Réjean Dubuc

Spinal pattern generation

Recent research in the field of spinal pattern generation has concentrated on three main areas: the effects of various transmitters on spinal rhythmic patterns in reduced preparations (neonatal rats, chick embryos, tadpole embryos, lampreys); the changes in membrane properties of different elements of the generating circuits; and the interactions between central generating mechanisms and afferent inputs. The important message is that new properties of neural membranes, as well as new reflex responses, have been identified that could not have been predicted in the absence of such rhythmic activity.

Stimulation of the mesencephalic locomotor region elicits controlled swimming in semi‐intact lampreys

The role of the mesencephalic locomotor region (MLR) in initiating and controlling the power of swimming was studied in semi‐intact preparations of larval and adult sea lampreys. The brain and the rostral portion of the spinal cord were exposed in vitro, while the intact caudal two‐thirds of the body swam freely in the Ringers‐containing chamber. Electrical microstimulation (2–10 Hz; 0.1–5.0 μA) within a small periventricular region in the caudal mesencephalon elicited well‐coordinated and controlled swimming that began within a few seconds after the onset of stimulation and lasted throughout the stimulation period. Swimming stopped several seconds after the end of stimulation. The power of swimming, expressed by the strength of the muscle contractions and the frequency and the amplitude of the lateral displacement of the body or tail, increased as the intensity or frequency of the stimulating current were increased. Micro‐injection of AMPA, an excitatory amino acid agonist, into the MLR also elicited active swimming. Electrical stimulation of the MLR elicited large EPSPs in reticulospinal neurons (RS) of the middle rhombencephalic reticular nucleus (MRRN), which also displayed rhythmic activity during swimming. The retrograde tracer cobalt‐lysine was injected into the MRRN and neurons (dia. 10–20 μm) were labelled in the MLR, indicating that this region projects to the rhombencephalic reticular formation. Taken together, the present results indicate that, as higher vertebrates, lampreys possess a specific mesencephalic region that controls locomotion, and the effects onto the spinal cord are relayed by brainstem RS neurons.

The multifunctional mesencephalic locomotor region.

In 1966, Shik, Severin and Orlovskii discovered that electrical stimulation of a region at the junction between the midbrain and hindbrain elicited controlled walking and running in the cat. The region was named Mesencephalic Locomotor Region (MLR). Since then, this locomotor center was shown to control locomotion in various vertebrate species, including the lamprey, salamander, stingray, rat, guinea-pig, rabbit or monkey. In human subjects asked to imagine they are walking, there is an increased activity in brainstem nuclei corresponding to the MLR (i.e. pedunculopontine, cuneiform and subcuneiform nuclei). Clinicians are now stimulating (deep brain stimulation) structures considered to be part of the MLR to alleviate locomotor symptoms of patients with Parkinsons disease. However, the anatomical constituents of the MLR still remain a matter of debate, especially relative to the pedunculopontine, cuneiform and subcuneiform nuclei. Furthermore, recent studies in lampreys have revealed that the MLR is more complex than a simple relay in a serial descending pathway activating the spinal locomotor circuits. It has multiple functions. Our goal is to review the current knowledge relative to the anatomical constituents of the MLR, and its physiological role, from lamprey to man. We will discuss these results in the context of the recent clinical studies involving stimulation of the MLR in patients with Parkinsons disease.

