Pons and medulla. Sites tested in the pons and medulla are nucleus of the solitary tract. The responsiveness of the PnC shown in Fig. 8. The medial aspects of the PnC are shown in and the Gi likewise did not appear to extend ventrally to the the transverse drawing for AP 9.3 in the upper left and in the magnocellular nuclei. Note, in all panels of Fig. 8, the general sagittal drawing for ML 0.1. The medial aspects of the Gi are decline in responses in the ventral regions, which include the illustrated in the transverse drawing for AP 12.3 and in both of ventral pontine reticular, and the anterior and ventral Gi. the sagittal representations. As may be seen in Table 1, these two regions were the most responsive of any tested region. The Gi had a higher incidence of Type I primed responses DISCUSSION than any other region. It also had high incidences of decrease The results show the regional distribution of neurons with responses and of declining II 1 responses that showed activity patterns correlated with both the onset of stepping negative priming. The responsiveness of the PnC was (Type I increase) and the facilitated stepping produced by attributable to high incidences of constant II 1 primed priming stimulation. Neurons showing the conjunction of responses, declining II 1 responses with negative priming, these independent properties should include, but not necessarily decrease responses and antidromic responses. Lateral aspects be restricted to, neurons with a role in locomotion initiation. of the Gi were sampled at posterior levels, shown at AP 12.3 Neurons with similar Type I primed patterns rarely showed in Fig. 8. The area appeared to be as responsive as the medial activity related to the phase of stepping and were found in regions but, contrary to the pattern in other regions, most of widespread regions of the brainstem. They were prevalent the Type I responses did not show priming effects. The as expected in the Gi of the medulla, a region previously ventrolateral aspects of the Gi also showed high incidences implicated in locomotion, and they were also prevalent in of decrease responses. regions not generally considered to be locomotor regions, The regions dorsal to the PnC and Gi were notable for the the ADT and PnO. Conversely, they were relatively sparse rarity of Type I responses. Dorsally, the unresponsive regions in other regions, such as the ventral tegmental area and included the caudal interstitial nucleus of the medial ªmidbrain locomotor areaº, which have been implicated in longitudinal fasciculus and the dorsal paragigantocellular locomotion by studies using other preparations. nucleus. At AP 12.3, the caudal interstitial nucleus of the medial longitudinal fasciculus contained a few Type I units and, at more anterior levels, it contained a variety of other Interpretation of response types response types. Type I increases were virtually absent in the Multiple processes are activated during the initiation of dorsal regions associated with sensory functions, including stepping. They include postural changes to provide antigravity the inferior colliculus (central and caudal), the parabrachial support,52 the arrest of antagonistic behaviors, orientation region (lateral), the vestibular complex (medial) and the of the head and anterior torso,81 shifting the center of mass 84 H. M. Sinnamon et al.

