GABA injected into the anterior dorsal tegmentum ADT of the midbrain blocks stepping initiated by stimulation of the hypothalamus

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Ž. Brain Research 766 1997 271 275 Short communication Ž. GABA injected into the anterior dorsal tegmentum ADT of the midbrain blocks stepping initiated by stimulation of the hypothalamus H.M.

1 Ž. Brain Research Short communication Ž. GABA injected into the anterior dorsal tegmentum ADT of the midbrain blocks stepping initiated by stimulation of the hypothalamus H.M. Sinnamon ), M. Benaur Neuroscience and BehaÕior Program, Wesleyan UniÕersity, Middletown, CT , USA Accepted 3 June 1997 Abstract Previous work showed that the activity rates of certain neurons in the anterior dorsal tegmentum Ž ADT. of the midbrain correlated with the onset of stepping elicited by hypothalamic stimulation. This study determined if reversible inactivation of the ADT would block locomotion elicited by hypothalamic stimulation of anesthetized rats Ž urethane, 800 mgrkg.. GABA Žconcentrations mgrml in saline. were injected in 52 sites in 21 rats. GABA at volumes of 0.1 or 0.2 ml blocked hindlimb stepping in 18 cases. Locomotor blocks occurred within 5 min of the injection, and typically recovered within min. The effective blocking sites were clustered around the interstitial nucleus of the medial longitudinal fasciculus. Sites more dorsal and more anterior were not as effective as sites in and ventral to this nucleus. The data are consistent with a role for the ADT of the midbrain in locomotor initiation. q 1997 Elsevier Science B.V. Keywords: Locomotion; Anterior dorsal tegmentum; Midbrain; GABA; Reticular formation; Hypothalamus In the anesthetized rat, electrical or chemical stimulation of the perifornical hypothalamus elicits sequences of controlled hindlimb stepping w13 x. A region in the midbrain, the anterior dorsal tegmentum Ž ADT., contains a relatively high density of cells with activity correlated with onset of stepping wx 5. There is some evidence from prior studies that supports the notion of an ADT role in locomotion Ž see below., but there has been no systematic approach to the question. The purposes of this study were to determine whether inactivation of neurons in the anterior midbrain would impair the initiation of locomotor stepping produced by hypothalamic stimulation, and to determine if the distribution of effective sites corresponds to the ADT region implicated in the recording study. We used the anesthetized rat in which brief bouts of hindlimb stepping were elicited by hypothalamic stimulation presented in trials at 1-min intervals. Injections of GABA were used to reversibly inactivate sites in and around the ADT because their brief effects facilitated the testing of multiple sites and doses in the same preparation. Male Sprague Dawley rats Ž n s 21. were initially anesthetized with 2% halothane in oxygen for 5 10 min, then ) Corresponding author. Neuroscience and Behavior Program, Judd Hall, 207 High Street, Wesleyan University, Middletown, CT , USA. Fax: q1 Ž ; hsinnamon@wesleyan.edu given an initial intraperitoneal injection of 500 mgrkg urethane. The appropriate anesthetic level was achieved by additional intraperitoneal injections of 100 mgrkg urethane over approximately the first hour of the experiment. Injections were given whenever the rat showed increases in respiration, vibrissae movement, mouth movement, or limb movement in the absence of brain stimulation. When the appropriate urethane level was reached, the rat would maintain an anesthetized state for several hours without a supplemental injection. Typically, the cumulative dose of urethane administered was 800 mgrkg. Body temperature was maintained by means of a heat lamp. The surgical preparation, stimulation parameters, and methods for meaw17 x. When the stimulation electrode was fixed in a hypotha- suring hindlimb stepping have been described lamic locomotor site, the site was stimulated on a 1- trialrmin schedule that was maintained for the duration of the experiment. Each trial consisted of two 5-s trains of stimulation, the control and test, which were separated by 10 s. For a test of an injection site, current was fixed at a current between 25 and 70 ma. GABA Ž Sigma. was injected after the baseline for stepping had been established for at least 10 min. GABA was delivered in saline at a concentration of mgrml at a volume of ml. The GABA was injected through a glass pipette Ž mm tip diameter. which was connected by PE 20 tubing to a 1-ml syringe. The injection was made at a rate