GABA distribution in lamprey is phylogenetically conserved

The localization of γ‐aminobutyric acid (GABA) has been well described in most classes of vertebrates but not in adult lampreys. The question if the GABA distribution is similar throughout the vertebrate subphylum is therefore still to be addressed. We here investigate two lamprey species, the sea lamprey, Petromyzon marinus, and the river lamprey, Lampetra fluviatilis, and compare the GABA pattern with that of other vertebrates. The present immunohistochemical study provides an anatomical basis for the general distribution and precise localization of GABAergic neurons in the adult lamprey forebrain and brainstem. GABA‐immunoreactive cells were organized in a virtually identical manner in the two species. They were found throughout the brain, with the following regions being of particular interest: the granular cell layer of the olfactory bulb, the nucleus of the anterior commissure, the septum, the lateral and medial pallia, the striatum, the nucleus of the postoptic commissure, the thalamus, the hypothalamus, and pretectal areas, the optic tectum, the torus semicircularis, the mesencephalic tegmentum, restricted regions of the rhombencephalic tegmentum, the octavolateral area, and the dorsal column nucleus. The GABA distribution found in cyclostomes is very similar to that of other classes of vertebrates, including mammals. Since the lamprey diverged from the main vertebrate line around 450 million years ago, this implies that already at that time the basic vertebrate plan for the GABA innervation in different parts of the brain had been developed. J. Comp. Neurol. 503:47–63, 2007.

Rhythmic antidromic discharges of single primary afferents recorded in cut dorsal root filaments during locomotion in the cat

Single units recorded in the proximal stump of cut dorsal root filaments were found to antidromically discharge rhythmically during fictive locomotion in decorticate and paralyzed cats. Some units fired throughout the period of flexor or extensor nerve activity, whereas other units discharged near the transitional phases. Similar findings were made in acutely spinalized and paralyzed cats injected with L-DOPA, as well as in non-paralyzed decorticate cats walking on a treadmill. These results suggest that different types of primary afferents may be depolarized cyclically at different specific time in the step cycle by the central pattern generator for locomotion, and that this central control of the primary afferents may be involved in the modulation of the reflex transmission observed during locomotion.

Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys

In lampreys as in other vertebrates, brainstem centres play a key role in the initiation and control of locomotion. One such centre, the mesencephalic locomotor region (MLR), was identified physiologically at the mesopontine border. Descending inputs from the MLR are relayed by reticulospinal neurons in the pons and medulla, but the mechanisms by which this is carried out remain unknown. Because previous studies in higher vertebrates and lampreys described cholinergic cells within the MLR region, we investigated the putative role of cholinergic agonists in the MLR‐controlled locomotion. The local application of either acetylcholine or nicotine exerted a direct dose‐dependent excitation on reticulospinal neurons as well as induced active or fictive locomotion. It also accelerated ongoing fictive locomotion. Choline acetyltransferase‐immunoreactive cells were found in the region identified as the MLR of lampreys and nicotinic antagonists depressed, whereas physostigmine enhanced the compound EPSP evoked in reticulospinal neurons by electrical stimulation of this region. In addition, cholinergic inputs from the MLR to reticulospinal neurons were found to be monosynaptic. When the brainstem was perfused with d‐tubocurarine, the induction of swimming by MLR stimulation was depressed, but not prevented, in a semi‐intact preparation. Altogether, the results support the hypothesis that cholinergic inputs from the MLR to reticulospinal cells play a substantial role in the initiation and the control of locomotion.

Trigeminal inputs to reticulospinal neurones in lampreys are mediated by excitatory and inhibitory amino acids

Reticulospinal (RS) neurones integrate sensory inputs from several modalities to generate appropriate motor commands for maintaining body orientation and initiation of locomotion in lampreys. As in other vertebrates, trigeminal afferents convey sensory inputs from the head region. The in vitro brainstem/spinal cord preparation of the lamprey was used for characterizing trigeminal inputs to RS neurones as well as the transmitter systems involved. The trigeminal nerve on each side was electrically stimulated and synaptic responses, which consisted of mixed excitation and inhibition, were recorded intracellularly in the middle and posterior rhombencephalic reticular nuclei. The EPSPs were mediated by activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors. An increase in the late phase of the excitatory response occurred when Mg2+ ions were removed from the Ringers solution. This effect was antagonized by 2-amino-5-phosphonopentanoate (2-AP5) or reversed by restoring Mg2+ ions to the perfusate suggesting the activation of N-methyl-D-aspartate (NMDA) receptors. IPSPs were mediated by glycine. These findings are similar to those reported for other types of sensory inputs conveyed to RS neurones, where excitatory and inhibitory amino acid transmission is also involved.

An immunocytochemical and autoradiographic investigation of the serotoninergic innervation of trigeminal mesencephalic and motor nuclei in the rabbit

The results of a previous experiment suggest that the cell bodies of many jaw closing muscle spindle afferents in the trigeminal mesencephalic nucleus of the rabbit are phasically inhibited during fictive mastication. The aim of this study was to investigate one possible neurotransmitter system that could be involved in this modulation, serotonin, by use of receptor autoradiography techniques and immunofluorescence combined with retrograde labelling of masseteric spindle afferents and motoneurons. A second objective was to compare the serotonin innervation of neurons in the trigeminal mesencephalic nucleus with that of masseteric motoneurons. Serotoninergic fibres were seen surrounding labelled masseteric spindle afferents, as well as unlabelled neurons, in the trigeminal mesencephalic nucleus. These fibres were close to the cell bodies and sometimes to the axon hillocks of the neurons. Although it has been reported that many neurons of the trigeminal nucleus are multipolar in some species, none of the labelled spindle afferent in this study had more than one process. Throughout the motor trigeminal nucleus, serotonin fibres were found in close proximity with cell bodies and with the proximal portions of axons and dendrites of labelled and unlabelled motoneurons. Serotonin fibres were also seen adjacent to cell bodies and processes of efferent neurons in cell group k. Autoradiography with several tritiated ligands was used to reveal the presence of receptors for serotonin as well as its uptake sites. Only serotonin2 receptors were found to be abundant in the trigeminal mesencephalic nucleus. The motor nucleus and cell group k contained serotonin2 and serotonin3 receptors, as well as serotonin uptake sites. Serotonin1A receptors appear to be absent from both nuclei. The findings suggest that release of serotonin from fibres in close proximity to trigeminal primary afferent somata could modify the transmission of action potentials from muscle spindle receptors during mastication through an action on serotonin2 receptors. In the motor nucleus and cell group k, serotonin may alter neuronal properties through actions on at least two receptor subtypes (serotonin2 and serotonin3).

Descending GABAergic projections to the mesencephalic locomotor region in the lamprey Petromyzon marinus.

The mesencephalic locomotor region (MLR) plays a significant role in the control of locomotion in all vertebrate species investigated. Forebrain neurons are likely to modulate MLR activity, but little is known about their inputs. Descending GABAergic projections to the MLR were identified by double‐labeling neurons using Neurobiotin injected into the MLR combined with immunofluorescence against GABA. Several GABAergic projections to the MLR were identified in the telencephalon and diencephalon. The most abundant GABAergic projection to the MLR came from the caudal portion of the medial pallium, a region that may have similarities with the amygdala of higher vertebrates. A small population of GABAergic cells projecting to the MLR was found in the striatum and the ventral portion of the lateral pallium, which could respectively correspond to the input and output components of the basal ganglia thought to be involved in the selection of motor programs. Other GABAergic projections were found to come from the thalamus and the hypothalamus, which could take part in the motivational aspect of motor behavior in lampreys. Electrophysiological experiments were also carried out to examine the effects of GABA agonists and antagonists injected into the MLR in a semi‐intact lamprey preparation. The GABA agonist inhibited locomotion, whereas the GABA antagonist initiated it. These results suggest that the GABAergic projections to the MLR modulate the activity of MLR neurons, which would be inhibited by GABA at rest. J. Comp. Neurol. 501:260–273, 2007.

A Novel Neural Substrate for the Transformation of Olfactory Inputs into Motor Output

Anatomical and physiological experiments in the lamprey reveal the neural circuit involved in transforming olfactory inputs into motor outputs, which was previously unknown in a vertebrate.