Activation of Brainstem Neurons During Mesencephalic Locomotor Region-Evoked Locomotion in the Cat.

The distribution of locomotor-activated neurons in the brainstem of the cat was studied by c-Fos immunohistochemistry in combination with antibody-based cellular phenotyping following electrical stimulation of the mesencephalic locomotor region (MLR) - the anatomical constituents of which remain debated today, primarily between the cuneiform (CnF) and the pedunculopontine tegmental nuclei (PPT). Effective MLR sites were co-extensive with the CnF nucleus. Animals subject to the locomotor task showed abundant Fos labeling in the CnF, parabrachial nuclei of the subcuneiform region, periaqueductal gray, locus ceruleus (LC)/subceruleus (SubC), Kölliker-Fuse, magnocellular and lateral tegmental fields, raphe, and the parapyramidal region. Labeled neurons were more abundant on the side of stimulation. In some animals, Fos-labeled cells were also observed in the ventral tegmental area, medial and intermediate vestibular nuclei, dorsal motor nucleus of the vagus, n. tractus solitarii, and retrofacial nucleus in the ventrolateral medulla. Many neurons in the reticular formation were innervated by serotonergic fibers. Numerous locomotor-activated neurons in the parabrachial nuclei and LC/SubC/Kölliker-Fuse were noradrenergic. Few cholinergic neurons within the PPT stained for Fos. In the medulla, serotonergic neurons within the parapyramidal region and the nucleus raphe magnus were positive for Fos. Control animals, not subject to locomotion, showed few Fos-labeled neurons in these areas. The current study provides positive evidence for a role for the CnF in the initiation of locomotion while providing little evidence for the participation of the PPT. The results also show that MLR-evoked locomotion involves the parallel activation of reticular and monoaminergic neurons in the pons/medulla, and provides the anatomical and functional basis for spinal monoamine release during evoked locomotion. Lastly, the results indicate that vestibular, cardiovascular, and respiratory centers are centrally activated during MLR-evoked locomotion. Altogether, the results show a complex pattern of neuromodulatory influences of brainstem neurons by electrical activation of the MLR.

Locomotor-activated neurons of the cat. II. Noradrenergic innervation and colocalization with NEα 1a or NEα 2b receptors in the thoraco-lumbar spinal cord.

Norepinephrine (NE) is a strong modulator and/or activator of spinal locomotor networks. Thus noradrenergic fibers likely contact neurons involved in generating locomotion. The aim of the present study was to investigate the noradrenergic innervation of functionally related, locomotor-activated neurons within the thoraco-lumbar spinal cord. This was accomplished by immunohistochemical colocalization of noradrenergic fibers using dopamine-ß-hydroxylase or NEα(1A) and NEα(2B) receptors with cells expressing the c-fos gene activity-dependent marker Fos. Experiments were performed on paralyzed, precollicular-postmamillary decerebrate cats, in which locomotion was induced by electrical stimulation of the mesencephalic locomotor region. The majority of Fos labeled neurons, especially abundant in laminae VII and VIII throughout the thoraco-lumbar (T13-L7) region of locomotor animals, showed close contacts with multiple noradrenergic boutons. A small percentage (10-40%) of Fos neurons in the T7-L7 segments showed colocalization with NEα(1A) receptors. In contrast, NEα(2B) receptor immunoreactivity was observed in 70-90% of Fos cells, with no obvious rostrocaudal gradient. In comparison with results obtained from our previous study on the same animals, a significantly smaller proportion of Fos labeled neurons were innervated by noradrenergic than serotonergic fibers, with significant differences observed for laminae VII and VIII in some segments. In lamina VII of the lumbar segments, the degree of monoaminergic receptor subtype/Fos colocalization examined statistically generally fell into the following order: NEα(2B) = 5-HT(2A) ≥ 5-HT(7) = 5-HT(1A) > NEα(1A). These results suggest that noradrenergic modulation of locomotion involves NEα(1A)/NEα(2B) receptors on noradrenergic-innervated locomotor-activated neurons within laminae VII and VIII of thoraco-lumbar segments. Further study of the functional role of these receptors in locomotion is warranted.

Locomotor-activated neurons of the cat. I. Serotonergic innervation and co-localization of 5-HT7, 5-HT2A, and 5-HT1A receptors in the thoraco-lumbar spinal cord.

Monoamines are strong modulators and/or activators of spinal locomotor networks. Thus monoaminergic fibers likely contact neurons involved in generating locomotion. The aim of the present study was to investigate the serotonergic innervation of locomotor-activated neurons within the thoraco-lumbar spinal cord following induction of hindlimb locomotion. This was determined by immunohistochemical co-localization of serotonin (5-HT) fibers or 5-HT(7)/5-HT2A/5-HT1A receptors with cells expressing the activity-dependent marker c-fos. Experiments were performed on paralyzed, decerebrate cats in which locomotion was induced by electrical stimulation of the mesencephalic locomotor region. Abundant c-fos immunoreactive cells were observed in laminae VII and VIII throughout the thoraco-lumbar segments of locomotor animals. Control sections from the same segments showed significantly fewer labeled neurons, mostly within the dorsal horn. Multiple serotonergic boutons were found in close apposition to the majority (80-100%) of locomotor cells, which were most abundant in lumbar segments L3-7. 5-HT7 receptor immunoreactivity was observed on cells across the thoraco-lumbar segments (T7-L7), in a dorsoventral gradient. Most locomotor-activated cells co-localized with 5-HT7, 5-HT2A, and 5-HT1A receptors, with largest numbers in laminae VII and VIII. Co-localization of c-fos and 5-HT7 receptor was highest in the L5-L7 segments (>90%) and decreased rostrally (to approximately 50%) due to the absence of receptors on cells within the intermediolateral nucleus. In contrast, 60-80 and 35-80% of c-fos immunoreactive cells stained positive for 5-HT2A and 5-HT1A receptors, respectively, with no rostrocaudal gradient. These results indicate that serotonergic modulation of locomotion likely involves 5-HT(7)/5-HT2A/5-HT1A receptors located on the soma and proximal dendrites of serotonergic-innervated locomotor-activated neurons within laminae VII and VIII of thoraco-lumbar segments.