2 272 ( ) H.M. Sinnamon, M. BenaurrBrain Research of 0.5 mlrmin. Flow through the system was confirmed by observing the movement of an air bubble in the tubing. If there was no effect of the GABA injection, at least 20 min elapsed before the pipette was moved to a new site. After the deepest site was tested, the pipette was fixed in the brain with acrylic cement to aid localization. In 10 experiments, a single midbrain site was tested, and GABA injections were repeated, typically 3 times, to assess reliability or to test different volumes. In another 11 experiments, multiple sites were tested to localize effective regions. Usually a single injection was made in each site when multiple sites were tested. A maximum of four midbrain sites was tested in 5 rats. We tested GABA injections in the anterior midbrain of 21 rats in which hypothalamic stimulation produced bilateral hindlimb stepping. All injections were ipsilateral to the stimulation site or, if contralateral, within 0.5 mm of the midline. GABA injections Ž 0.1 or 0.2 ml. produced a reversible block of stepping at 18 of 52 sites. A block was defined as the complete absence of a step cycle by both hindlimbs for at least 4 consecutive trials. Blocks occurred within 5 min of the GABA injection Ž median 3 min.. There were no indications of graded effects in the onset of the blocks. When a block occurred, there was an initial series of trials in which stepping completely failed to appear during both the control or test trains of stimulation. Fig. 1. Representative case in which GABA Ž 100 mg in 0.1 ml saline. injected into one of four midbrain sites reversibly blocked hindlimb stepping produced by hypothalamic stimulation. A: schematic illustration of the location of the stimulation electrode in the perifornical Ž PeF. area of the hypothalamus plotted onto a drawing adapted from the atlas of Paxinos and Watson wx 9. The number at the lower left indicates the distance in mm posterior to bregma. B: representation of the injection sites in the anterior midbrain. GABA injections at the sites represented by the stippled circles produced no effect on stepping. The filled circle represents the location of a site at which GABA blocked stepping. C: accelerometer records from seven trials showing the onset of the GABA-induced block and the recovery of stepping for the effective site in panel B. The upper trace in each pair represents the accelerometer tracing for the right hindlimb Ž Rhl. and the lower trace represents the left hindlimb Ž Lhl.. Upward deflections represent flexion and downward deflections represent extension. The marker at the bottom indicates the control and test trains of hypothalamic stimulation which were separated by 10 s. The pair of trains was presented at 1-min intervals. The GABA injection was made over approximately 12 s beginning at the offset of the control train on the trial at 0 min. Note that the block of stepping appeared at the 4-min trial, recovery first appeared at the 18-min trial and was complete at the 26-min trial. See Fig. 2 for abbreviations.

3 ( ) H.M. Sinnamon, M. BenaurrBrain Research During recovery, graded effects did appear. Stepping usually appeared first in response to a test train which generally showed stronger stepping than the control train Žthe priming effect, w17 x.. The end of the block was defined to be at the trial at which bilateral stepping first reappeared on both the control and test trains. The median duration of the blocks was 13 min. The durations were variable Žrange 4 min to 2 h., even when the same site was retested with the same injection volume. Results of a representative experiment in which one injection of GABA was given at each of multiple midbrain sites are shown in Fig. 1. Panel A illustrates the location of the locomotor stimulation electrode in the perifornical hypothalamus. In other experiments, the stimulation sites were dorsal to the fornix in the lateral hypothalamus within 1 mm posterior to this level. In the case illustrated, stimulation at 45 ma produced bilateral hindlimb stepping which is illustrated by the accelerometer records of panel C. Panel B illustrates the locations of four GABA injection sites in the anterior midbrain. GABA Ž100 mg in 0.1 ml saline. produced a block of stepping at one ADT site Ž filled circle., but not at other sites Ž stippled circles.. Panel C shows the time course of the 22-min block for the effective site. Fig. 2 summarizes the locations of the midbrain injection sites. All sites were within an anterior-posterior range of 4.8 to 5.8 mm posterior to bregma. The sites at which GABA produced a reversible block are represented by the filled circles. The sites at which GABA produced no apparent effect on stepping are represented by the stippled circles. Most of the effective sites were within 0.5 mm of the interstitial nucleus of the medial longitudinal fasciculus Ž IMLFG, IMLF.. At the most anterior level of 4.8 mm, only 2 of 18 sites were effective and both were in the IMLF region. Sites dorsal to the IMLF region appeared to be the least effective at any anterior-posterior level. At anterior-posterior levels 5.3 and 5.8 mm, three sites at which more ventral GABA injections were also effective were found. These sites were in the medial red nucleus Ž R. and the ventral tegmental area Ž VTA.. Fig. 2. Schematic summary of the locations of the GABA injection sites in the anterior midbrain. The composite represents 52 sites from 21 rats. Filled circles indicate sites at which GABA blocked stepping elicited by hypothalamic stimulation; stippled circles represent sites at which GABA did not block stepping. A concentration of 250 mgrml was used at 10 sites, 500 mgrml at 2 sites, and 1 mgrml was used at 40 sites. Most sites were tested with a volume of 0.2 ml. Some blocks were produced by a volume of 0.1 ml. The anterior-posterior level in mm posterior to bregma is indicated by the number at the lower left. Note the proximity of many effective sites to the region containing the interstitial nucleus of the medial longitudinal fasciculus Ž IMLF, IMLFG.. Abbreviations: APT, anterior pretectal nucleus; CG, central gray; cp, cerebral peduncle; DK, Darksweizts nucleus; DM, dorsomedial nucleus; DpMe, deep mesencephalic nucleus; f, fornix; fr, fasciculus retroflexus; IF, intrafascicular nucleus; IMLFG, interstitial nucleus of the medial longitudinal fasciculus, gigantocellular; LH, lateral hypothalamus; MA3, medial accessory nucleus of the nerve 3; MCPC, magnocellular nucleus of the posterior commissure; ml, medial lemniscus; mt, mammillothalamic tract; pc, posterior commissure; Pcom, nucleus of the posterior commissure; PeF, perifornical nucleus of the hypothalamus; PR, prerubral nucleus; R, red nucleus; Rli, rostral linear nucleus; RPC, parvocellular red nucleus; SNC, substantia nigra compacta; SNR, substantia nigra reticulata; VM, ventromedial nucleus of hypothalamus; VTA, ventral tegmental area; ZI, zona incerta.