critical than its relative rate of occurrence in different regions. High occurrence rates of Type I primed patterns are at least consistent with a role for a region in the initiation of hypothalamic stepping. However, it is dif®cult to conceive of a mechanism by which a region showing a low rate of occurrence of Type I primed responses would importantly contribute to the onset of stepping. The focus here on Type I primed response patterns does not minimize the potential importance of other response patterns for the initiation of locomotion. For example, the early rise to an asymptote of constant Type II responses is consistent with neuronal patterns involved in the establishment of postural and exercise-related states, which precede locomotion. Type II responses had a regional distribution that was similar to, Fig. 4. Response patterns of an antidromically activated unit in the medial but wider than, Type I responses. The earlier responding Type PnC during trains of locomotor stimulation of the hypothalamus. Note the II neurons with positive priming are feasible candidates for nearly veridical response of the neuron to the 50-Hz stimulation and the providing excitatory drive for neurons showing Type I post-stimulation decrease in activity rate relative to baseline. The inset shows the unit response following each of three pulses at an interpulse patterns. Feasible proposals for functions in locomotor period of 3 ms. Rate calibration: ®ve spikes/100 ms. initiation can also be made for the other response patterns but, lacking the behavioral correlations that support the forward32,83 and cardiopulmonary adjustments appropriate Type I and II primed neurons, they would be unduly speculative. for exercise.17,90 In addition, hypothalamic locomotor stimu- The mechanisms underlying the priming effects on locomotor lation, even at minimal currents, activates non-locomotor stepping and on the neuronal responses studied here need processes. Therefore, neurons that respond to locomotor further study. The appearance of primed responses throughout stimulation will vary in their degree of relatedness to stepping the extent of the brainstem is consistent with the evidence onset. Some will be critical, some associated and some that priming re¯ects widespread processes. Stimulation at extraneous. The group of Type I primed neurons must include sites from the posterior hypothalamus to the preoptic area neurons that are critical to the onset of stepping. Extraneous produces apparently similar locomotor priming effects. neurons are no doubt included as well, and there were variations Stimulation at one locomotor site will prime stepping elicited in the Type I primed patterns that indicate that future studies by a second train at that same site, and at ipsilateral and will need to use additional criteria to improve speci®city. One contralateral sites as well.87 At longer Control±Test intervals interesting variation was a pattern appearing in one-third of than used here, suppressive effects of priming stimulation the cases, in which an increase in activity appeared at the in the hypothalamus can also appear for some sites.77 The offset of the stimulation train. Although this rise was usually amplitude and frequency of hippocampal rhythmic slow- brief and small, often appearing only on the averaged records, wave activity in the 3±6-Hz band generally co-vary with its occurrence at the offset of stimulation when stepping was the facilitation of stepping in the priming situation.78 In declining is dif®cult to reconcile with a simple role in loco- cases where the priming effect shows a monotonic decline, motor initiation. Other variations in the typical ramp-to- the correlation is positive, but in cases where priming asymptote Type I pattern were a delayed rise in activity effects are mixed, the correlation can be absent or negative.77 until just prior to the start of stepping and a downward in¯ec- In the awake and freely moving rat, the priming effect is tion early in the upward ramp that appeared in the averaged associated with a reduction in pre-locomotor head scanning records. The variations in the Type I primed pattern suggest movements.81 Although there is no direct evidence, it caution in attributing to the neurons displaying them a de®nite would seem likely that priming effects would also be role in locomotor initiation. For the purposes of this study, correlated with facilitated exercise-related, cardiopulmonary however, the speci®city of the Type I primed pattern was less processes.17,90

Fig. 5. Two units showing decreases in activity during locomotor stimulation. (A) Most common pattern, low or zero activity rate for several seconds preceding the ®rst step. (B) Progressive decrease in activity precedes the ®rst step. Locations of units: (A) dorsal raphe nucleus; (B) dorsal Gi. Rate calibration: ®ve spikes/100 ms. Priming of locomotion 85

Table 1. Responses to locomotor stimulation: regional comparison of the incidence rate of selected response types and priming effects

All responses Incidence (per 100 sites) of response types

Region Recording Frequency Incidence Type I1 Constant II1 Declining II1 Decreased Antidromic sites primed primed neg. prime

ADT 373 78 20.9* 4.6* 6.2* 0.5 4.8* 0.0 Ventral tegmental area 191 11 5.8* 0.0 0.5 0.0 3.7 0.0 Dorsolateral tegmentum 910 100 11.0 1.8 0.8 0.3 1.5 1.5* PPA/MLA 538 76 14.1* 0.9 1.1 0.9 4.5* 1.3* PnO 808 111 13.7* 3.8* 0.9 0.6 2.2 0.4 Median raphe 512 42 8.2 1.2 0.6 1.2* 2.3 0.4 PnC 150 38 25.3* 2.0 4.0* 4.0* 8.7* 1.3* Gi 344 83 24.1* 5.2* 1.7 2.3* 7.3* 0.3

Population 9310 959 10.3 1.5 1.2 0.6 2.6 0.5

*Signi®cant (P , 0.05) difference from remainder of population by chi-square or Fisher tests.

