Search

These studies were designed to investigate projections of the interstitial nucleus of Cajal (INC) to the region of the contralateral Probst's tract (PrTr). In electrophysiological experiments, INC neurons were antidromically activated from the contralateral PrTr, the medial longitudinal fasciculus (MLF) at medullary levels and the MLF at spinal cord levels. Some INC cells could be antidromically activated only from PrTr and others from both PrTr and the MLF. Anatomical experiments confirmed the existence of an INC projection into the region of the contralateral PrTr. Following injections of fluorescent dyes into the PrTr area, retrogradely labeled neurons were observed in the contralateral INC, with only occasional labeling ipsilaterally. Injections which included the medial vestibular nucleus labeled a greater number of INC cells ipsilaterally. After injections of dyes into the medullary MLF, retrogradely labeled cells were observed bilaterally in INC, although in greater numbers ipsilaterally. In experiments in which different dyes were injected into PrTr and the MLF, double labeled cells were found in the contralateral INC.

Long descending propriospinal (LDP) neurons (antidromically identified) having cell bodies of origin in the cervical enlargement and projecting axons at least as far as the L2 segment were studied. Extracellular recording of responses to natural and electrical stimuli was done in high-spinal cats. (1) A receptive field for natural stimuli was found for 123 LDP neurons. An additional 108 LDP cells were not activated by the natural stimuli used, but some of these fired spike potentials in response to electrical stimulation of peripheral nerves of the forelimb. There was no distinction between neurons activated and those not activated by natural stimuli on the basis of location or conduction velocity. (2) The most effective natural stimuli were mechanical manipulation of the skin (both low and high threshold), movement of joints of the paw, and pressure to the deep tissues, especially to the extensor side of the arm. These modalities of stimuli were most often excitatory, but could be inhibitory as well. (3) On the basis of modality, 4 subgroups of LDP cells were identified: those which were responsive only to mechanical-cutaneous, joint-movement, or deep-pressure stimuli, and those which responded to several of these modalities of stimuli, the multimodal group. These subgroups could not be distinguished on the basis of conduction velocity. (4) The receptive fields varied in size from small (one digit) to large (all of a forelimb). For single LDP cells they included ones with single and/or multimodal input from one or both forelimbs and various combinations of excitation and/or inhibition. However, those in the dorsal horn had only ipsilateral receptive fields, mainly of the mechanical-cutaneous type. Cells with bilateral receptive fields were mainly located medially in the ventral gray in laminae VII and VIII. (5) A comparison of the location of the subtypes of LDP cells revealed that neurons activated by mechanical-cutaneous stimuli were in laminae I and IV-VIII; whereas deep-pressure and multimodal activated neurons were almost exclusively in laminae VII and VIII. (6) LDP cells receiving input from deep-pressure receptors of the paw probably relay position or weight-bearing information about the forelimbs to the lumbosacral spinal cord. This arrangement suggests that LDP neruons function in interlimb coordination and would be active during locomotion. They probably participate also in other reflexes elicited by cutaneous and deep stimuli.

Ascending projections of long descending propriospinal tract (LDPT) cells were investigated using the technique of double retrograde labeling. In rat, injection of one fluorescent dye was made into either the reticular formation or the cerebellum, and a second dye was injected into the lumbosacral enlargement (LSE). In cat, injections were made into the reticular formation and into the lumbosacral enlargement. Using fluorescence microscopy, observation of neurons in the cervical enlargement (CE) revealed single- and double-labeled cells which were either spinoreticular or spinocerebellar tract cells and/or LDPT cells. In both cat and rat, the location of double-labeled LDPT-spinoreticular cells were in the ventromedial spinal gray matter of the CE and were coextensive with single-labeled LDPT and spinoreticular cells. The locations of double-labeled LDPT-spinocerebellar cells in rat were in the ventromedial gray and were coextensive with single-labeled LDPT cells, but not with single-labeled spinocerebellar cells. The latter group was located in central lamina VII and medial laminae V and VI. Overall, the mean number of double-labeled cells was 40% of rat and 7% of cat LDPT cells, indicating projections to either the brainstem reticular formation or cerebellum as well as to the lumbosacral enlargement. Thus, a subpopulation of LDPT cells apparently also serves as a spinoreticular (SR) and spinocerebellar (SC) projection system.