4 274 ( ) H.M. Sinnamon, M. BenaurrBrain Research The results show that GABA injections which presumably inactivate neurons in the ADT, block stepping produced by hypothalamic stimulation. Injections in the medial regions around the IMLF were particularly effective. The ADT is a complex region including several nuclear structures within the midbrain reticular tegmentum. The recording study wx 5 showed that neurons displaying activity correlated with stepping onset were distributed across nuclear boundaries within the ADT. The regions included the IMLF, the MCPC, the nucleus of the posterior commissure Ž Pcom., and the adjacent reticular formation Ž DpMe.. Within the limited localization possible with the drug injection method, there was general agreement with the recording data. Disruption of activity in the region showing single neuronal activity correlated with stepping did impair initiation. These findings do not prove that neurons of the ADT have a command function in locomotion. However, they provide support for the idea that the ADT plays a role at least related to the initiation of locomotion produced by hypothalamic stimulation. The presumed mechanism for the action of GABA injections is hyperpolarization of neurons located in the injection field, as opposed to passing axons of remotely located neurons. The effective size of the injection field is an important concern in this type of study. The distribution of effective sites shown in Fig. 2 shows that clusters of effective sites differed by approximately 0.5 mm on the anterior-posterior dimension from clusters of ineffective sites. Such limited spread may seem unreasonable considering the large concentrations of GABA used Žup to 1 mgrml.. However, the high concentrations of GABA rewx 8 probably reflect a quired to achieve functional blocks pervasive and efficient uptake mechanism for this widely distributed transmitter. However limited, this degree of spread and the 3-min typical latency for a locomotor block raise the possibility of spread of the drug to the contralateral ADT. The possibility that bilateral inactivation of the ADT is required to block stepping cannot be excluded. A few earlier reports have implicated the ADT in locomotion. Studies in this laboratory using lesions w15x and procaine injections wx 7 to block stepping showed effective sites in and near the ADT. The present findings extend these earlier studies by specifically implicating the region of the IMLF and showing that fibers of passage were not likely to be critical. In stimulation mapping studies w12,14 x, the ADT contained some positive sites, but the density was less than the ventromedial midbrain at the same anteriorposterior level. There is evidence for a locomotor role for the interstitial nucleus of Cajal Ž INC., which is nearby and often not distinguished from the IMLF. In the cat, axons from the INC project to the region of the pontine locomow16 x, and some activity in the nucleus has been tor strip reported to be phase-related to stepping wx 6. In the rat, stimulation in the region of the ICN Žat relatively high currents. elicited stepping in both intact-anesthetized and transected-unanesthetized preparations wx 2. The region projects to the medial medullary region which has been implicated in locomotion wx 3. Neurons of the ADT of the rat project to the cord, as the interstitiospinal or the reticulospinal systems wx 1. The ADT has afferents of possible locomotor significance. The medial part of the ADT receives an excitatory projection from the lateral deep nucleus of the cerebellum wx 4. The ADT is reciprocally connected to the laterodorsal tegmental nucleus w11 x; this nucleus is situated in a region that is densely positive for electrically elicited stepping w10 x. Thus the present findings can be placed in the context of other functional and anatomical evidence that is consistent with a role for the ADT in locomotor initiation. Acknowledgements Supported by NIH Grant 1R15NS We thank L. Vita, Jr. and C. Cavnor for assistance. References wx 1 F. Auclair, M.-C. Belanger, R. Marchand, Ontogenetic study of early brain stem projections to the spinal cord in the rat, Brain Res. Bull. 30 Ž wx 2 V. Berezovsky, Participation of the interstitial nucleus of Cajal in locomotion in cats and rats, Neirofiziologiia 23 Ž wx 3 V. Berezovsky, T. Kebkalo, Afferent projections of the bulbar locomotor region situated in the medial reticular formation of the brain stem, Neirofiziologiia 23 Ž wx 4 G. Bosco, A. Casabona, V. Perciavalle, Non-N-methyl-D-aspartate receptors mediate neocerebellar excitation at accessory oculomotor nuclei synapses of the rat, Arch. Ital. Biol. 132 Ž wx 5 D.R. Epuru, H.H. Liang, H.M. Sinnamon, Neural activity in the midbrain correlated with hindlimb extension initiated by locomotor stimulation of the hypothalamus of the anesthetized rat, Neuroscience 64 Ž wx 6 E. Garcia-Rill, R.D. Skinner, J.A. Fitzgerald, Mesencephalic locomotor region Ž MLR. unit activity during locomotion, Soc. Neurosci. Abstr. 8 Ž wx 7 D.I. Levy, H.M. Sinnamon, Midbrain areas required for locomotion initiated by electrical stimulation of the lateral hypothalamus in the anesthetized rat, Neuroscience 39 Ž wx 8 G.J. Mogenson, M. Wu, Disruption of food hoarding by injections of procaine into mediodorsal thalamus, GABA into subpallidal region and haloperidol into accumbens, Brain Res. Bull Ž wx 9 G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, w10x G.S. Ross, H.M. Sinnamon, Forelimb and hindlimb stepping by the anesthetized rat elicited by electrical stimulation of the pons and medulla, Physiol. Behav. 33 Ž w11x K. Satoh, H.C. Fibiger, Cholinergic neurons of the laterodorsal tegmental nucleus: efferent and afferent connections, J. Comp. Neurol. 253 Ž w12x H. Sinnamon, Forelimb and hindlimb stepping by the anesthetized rat elicited by electrical stimulation of the diencephalon and mesencephalon, Physiol. Behav. 33 Ž w13x H.M. Sinnamon, Preoptic and hypothalamic neurons and the initiation of locomotion in the anesthetized rat, Prog. Neurobiol. 41 Ž

