Phase-specific premotor inhibition modulates leech rhythmic motor output

Reviewer #1 (Public review):

Summary:

The Szczupak lab published a very interesting paper in 2012 (Rodriquez et al. J Neurophysiol 107:1917-1924) on the effects of the segmentally-distributed non-spiking (NS) cell on crawl-related motoneurons. As far as I can tell, the working model presented in 2012, for how the non-spiking (NS) cell impacts the crawling motor pattern, is the same functional model presented in this new paper. Unfortunately, the Discussion does not address any of the findings in the previous paper or cite them in the context of NS alterations of fictive crawling. Aside from different-looking figures and some new analyses, the results and conclusions are the same.

Strengths:

The figures are well illustrated.

Weaknesses:

The paper is a mix of what appears to be two different studies and abruptly switches gears to examine how closely the crawl patterning is in the intact animal as compared to the fictive crawl patterning in the intact animal. Unfortunately, previous studies in other labs are not cited even though identical results have been obtained and similar conclusions were made. Thus, the novelty of the results is missing for those who are familiar with the leech preparation. The lack of appropriate citations and discussion of previous studies also deprives the scientific community of fully comprehending the impact of the data presented and the science it was built upon.

(1) Results, Lines 167-170: "While multiple extracellular recordings have been performed previously (Eisenhart et al., 2000), these results present the first quantitative analysis of motor units activated throughout the crawling cycle. The In-Phase units are expected to control the contraction stage by exciting or inhibiting the longitudinal or circular muscles, respectively, and the Anti-Phase units to control the elongation stage by exciting or inhibiting the circular or longitudinal muscles, respectively."

The first line above is misleading. The study by Puhl and Mesce (2008, J. Neurosci, 28:4192- 420) contains a comprehensive analysis of the motoneurons active during fictive crawling with the aim of characterizing their roles and phase relationships and solidifying the idea that the oscillator for crawling resides in a single ganglion. Intracellular recordings from a number of key crawl-related motoneurons were made in combination with extracellular recordings of motoneuron DE-3, a key monitor of crawling. In their paper, it was shown that motoneurons AE, VE-4, DI-1, VI-2, and CV were all correlated with crawl activity, and fired repeatedly either in phase or out-of-phase with DE-3. They were shown to be either excitatory or inhibitory.

At a minimum, the above paper should be cited. The submitted paper would be strengthened if some of these previously identified motoneurons were again recorded with intracellular electrodes and concomitant NS cell stimulation. The power of the leech preparation is that cells can be identified as individuals with dual somatic (intracellular) and axonal recordings (extracellular). The shortfall of this aspect of the study (Figure 5) is that the extracellular units have not been identified here. In fact, these units might not even be motoneurons. They could represent activity from the centrally located sensory neurons, dopamine-modulated afferent neurons or peripherally projecting modulatory neurons. Essentially, they may not have much to do with the crawl motor pattern at all.

(2) Results Lines 206-210: "with the elongation and contraction stages of in vivo behavior. However the isometric stages displayed in vivo have no obvious counterpart in the electrophysiological recordings. It is important to consider that the rhythmic movement of successive segments along the antero-posterior axis of the animal requires a delay signal that allows the appropriate propagation of the metachronal wave, and this signal is probably absent in the isolated ganglion."

The so-called isometric stages, indeed, have an electrophysiological counterpart due in part to the overlapping activities across segments. This submitted paper would be considerably strengthened if it referred to the body of work that has examined how the individual crawl oscillators operate in a fully intact nerve cord, excised from the body but with all the ganglia (and cephalic ganglion) attached. Puhl and Mesce 2010 (J. Neurosci 30: 2373-2383) and Puhl et al. 2012 (J. Neurosci, 32:17646 -17657) have shown that "appropriate propagation of the metachronal wave" requires the brain, especially cell R3b-1. They also show that the long-distance projecting cell R3b-1 synapses with the CV motoneuron, providing rhythmic excitatory input to it.

For this and other reasons, the paper would be much more informative and exciting if the impacts of the NS cell were studied in a fully intact nerve cord. Those studies have never been done, and it would be exciting to see how and if the effects of NS cell manipulation deviated from those in the single ganglion.

(3) Discussion Lines 322-324. "The absence of descending brain signals and/or peripheral signals are assumed as important factors in determining the cycle period and the sequence at which the different behavioral stages take place."

The authors could strengthen their paper by including a more complete picture of what is known about the control of crawling. For example, Puhl et al. 2012 (J Neurosci, 32:17646-17657) demonstrated that the descending brain neuron R3b-1 plays a major role in establishing the crawl-cycle frequency. With increased R3b-1 cell stimulation, DE-3 periods substantially shortened throughout the entire nerve cord. Thus, the importance of descending brain inputs should not be merely assumed; empirical evidence exists.

(4) Discussion Lines 325-327: "the sequence of events, and the proportion of the active cycle dedicated to elongation and contraction were remarkably similar in both experimental settings. This suggests that the network activated in the isolated ganglion is the one underlying the motor behavior."

The results and conclusions drawn in the current manuscript mirror those previously reported by Puhl and Mesce (2008, J. Neurosci, 28:4192- 420) who first demonstrated that the essential pattern-generating elements for leech crawling were contained in each of the segmental ganglia comprising the nerve cord. Furthermore, the authors showed that the duty cycle of DE-3, in a single ganglion treated with dopamine, was statistically indistinguishable from the DE-3 duty cycle measured in an intact nerve cord showing spontaneous fictive crawling, in an intact nerve cord induced to crawl via dopamine, and in the intact behaving animal. What was statistically significant, however, was that the DE-3 burst period was greatly reduced in the intact animal (i.e., a higher crawl frequency), which was replicated in the submitted paper.

In my opinion, the novelty of the results reported in the submitted manuscript is diminished in the light of previously published studies. At a minimum, the previous studies should be cited, and the authors should provide additional rationale for conducting their studies. They need to explain in the discussion how their approach provided additional insights into what has already been reported.

Reviewer #2 (Public review):

The paper is well-written overall. The findings are clearly presented, and the data seems solid overall. I do have, however, a few major and some minor comments representing some concerns. My major comments are below.

(1) This may seem somewhat semantic, yet, it has implications on the way the data is presented and moreover on the conclusions drawn - a single ganglion cannot show fictive crawling. It can demonstrate rhythmic patterns of activity that may serve in the (fictive) crawling motor pattern. The latter is a result of the intrinsic within single-ganglion connectivity AND the inter-ganglia connections and interactions (coupling) among the sequential ganglia. It may be affected by both short-range and long-range connections (e.g., descending inputs) along the ganglia chain.

(2) The point above is even more critical where the authors set to compare the motor pattern in single ganglia with the intact animals. It would have made much more sense to add a description of the motor pattern of a chain of interconnected ganglia. The latter would be expected to better resemble the intact animal. Furthermore, this project would have benefitted from a three-way comparison (isolated ganglion-interconnected ganglia-intact animal.

(3) Two previous studies by the same group are repeatedly mentioned (Rela and Szczupak, 2003; Rodriguez et al., 2009) and serve as a basis for the current work. The aim of one of these previous studies was to assess the role of the NS neurons in regulating the function of motor networks. The other (Rodriguez et al., 2009) reported on a neuron (the NS) that can regulate the crawling motor pattern. LL 71-74 of the current report presents the aim of this study as evaluating the role of the known connectivity of the premotor NS neuron in shaping the crawling motor pattern. The authors should make it very clear what indeed served as background knowledge, what exactly was known about the circuitry beforehand, and what is different and new in the current study.