These studies demonstrate the presence of the MLR in the rat brain. Controlled locomotion on a treadmill could be induced by low level stimulation (less than 50 microA) of an area in the posterior midbrain following a precollicular-prenigral brainstem transection. This area included the lateral part of the cuneiform nucleus and anterior as well as posterior portions of the pedunculopontine nucleus. In addition, the presence of a subthalamic locomotor region in the fields of Forel was determined in rats after prethalamic transections.

Electrical stimulation of the brainstem was used to induce locomotion in the in vitro brainstem-spinal cord preparation. Recordings of electromyograms in 'limb-attached' preparations revealed an apparently adult-like step cycle. The lowest threshold sites were localized in the posterior midbrain (reticular formation in the peribrachial area and in the ventral reticular formation) and in the medioventral medulla (dorsolateral to the pyramids).

Retrograde labeling and electrophysiological techniques were used to determine the organization of efferent projections of the mesencephalic locomotor region (MLR) in the rat. Injections of horseradish peroxidase (HRP) or fluorescent dyes were made into suspected terminal zones of the MLR. After allowing for retrograde transport, the same animals underwent precollicular transection under anesthesia. The MLR was identified physiologically by the induction of controlled locomotion on a treadmill at low current (less than 50 microA) levels. Stimulation sites were marked and the brain processed accordingly. Retrogradely labeled cells in proximity (less than 0.5 mm) to locomotion-inducing stimulation sites were considered to be putative MLR neurons and were plotted according to stereotactic coordinates. These studies revealed the intrinsic organization of MLR neurons projecting to various brainstem sites. This organization may be related to the different types of transmitter systems involved in mediating MLR function.

These studies were designed to determine whether or not substantia nigra (SN) neurons project to the mesencephalic locomotor region (MLR). An attempt was made to activate SN neurons antidromically from the same site which induced locomotion on a treadmill following a precollicular-postmammillary transection in the same animal. Less than 10% of posterior SN neurons were activated antidromically from the physiologically-identified MLR. These results support previous anatomical findings describing a sparse projection from the SN to the MLR [5]. Locomotion on a treadmill was elicited at low current strengths (20-50 muA) from an area around the cuneiform nucleus in the posterior mesencephalon. This area included the lateral central gray, mesencephalic trigeminal root, dorsal brachium conjunctivum and nucleus tegmenti pedunculopontinus (NTPP) and perhaps anterodorsal locus coeruleus. Stimulation of the area just described induced a complete stepping cycle with a flexion phase and a three-part extension phase. Stimulation of the posterior SN produced spastic locomotion on a treadmill at higher current strengths (70 muA) in cats with a precollicular-postmammillary transection.

Electrical stimulation of the mesencephalic locomotor region (MLR) in the precollicular-postmammillary transected cat is known to induce controlled locomotion on a treadmill. We have been able to induce and block locomotion in this preparation by using localized infusions of transmitters and their agonists and antagonists. Infusions of the GABA antagonists bicuculline and picrotoxin into the MLR elicit locomotion at low concentration (5 mM). Applications of muscimol (5 mM) or GABA (0.5 M) were found to block chemically-induced locomotion, as well as electrically-elicited and spontaneous walking. Priming infusions of Diazepam amplified the blockage of locomotion by GABA. On the other hand, applications of strychnine (10 mM) were ineffective in inducing stepping, as were infusions of the excitatory agents glutamic acid, acetylcholine and norepinephrine. These findings suggest that the MLR is under inhibitory GABAergic input. The substantia nigra is the only known afferent to the MLR located posterior to the brainstem transection, and is a likely source for this input. A model is proposed to account for our results, as well as those of others, and it provides a working hypothesis for the neurochemical events occurring in brainstem centers which modulate locomotor events.

Single neurons were recorded extracellularly in an area of the medioventral medulla known to receive descending mesencephalic locomotor region (MLR) input. A large number (47%) of these cells were found to receive short-latency orthodromic input following stimulation of the physiologically identified MLR. Of the medioventral medulla neurons studied, 34% were found to project to the spinal cord (determined by antidromic activation following stimulation of the ventrolateral funiculus). Approximately one-half (17% of the total population) of these reticulospinal cells were found to receive short-latency orthodromic input from the MLR. The regional distribution of this group of reticulospinal cells corresponded with the area found to receive descending projections from the MLR in previous anatomical studies. In addition, electrical and chemical activation of the same region was found to elicit locomotion on a treadmill in a companion study. The present findings demonstrate that descending MLR projections influence a large number of medioventral medulla cells, some of which have direct spinal projections, and suggest that this area is a primary relay in the manifestation of MLR function.

The activity of single neurons in the mesencephalic locomotor region (MLR) was recorded extracellularly in cats during spontaneous locomotion on a treadmill. Although stimulation of the MLR is required to induce locomotion on a treadmill after a precollicular-postmamillary brain stem transection in the cat, spontaneous locomotion may occur after a precollicular-premamillary transection. The activity of flexor and extensor muscles of each limb also was recorded by EMG. Nearly 50% of the MLR neurons exhibited rhythmic firing patterns during locomotion. In about one-half of those cells, unit firing patterns could be correlated with the EMG activity in one or more muscles by using spike-triggered averaging. Single MLR neurons were found to be correlated to EMG activity in a single limb, and others were related to the EMG from muscles in two limbs or in all four limbs. Passive movement or stoppage of the limb(s) did not abolish rhythmicity in these neurons. In addition, somatosensory stimulation did not appear to affect the firing patterns of MLR neurons. Averaged EMGs of correlated forelimb muscles revealed a postspike mean latency of 7.1 ms. These measurements agreed well with reports of a 1- to 1.5-ms delay in MLR projections to reticulospinal neurons and a 5- to 6-ms delay (postspike) in reticulospinal activity correlated to EMGs during locomotion. These findings suggest that (a) MLR neurons are rhythmically active during locomotion, (b) the activity of MLR neurons can be correlated with that of EMGs in one or more limbs, (c) rhythmicity in MLR neurons may be independent of phasic sensory input, and (d) the downstream influence of the MLR may be relayed, at least in part, via reticulospinal neurons.

Recordings of single unit activity in the posterior midbrain of the cat were carried out in the "fictive spontaneous locomotion" preparation. Neuronal activity was studied in relation to the onset, alternation and termination of cyclic hindlimb neurographic activity in the precollicular-postmammillary transected animal. Histochemical identification of pedunculopontine (nicotinamide adenine dinuceotide phosphate-diaphorase positive) neurons allowed the localization of recording sites in relation to this nucleus. Neurons located in the area of the cuneiform nucleus dorsal to the pedunculopontine nucleus were found to be related preferentially to cyclic (bursting) neurographic activity, while neurons in the area of the pedunculopontine were found to be related preferentially to the onset ("on") or termination ("off") of cycling episodes. Different populations of cells in the area appeared to be related to the frequency of alternation (bursting) compared with the duration of the cyclic episodes (on/off). While the area of the cuneiform-pedunculopontine nucleus has been found to be equivalent to the mesencephalic locomotor region, the same area has been found to be related to other rhythmic activities (e.g. respiratory, masticatory, sleep cycle, pressor, vesico-motor, etc.). A hypothesis is proposed to account for the weight of evidence implicating the same region in a host of distinct rhythmic activities. This hypothesis suggest that an oscillatory reverberation between cholinergic (pedunculopontine, laterodorsal tegmental nuclei) and aminergic (locus coeruleus, substantia nigra) centers is responsible for generating the various function-related "frequencies" (bursting) or "states" (on/off) of activity.

The entopeduncular nucleus (EN) in the cat, homologue of the primate internal globus pallidus and main output of the basal ganglia, is known to project to the mesencephalic tegmentum. We have been able to elicit antidromic responses in single EN neurons from a site in the posterior mesencephalon, then transect the brainstem (precollicular-postmamillary) and elicit locomotion and rhythmic movements of the limbs by stimulation of the same site in the same animal. These studies demonstrate the existence of a direct projection from the EN to the mesencephalic locomotor region (MLR). However, this is not a particularly large pathway since fewer than 5% of the EN cells appear to project to the MLR. In a parallel series of anatomical experiments, injections of fluorescent dyes into the area of the MLR induced retrograde labeling of cell bodies in the EN and motor cortex. Injections of tritiated amino acids into the motor cortex resulted in labeling in the area anterior to the MLR. We assume that these connections may be involved, in part, in the sequencing and ordering of series of voluntary movements in which locomotion is involved.

Nicotinamide adenine dinucleotide phosphate (NAD-PH) diaphorase histochemistry was used to localize cholinergic neurons in the pedunculopontine nucleus of neonatal and adult rats. Measurements of cell body areas revealed an average area around 200 microns2 at birth, followed by a significant increase to approximately 500 microns2 by 2 weeks of age. Thereafter, there was a decrease in cell area such that by 5 weeks of age the neurons had attained their adult size of around 300 microns2. The marked increase in cell size at the end of 2 weeks of age is discussed in relation to significant events in the development of locomotor and other rhythmic function control systems.