5 ( ) H.M. Sinnamon, M. BenaurrBrain Research w14x H.M. Sinnamon, R.N. Ginzburg, G.A. Kurose, Midbrain stimulation in the anesthetized rat: direct locomotor effects and modulation of locomotion produced by hypothalamic stimulation, Neuroscience 20 Ž w15x H.M. Sinnamon, S. Lee, D.B. Adams, C.K. Stopford, Locomotor stepping elicited by electrical stimulation of the lateral hypothalamus requires an ipsilateral descending pathway, Physiol. Behav. 33 Ž w16x R.D. Skinner, E. Garcia-Rill, S. Griffin, R. Nelson, J.A. Fitzgerald, Interstitial nucleus of Cajal Ž INC. projections to the region of Probst s tract, Brain Res. Bull. 13 Ž w17x M.C. Tresch, C.L. Miller, H.M. Sinnamon, Priming of locomotor initiation by electrical stimulation in the hypothalamus and preoptic region in the anesthetized rat, Physiol. Behav. 57 Ž

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

BRAINSTEM REGIONS WITH NEURONAL ACTIVITY PATTERNS CORRELATED WITH PRIMING OF LOCOMOTOR STEPPING IN THE ANESTHETIZED RAT Pergamon www.elsevier.com/locate/neuroscience Priming of locomotion Neuroscience Vol. 99, No. 1, pp. 77±91, 2000 77 q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved