Journal Club: Fast Silencing Reveals a Lost Role for Reciprocal Inhibition in Locomotion

By Peter R. Moult, Glen A. Cottrell, and Wen-Chang Li
Neuron Volume 77, Issue 1, 9 January 2013, Pages 129-140

Reciprocal inhibition is considered as a fundamental building block in neural circuits for rhythmic motor pattern generation. Despite commonality in many rhythmic systems, however, the reciprocal inhibition is often found to be not a necessary component for rhythmogenesis. This is because a network is often capable of exhibiting rhythmic activity even when the reciprocal inhibition was removed mechanically or pharmacologically. The excitatory synaptic components within each half of the network are more crucial.  In this study, Wen-Chang Li's group challenged this idea by using optogenetics to hyperpolarize the entire population of neurons in one side of the spinal cord in a tadpole.

When the entire half of the spinal cord was suppressed by flashing yellow light, the other half also stopped bursting. This indicates that, in this reciprocally inhibitory network, the excitation in one side is necessary for the excitation of the other. Firing in one side would cause a bombardment of IPSPs, which induce post-inhibitory rebound. The authors also get a similar result by giving a strong hyperpolarizing current into one of the reciprocally inhibitory neurons. They concluded that the reciprocal inhibition is necessary in the tadpole swim circuit; it induces rebound excitation in the contralateral neurons.

In this paper, it is noteworthy that the author also mentioned about the "functional homeostasis" of the neural circuit. They said the rhythmic activity recovers after half an hour by itself without the reciprocal inhibition. The mechanism of the recovery still unknown.

Organization of spinal circuitry for rodent locomotion

Neuronal activity in the isolated mouse spinal cord during spontaneous deletions in fictive locomotion: Insights into locomotor CPG organization

by Guisheng Zhong, Natalia A Shevtsova, Ilya A Rybak, Ronald M Harris-Warrick

Journal of Physiology (2012)

Deletions are spontaneous errors in the rhythmic locomotor pattern when a set of synergist motoneurons (for example, flexor motoneurons on one side) loses rhythmic firing or falls silent during a time period when they are normally active. 

In the non-resetting deletions, the phase of the rhythm after the deletion did not change. The resetting deletions show rhythm resetting which was recognized by a shift in the phase of the motor bursts after the deletion. This study follows Rybak-McCrea model of the locomotor CPG, which has two functional levels: a half-center rhythm-generator and pattern formation networks. 

Deletions occur simultaneously in motor activity across more than one spinal segment. Spontaneous non-resetting deletions on one side are independent of the other side. The CPG can be functional within an isolated hemisegment. The reduction in locomotor frequency after simulated hemisection results mainly from the elimination of excitatory input to the rhythm-generating ipsilateral RG-F population from the contralateral RG-E population. The resumption of activity an integer number of cyces later does not require input from other parts of the spinal cord. Each hemicord contains an independent rhythmogenic network that can function in the absence of the other hemicord, although the left and right networks are normally coupled via commissural interneurons.

Among V2a interneurons, there are deletion-sensitive types and insensitive type (type I and II V2a interneurons). Type I V2a does not respond to non-locomotory firing in iL2, whereas type II V2a does. Both neurons were depolarized by synaptic drive. The type I V2a interneurons are involved in rhythm generation and/or coordination between left and right networks via the CINs. In contrast, the type II V2a interneurons do not belong to rhythm generator networks, but can be components of the pattern gormation network and/or last-order interneurons that directly project to motoneurons. Commissural interneurons (CINs) send their axons to the opposite side of the cord and coordinate left-right alternation. CINs were not affected by motoneuronal deletion. There was asymmetry in deletion: During all of the flexor deletions, the ipsilateral extensor root showed sustained activity with no interruptions at the times of the missing flexor bursts. In contrast, during all extensor deletions, the ipsilateral flexor root continued unperturbed bursting.

Their computational model combines the Rybak-McCrea concept of the two-level locomotor CPG (Rybak et al., 2006a,b; McCrea and Rybak, 2007,2008) with the Duysens-Pearson concept of an asymmetric rhythm generator with a dominant flexor half-centre (Pearson and Duysens, 1976).

Re-expression of Locomotor Function After Partial Spinal Cord Injury

S. Rossignol, G. Barrière, O. Alluin and A. Frigon

The CPG is defined as a spinal network of neurons capable of generating a rhythmic pattern consisting of alternating activity between flexor and extensor motoneurons on the same side with reciprocal activation of homologous motoneurons in the other limb of
the same girdle. In general, during walking or trotting, this network ensures that flexor motoneurons on one side are active with contralateral extensors and vice versa for extensor motoneurons.

...In such a preparation, rhythmic activity, evoked by injecting the noradrenaline precursor l-dihydroxyphenylalanine (L-DOPA), is recorded from peripheral muscle nerves and is termed “fictive locomotion.”

...Indeed, electrical stimulation of a circumscribed brain stem region called the mesencephalic locomotor region (MLR), ... Various gait patterns, such as walk, trot, and gallop, can be
evoked with increasing stimulation intensity.

...Other descending pathways release specific neurotransmitters, which are synthesized by cells in well defined brain stem nuclei (e.g., noradrenaline in the locus coeruleus and serotonin in the raphe and parapyramidal nuclei). These neurotransmitters exert powerful effects on the spinal circuitry and can change characteristics of the locomotor pattern.

