Reticulospinal neurons of the lamprey brain stem receive rhythmic input from the spinal cord during locomotor activity. The goal of the present study was to determine whether such spinal input has a direct component to reticulospinal neurons or depends on brain stem interneurons. To answer this question, an in vitro lamprey brain stem-spinal cord preparation was used with a diffusion barrier placed caudal to the obex, separating the experimental chamber into two baths. Locomotor activity was induced in the spinal cord by perfusion of d-glutamate or N-methyl-dl-aspartate into the spinal cord bath. The brain stem bath was first perfused with normal Ringer solution followed by a high-Ca(2+), -Mg(2+) solution, which reduced polysynaptic transmission. The amplitudes of membrane potential oscillations of reticulospinal neurons in the posterior and middle rhombencephalic reticular nuclei (PRRN and MRRN, respectively) recorded with sharp intracellular microelectrodes did not significantly change from normal to high-divalent solution. This finding suggests a large part of the spinal input creating the oscillations is direct to the reticulospinal neurons. Application of strychnine to the high-Ca(2+), -Mg(2+) solution decreased membrane potential oscillation amplitude, and injection of Cl(-) reversed presumed inhibitory postsynaptic potentials, indicating a role for direct spinal inhibitory inputs. Although reduced, the persistence of oscillations in strychnine suggests that spinal excitatory inputs also contribute to the oscillations. Thus it was concluded that both excitatory and inhibitory spinal neurons provide direct rhythmic inputs to reticulospinal cells of the PRRN and MRRN during locomotor activity. These inputs provide reticulospinal cells with information regarding the activity of the spinal locomotor networks.

Einum, James F. and James T. Buchanan. Membrane potential oscillations in reticulospinal and spinobulbar neurons during locomotor activity. J Neurophysiol 94: 273-281, 2005. First published March 2, 2005 doi:10.1152/jn.00695.2004. Feedback from the spinal locomotor networks provides rhythmic modulation of the membrane potential of reticulospinal (RS) neurons during locomotor activity. To further understand the origins of this rhythmic activity, the timings of the oscillations in spinobulbar (SB) neurons of the spinal cord and in RS neurons of the posterior and middle rhombencephalic reticular nuclei were measured using intracellular microelectrode recordings in the isolated brain stem-spinal cord preparation of the lamprey. A diffusion barrier constructed just caudal to the obex allowed induction of locomotor activity in the spinal cord by bath application of an excitatory amino acid to the spinal bath. All of the ipsilaterally projecting SB neurons recorded had oscillatory membrane potentials with peak depolarizations in phase with the ipsilateral ventral root bursts, whereas the contralaterally projecting SB neurons were about evenly divided between those in phase with the ipsilateral ventral root bursts and those in phase with the contralateral bursts. In the brain stem under these conditions, 75% of RS neurons had peak depolarizations in phase with the ipsilateral ventral root bursts while the remainder had peak depolarizations during the contralateral bursts. Addition of a high-Ca 2ϩ , Mg 2ϩ solution to the brain stem bath to reduce polysynaptic activity had little or no effect on oscillation timing in RS neurons, suggesting that direct inputs from SB neurons make a major contribution to RS neuron oscillations under these conditions. Under normal conditions when the brain is participating in the generation of locomotor activity, these spinal inputs will be integrated with other inputs to RS neurons.

Locomotor networks in the spinal cord are controlled by descending systems which in turn receive feedback signals from ascending systems about the state of the locomotor networks. In lamprey, the ascending system consists of spinobulbar neurons which convey spinal network signals to the two descending systems, the reticulospinal and vestibulospinal neurons. Previous studies showed that spinobulbar neurons consist of both ipsilaterally and contralaterally projecting cells distributed at all rostrocaudal levels of the spinal cord, though most numerous near the obex. The axons of spinobulbar neurons ascend in the ventrolateral spinal cord and brainstem to the caudal mesencephalon and within the dendritic arbors of reticulospinal and vestibulospinal neurons. Compared to mammals, the ascending system in lampreys is more direct, consisting of excitatory and inhibitory monosynaptic inputs from spinobulbar neurons to reticulospinal neurons. The spinobulbar neurons are rhythmically active during fictive locomotion, representing a wide range of timing relationships with nearby ventral root bursts including those in phase, out of phase, and active during burst transitions between opposite ventral roots. The spinobulbar neurons are not simply relay cells because they can have mutual synaptic interactions with their reticulospinal neuron targets and they can have synaptic outputs to other spinal neurons. Spinobulbar neurons not only receive locomotor inputs but also receive direct inputs from primary mechanosensory neurons. Due to the relative simplicity of the lamprey nervous system and motor control system, the spinobulbar neurons and their interactions with reticulospinal neurons may be advantageous for investigating the general organization of ascending systems in the vertebrate.

An in vitro preparation of the nervous system of the lamprey, a lower vertebrate, was used to characterize the properties of spinal neurons with axons projecting to the brain stem [i.e., spinobulbar (SB) neurons)]. To identify SB neurons, extracellular electrodes on each side of the spinal cord near the obex recorded the axonal spikes of neurons impaled with sharp intracellular microelectrodes in the rostral spinal cord. The ascending spinal neurons (n ϭ 144) included those with ipsilateral (iSB) (63/144), contralateral (cSB) (77/144), or bilateral (bSB) (4/144) axonal projections to the brain stem. Intracellular injection of biocytin revealed that the SB neurons had small-to medium-size somata and most had dendrites confined to the ipsilateral side of the cord, although about half of the cSB neurons also had contralateral dendrites. Most SB neurons had multiple axonal branches including descending axons. Electrophysiologically, the SB neurons were similar to other lamprey spinal neurons, firing spikes throughout long depolarizing pulses with some spike-frequency adaptation. Paired intracellular recordings between SB and reticulospinal (RS) neurons revealed that SB neurons made either excitatory or inhibitory synapses on RS neurons and the SB neurons received excitatory input from RS neurons. Mutual excitation and feedback inhibition between pairs of RS and SB neurons were observed. The SB neurons also received excitatory inputs from primary mechanosensory neurons (dorsal cells), and these same SB neurons were rhythmically active during fictive swimming, indicating that SB neurons convey both sensory and locomotor network information to the brain stem.

The biogenic amines serotonin (5-HT) and noradrenaline (NA) are well known modulators of central pattern-generating networks responsible for vertebrate locomotion. Here we have explored monoaminergic modulation of the spinal circuits that generate two distinct modes of locomotion in the metamorphosing frog Xenopus laevis. At metamorphic climax when propulsion is achieved by undulatory larval tail movements and/or by kicking of the newly developed adult hindlimbs, the underlying motor networks remain spontaneously active in vitro, producing either separate fast axial and slow appendicular rhythms or a single combined rhythm that drives coordinated tail-based and limb-based swimming in vivo. In isolated spinal cords already expressing distinct axial and limb rhythms, bath-applied 5-HT induced coupled network activity through an opposite slowing of axial rhythmicity (by increasing motoneuron burst and cycle durations) and an acceleration of limb rhythmicity (by decreasing burst and cycle durations). In contrast, in preparations spontaneously expressing coordinated fictive locomotion, exogenous NA caused a dissociation of spinal activity into separate faster axial and slower appendicular rhythms by decreasing and increasing burst and cycle durations, respectively. Moreover, in preparations from premetamorphic and postmetamorphic animals that express exclusively axial-based or limb-based locomotion, 5-HT and NA modified the developmentally independent rhythms in a similar manner to the amines' opposing effects on the coexisting circuits at metamorphic climax. Thus, by exerting differential modulatory actions on one network that are opposite to their influences on a second adjacent circuit, these two amines are able to precisely regulate the functional relationship between different rhythmogenic networks in a developing vertebrate's spinal cord.