The medial longitudinal fasciculus (MLF) and immediately adjacent reticular formation were stimulated electrically just caudal to the trochlear nucleus in the cat. In the pontomedullary reticular formation within 1.5 mm of the midline, antidromically excited neurons were detected in (1) the contralateral abducens nucleus, (2) the contralateral reticular formation just caudal and caudoventral to the abducens nucleus, and (3) the ipsilateral nucleus reticularis pontis caudalis beneath and rostral to the abducens nucleus. The possible involvement of these neurons in eye or body movements is discussed.

Injections of a tritiated amino acid-fluorescent dye mixture were made unilaterally into the area of the mesencephalic locomotor region (MLR). After allowing for retrograde and anterograde transport, the same site was electrically stimulated to induce locomotion on a treadmill following a precollicular-postmamillary transection. The tritiated amino acid transported anterogradely primarily was found autoradiographically to descend in the area of Probst's tract and to ascend to the centremedian nucleus (CM) of the thalamus. Neurons labeled retrogradely by the fluorescent dye in the same injection-stimulation site were observed in the substantia nigra, entopeduncular nucleus, sub- and hypothalamus and amygdala. In subsequent experiments, injections of fluorescent tracers were made into the area of Probst's tract and CM. Neurons in the mesencephalic trigeminal root, cuneiform nucleus, nucleus tegmenti pedunculopontinus (NTPP), dorsal locus coeruleus and lateral central gray were labeled from Probst's tract injections. Neurons in medial and lateral central gray, as well as NTPP, were labeled from CM injections.

Previous anatomical studies from our laboratories have demonstrated descending projections of the physiologically identified mesencephalic locomotor region (MLR) to the medioventral medulla. In the present study, this area was activated electrically and chemically to determine the nature of locomotor events elicited in the precollicular-postmammillary transected cat. Low-amplitude (less than 70 microA), high-frequency (5-60 Hz) stimulation of a specific region in the medioventral medulla elicited controlled episodes of locomotion on a treadmill. Injections of cholinergic agonists into this area also were found to elicit short episodes of locomotion. Injections of cholinergic antagonists were found to block locomotion elicited by: electrical stimulation of the same area; injections of cholinergic agonists; or electrical stimulation of the MLR. Injections of substance P into the medioventral medulla were found to induce locomotion for longer periods of time than injections of cholinergic agonists. These findings demonstrate physiologically that a primary caudal termination site of the MLR is located in the medioventral medulla, and suggest that at least a portion of descending MLR projections employ acetylcholine and substance P as neurotransmitters.

Retrograde transport of fluorescent tracers and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemical techniques were combined in a study of septohippocampal projections in the rat. The dorsal (DH) and ventral (VH) hippocampus were simultaneously injected with different tracers (Fast Blue or Fluoro-Gold). Histochemical procedures revealed many NADPH-d positive cells located in the medial septum and the horizontal limb of the diagonal band. In the medial septum, NADPH-d positive neurons were mostly located lateral to the midline region and some of these were double-labeled by the tracer injected into the VH. Also, NADPH-d positive cells were found in the horizontal diagonal band and some of these were double-labeled following injections into the DH. No fluorescence/NADPH-d double-labeled neurons were observed in other structures known to project to the hippocampus.

In decerebrate cats long descending propriospinal (LDP) neurons were recorded extracellularly in the cervical enlargement. They were identified antidromically by spinal cord stimulation at the L1-L2 level. Inputs to these cells were tested by stimulating the medial longitudinal fasciculus (MLF) 5 to 6 mm rostral to the obex, the lateral vestibular nucleus (LVN), the upper MLF 1 mm caudal to the trochlear nucleus, and the medial vestibular nucleus (MVN), all on the ipsilateral side. Action potentials were elicited in 44% (64/144) of LDP neurons in the ventral horn (laminae VII, VIII) at a segmental latency of 1 ms or less following brain stem stimulation. This was considered to be a monosynaptic latency. The most effective stimulation sites were the MLF and the LVN. MLF stimulation accounted for about two-thirds of the monosynaptically elicited action potentials and LVN for about one-third. Another 22% of LDP neurons responded at longer latencies, but some of those responses may also have been monosynaptic. Stimulation of the upper MLF and the MVN were much less effective, indicating that the MLF input was predominantly from fibers originating in the medullary and/or pontine reticular formation.