Regional differences in response patterns for the ADT in processes related to locomotor initiation. Reversible inactivation of neurons in the region using Neurons in the ADT are known to show Type I patterns18 GABA injections blocked stepping produced by hypothalamic and this study demonstrates that most of these neurons also stimulation.78 Stimulation in the region produces stepping in show priming effects. These patterns are consistent with both intact-anesthetized and brain-transected rats.5 In ®sh, functional and anatomical evidence that indicates a role electrical or glutamate activation of the region elicits swimming

Fig. 6. Schematic representation of the locations of the units in the anterior midbrain showing increases in activity during locomotor stimulation. The grey lines represent the recording tracks and indicate the sampling density. All units were ipsilateral to the side of stimulation or on the midline, but Type I units are represented on the left, and Type II and antidromic (stars) units on the right. Two Type II variants are represented. The smaller circles represent the constant II 1 (e.g. Fig. 3A±C) and the squares indicate the declining II 1 (e.g. Fig. 3D). Filled circles indicate positive priming effects; open circles indicate no priming effects or negative priming effects. Stippled squares indicate negative priming effects for the declining II 1 units. The grey rectangle indicates the boundaries of the ADT. The numbers to the left of each drawing indicate the distance in mm posterior to bregma. 86 H. M. Sinnamon et al.

Fig. 7. Schematic representation of the locations of the units in the posterior midbrain showing Type I and Type II increases and antidromic responses. Format similar to Fig. 6. One track located at AP 6.7 is not represented. The grey polygons indicate the boundaries of the dorsolateral tegmentum, the PPA/MLA, the PnO and the median raphe nucleus. movements.6,88 In the cat, neural activity phase-related to area elicits stepping in the awake63 and anesthetized rat.74,79 stepping was found in the interstitial nucleus of Cajal.29 The Lesions82 and procaine injections44 there block stepping ADT has been implicated in the control of vertical eye and elicited by perifornical stimulation, possibly by interrupting head movements in cats and monkeys,20,21 and in the awake ®bers of passage. The locomotor effects of drugs applied to rat, vertical head movements and related postural adjustments this region in the awake rat appear to depend on the ascending are a prominent component of locomotor initiation.81,83 The projections of dopamine neurons.36,51 In the anesthetized rat, presence of Type I primed neurons in this region suggests a large lesions in the anterior hypothalamus, which would possible role in the interaction between locomotion and either interrupt these ®bers, do not block locomotion elicited by neck posture or head scanning. stimulation in the perifornical hypothalamus.75 Neither the In the ventral tegmental area, the most common response present data nor previous ®ndings provide evidence that the was a decrease, but overall few responses were seen. Recordings neurons in the ventral tegmental area or their ascending axons there contrasted with those immediately dorsal in the red directly contribute to the locomotion studied here. nucleus, which was similarly unresponsive but showed The PPA/MLA, which includes both the pedunculopontine more baseline activity. Stimulation in the ventral tegmental tegmental and the cuneiform nuclei, showed few Type I Priming of locomotion 87

Fig. 8. Schematic representation of the locations of units in the pons and medulla showing Type I and Type II increases and antidromic responses. Format similar to Fig. 6, except the two sagittal drawings cover the region between the transverse levels at AP 9.3 and 11.8. The sagittal levels are represented twice, on the left for Type I, and the right for Type II and antidromic. The numbers to the left of sagittal drawings indicate mm from midline. The transverse drawing at AP 12.3 includes units that were located at AP 11.8 and 12.8. Four tracks that were too lateral to be represented on the sagittal drawings are not shown. The grey polygons represent the boundaries of the Gi and the PnO. primed responses. Decreases were common, and antidromic evidence for a direct role for pedunculopontine neurons in responses appeared in and around the region. The cuneiform the initiation of locomotion.7,22,23 However, recent studies nucleus is one of several midbrain sites where electrical have raised complexities. Lesions do not reduce open ®eld stimulation elicits stepping in the anesthetized rat.13,45,67 locomotion57,86 or locomotion elicited by drugs applied However, the relative scarcity of primed responses is to limbic structures.34,57,86 Neither do lesions block either consistent with the evidence that cuneiform neurons are not locomotor avoidance or escape patterns.19 The cholinergic essential for locomotion initiated by forebrain systems. neurons of the pedunculopontine tegmental nucleus have Lesions of the nucleus in the thalamic cat do not block the been proposed to be particularly important in locomotor locomotor effects of diencephalic stimulation.58 Nor do initiation.22 Although the nucleus itself was well sampled, procaine injections44 or electrolytic lesions84 in the anesthetized what proportion of the recordings was from cholinergic rat block stepping elicited by perifornical stimulation. In the cells is not known. Another reason for caution in interpreting awake rat, somatotoxic lesions do not reduce either open ®eld negative ®ndings for this nucleus is that its rostral extension, locomotion or locomotion elicited by amphetamine injections anterior to AP 7.3, was not sampled. The region, as well as into the nucleus accumbens.1 The cuneiform nucleus appears the cuneiform nucleus, may have a modulatory role in to be part of a system involved in defensive behavior that locomotion26 that would not be apparent in the present includes aversively motivated locomotion.13,14,46 study because of a relatively greater sensitivity to anesthetics. The region in and around the pedunculopontine tegmental The dorsolateral tegmentum and CG at AP 7.8 showed a nucleus also showed a low density of Type I primed variety of responses to locomotor stimulation, including responses. In the anesthetized rat67,79 and in the midbrain- Type I and II primed patterns, but only the incidence rate of transected rat,12 stimulation is not as effective there as it is antidromic responses was greater than the general population. in more dorsal areas. In the unanesthetized rat, there is The ventrolateral CG showed the highest density of antidromic 88 H. M. Sinnamon et al. responses of any region. Stimulation in the dorsolateral Gi. In the present study, these magnocellular nuclei were tegmentum can produce locomotion in the awake rat, but it remarkably sparse in Type I responses and priming effects. is often mixed with crouching, vocalization and leaping, Given the high incidence of responses in the gigantocellular which suggests aversion.63,66 This region is not a source of region, it seems unlikely that recordings in the same tracks projections to the spinal cord, and lesions or procaine would have missed responsive cells in the immediately injections here do not block stepping elicited by perifornical ventral magnocellular nuclei. stimulation.44,84 Posterior to AP 7.8 in the CG, there was a There were no indications that caudal brainstem responses sharp reduction in increased responses, and most responses had longer latencies or peaks of activity closer in time to were decreases or declining increases. The posterior CG has stepping onset compared to midbrain regions. Type I patterns been implicated in fear-related immobility.10 in general were more common in the Gi than any other region, Type I primed responses were the dominant pattern in and but the temporal characteristics of both the late- and early- around the PnO, and their prevalence is consistent with onset patterns appeared similar to those of more anterior evidence suggesting a role for the region in the control of regions. The earliest synaptic responses, the Type II stepping. Oral pontine neurons project directly to the lumbar increases, also appeared to be similar in the Gi and in the cord4,43,69 and to reticulospinal neurons of the medulla.56 In midbrain. These impressions need to be evaluated more the anesthetized rat, stimulation applied to the lateral and rigorously with simultaneous recordings, but at this time the ventral aspects of the nucleus elicits stepping.67,79 Procaine ®ndings seem at odds with a simple hierarchical model injections throughout the nucleus blocked stepping elicited by postulating a serial ¯ow of information from forebrain perifornical stimulation in the anesthetized rat.44 Lesions of systems to a single midbrain locomotor region, and then the nucleus in awake rats produced de®cits in hindlimb to a posterior brainstem region which controls the cord. control during locomotion.11,30,39,53 The PnO is large and Alternatively, the ®ndings are consistent with a model that may have multiple, possibly antagonistic, roles in stepping postulates multiple locomotor circuits organized as a distributed control. In the anesthetized rat, stimulation in the central bidirectional network in which various specialized components region disrupts stepping elicited by perifornical stimulation.79 act concurrently on the cord. Redundant and distributed In the cat, stimulation in a single oral pontine site both circuitry would be appropriate to mediate highly adaptable suppressed muscle tone and elicited stepping movements.42 forms of locomotion initiated in diverse motivational Lesions in the decerebrated cat resulted in spontaneous contexts. rhythmic hindlimb movements.41 The median raphe, rostral and caudal linear nuclei showed Activity patterns related to phase of stepping low responsiveness. Only the declining II 1 response pattern had a relatively high incidence, and neurons with this There was no tendency for Type I primed units to show pattern could play a role in the locomotor suppressive phase-related activity. The overall occurrence of phase- function that has been suggested for this general region. In related activity was lower than expected from ®ndings with the anesthetized rat, stepping elicited by perifornical stimula- non-anesthetized preparations. It was more common in the tion is suppressed by concurrent ventromedial stimulation79 reticular regions, as expected from previous studies,15,37,59,65,73 and it is facilitated by GABA injections in the region.80 In the but rare or absent in other regions, such as the red nucleus3,60 awake cat, stimulation in the median raphe stops spontaneous and .61 The dearth of phase-related activity in locomotion and reduces postural tone.40 In the awake rat, the red nucleus was surprising. Recordings in the nucleus stimulation in the region produces behavioral suppression31 typically showed highly active units that were unresponsive and blocking raphe neural activity increases open ®eld to locomotor stimulation. It is possible that the anesthesia locomotion.92 depressed cerebellar activity which, together with the In agreement with evidence implicating the Gi in the spinorubral paths, provide the major phase-related in¯uences initiation and control of locomotion, the region was highly to the red nucleus.2,60,89 If this speculation is correct, it responsive to locomotor stimulation and had the highest underscores one of the limitations of the anesthetized rat as incidence of Type I patterns. Type I primed response patterns a locomotor model. were prevalent throughout the anterior and posterior extent of the Gi, but they were rare in the adjacent dorsal and ventral large cell regions. The gigantocellular Type I neurons in the CONCLUSIONS rat are similar in location and activity pattern to hindlimb- This study identi®ed brainstem regions that have high related neurons in the cat, which show tonic but not phase- incidences of neurons with activity correlated with the onset related activity during stepping.15,16,65 There appear to be and facilitation of locomotor stepping in the anesthetized signi®cant differences between the gigantocellular location intact rat (Type I primed pattern). Type I primed neurons, of Type I neurons and the locations of locomotor-related with generally similar response pro®les, were common in units reported in other studies. The gigantocellular Type I certain tegmental reticular regions that project to the spinal neurons appear to be ventral and caudal to neurons in the cord. They included a posterior region, the Gi, which has cat that jointly responded to stimulation in the midbrain and an established involvement in locomotor initiation, and two hypothalamic locomotor regions.62 However, they were anterior regions, the ADT and PnO. Few Type I primed located more dorsally than the region in the cat, described neurons showed a modulation of activity related to the step as containing neurons responding to midbrain locomotor cycle. They therefore do not appear to contribute to the stimulation.24 A report38 from the latter laboratory described control of the stepping pattern itself. In general, Type I this region in the transected rat as most effective for chemically primed neurons mingled with neurons showing other response and electrically elicited stepping, and speci®ed its location patterns, including decreases. Certain regions, the cuneiform as the gigantocellular alpha at its transition with the ventral nucleus, the pedunculopontine region and the ventral Priming of locomotion 89 tegmental area, that have been implicated in locomotion did AcknowledgementsÐThis work was supported by NIH grant not show the prevalence of Type I primed responses expected 1R15NS34118-01, grants from Wesleyan University, the Hughes Medical Foundation and the McNair Foundation. We thank J. S. in regions critical for the stepping elicited by perifornical Lapsansky, A. W. Salyapongse, A. Fuller, B. Hasler, R. Slotnick and stimulation. If these regions have a role in locomotion in S. Wilson for help in collecting data. this preparation, it is indirect.

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(Accepted 7 April 2000)