...it is important to know that, after a complete spinal transection, most quadruped mammals will
recover some degree of locomotor function in the limbs below the lesion. Cats, rats, and mice can re-express hindlimb locomotion provided the spinal cord below the complete lesion is properly
stimulated, either pharmacologically or through locomotor training.

...locomotion is controlled at multiple levels of the central nervous system, and a subtle and intricate balance is established between these levels of control. This then leads to the question of how an optimal equilibrium is re-established when this exquisite balance is perturbed following lesions of the spinal cord.

...Instead of taking over lost spinal functions, remnant descending pathways or regenerating pathways could direct the reorganization of the spinal circuitry so that it can function optimally and with a greater level of independence so that, after the complete section, the full pattern of hindlimb locomotion can be expressed by an already autonomous CPG.

The main conclusion of this brief review of multiple types of lesions is that there are several ways through which the CNS and peripheral afferent inputs can access the spinal locomotor circuitry. This apparent redundancy points to the fact that the rhythm is generated at the spinal level and that various degrees of control levels can modulate this spinal circuitry through multiple pathways. The removal of certain pathways produces specific locomotor deficits, but the
spinal circuitry and other intact pathways are still able to optimize remnant locomotor functions.

Prominent Role of the Spinal Central Pattern Generator in the Recovery of Locomotion after Partial Spinal Cord Injuries

Grégory Barrie`re, Hugues Leblond, Janyne Provencher, and Serge Rossignol

The general accepted model of locomotor control is tripartite.
1) CPG
2) sensory feedback
3) descending pathways

After partial spinal cord injury (SCI), this optimal balance is perturbed because communication between the brain and the spinal CPG is altered (Barbeau and Rossignol, 1994).

Q: Are there lastic changes within descending pathways?
Or, the spinal CPG retains its function and that changes in descending commands aim at maintaining an optimal control of the spinal locomotor network?

The role of the CPG in the recovery of locomotion after incomplete spinal cord lesion is mostly unknown.

RESULT
The first step consisted of an incomplete section of the spinal cord at the thoracic level T10 or T11.
The second step was a complete transection of the spinal cord at T13 or L1.

1) The recovery of quadrupedal locomotion after partial lesions is mostly the result of an intrinsic reorganization of the spinal locomotor network below the lesion.

2) Locomotor training is a major factor in facilitating the recovery process because cats intensively trained after the partial lesion expressed a very high locomotor
performance bilaterally within hours of the complete spinalization, whereas in untrained animals only a unilateral locomotion was observed in the limb ipsilateral to the partial lesion.

3) Plastic reorganization of the spinal CPG may still occur after the complete spinal section because bilateral locomotor performance improved with training over time in all cats after complete spinalization.

Altogether, this work highlights the importance of promoting spinal neuroplasticity in rehabilitation strategies in SCI patients, especially to maintain the spinal circuitry in an optimal condition to generate locomotion.

Jing's CC9&10 paper (2008)

They generated rat antibody for 5-HT.
Briefly, the antigen was prepared by coupling 2 mg serotonin oxalate to 10 mg BSA (Sigma-Aldrich) in 1 ml of 50 mM NaH2PO4, pH 7.2, using 100 microL 16% paraformaldehyde (EMS). Followingovernight incubation at 4°C, coupled antigen was purified from the reaction using a Microcon-30 spinning at 13,800 x g for 30 min at 4°C. After washing the retentate four times with 0.4 ml of 50 mM NaH2PO4, it was resuspended in 0.5 ml of the same buffer and transferred to a new tube...
Tissues were fixed in 4% paraformaldehyde, 0.2% picric acid, 25% sucrose, and 0.1MNaH2PO4, pH 7.6, for either 3 h at room temperature or overnight at 4°C.
Washing buffer (WB; 2% Triton X-100, 1% BSA, 154 mM NaCl, 50 mM EDTA, 0.01% thimerosal, and 10mM Na2HPO4, pH 7.4).

Both the dorsal and the ventral surfaces of the cerebral ganglion were desheathed. The cerebral ganglion was then twisted at the commissure to make it possible to access CC9/10 on the dorsal surface and contralateral CBIs on the ventral surface.

Frequent IPSPs are observed simultaneously in both CC9/10 cells (Fig. 2B). Activation of one CC9/10 can induce polysynaptic inhibition in the other CC9/10 (Fig. 2C1).

CC9/10 responds to noxious stimulus by tonic firing.
CC9/10 firing evokes locomotion.
The locomotion can be seen very well from PPCN (para-pedal commissural nerve).
The frequency of the locomotory rhythm is different depending on whether you stimulated P9 or AT4 nerve.

The cycle frequency of locomotor activity reflects the firing frequency of CC9/10 activity.
CC9 and CC10 may act as locomotion initiators.

One function of CC9/10 is to provide excitation to serotonergic modulatory neurons of the pedal ganglion.
It enhances the induction of the locomotory response.

CC9/10 weakly excites contralateral MCC and CBI-2. MCC>CBI-2

CC9/10 increase excitability (?) of MCC, but this could be just because of depolarization.

CC9–10 are broadly activated and their responses are not site-specific.

Aplysia locomotes, and then eats.


The Journal of Neuroscience, November 19, 2008 • 28(47):12349 –12361
Neural Analog of Arousal: Persistent Conditional Activation of a Feeding Modulator by Serotonergic Initiators of Locomotion
Jian Jing, Ferdinand S. Vilim, Elizabeth C. Cropper, and Klaudiusz R. Weiss