Coupling between fast and slow oscillator circuits in Cancer borealis is temperature-compensated

  1. Daniel Powell
  2. Sara A Haddad
  3. Srinivas Gorur-Shandilya
  4. Eve Marder

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Abstract

Coupled oscillatory circuits are ubiquitous in nervous systems. Given that most biological processes are temperature-sensitive, it is remarkable that the neuronal circuits of poikilothermic animals can maintain coupling across a wide range of temperatures. Within the stomatogastric ganglion (STG) of the crab, Cancer borealis , the fast pyloric rhythm (~1 Hz) and the slow gastric mill rhythm (~0.1 Hz) are precisely coordinated at ~11°C such that there is an integer number of pyloric cycles per gastric mill cycle (integer coupling). Upon increasing temperature from 7°C to 23°C, both oscillators showed similar temperature-dependent increases in cycle frequency, and integer coupling between the circuits was conserved. Thus, although both rhythms show temperature-dependent changes in rhythm frequency, the processes that couple these circuits maintain their coordination over a wide range of temperatures. Such robustness to temperature changes could be part of a toolbox of processes that enables neural circuits to maintain function despite global perturbations.

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  1. Version published to 10.7554/elife.60454 on eLife
  2. eLife

    ###Reviewer #3:

    The authors examine the robustness of coupling of distinct oscillatory circuits of different frequencies across a range of temperatures. The two circuits have different means of generating oscillations and could therefore, potentially, be impacted to different degrees by temperature perturbations. Across all temperatures tested the two distinct rhythms increased their frequency but remained coordinated. The coordination was in the form of the previously-described integer-coupling where the cycle period of the slow rhythm was an integer multiple of that of the fast one. This is due to the fact that the slower rhythm was most likely to start at a given phase within the faster oscillation cycle. The temperature robustness of this coupling is an interesting and important result and the description and analysis are both well done.

    Major comments:

    The main finding of the paper is that a previously-described integer-coupling between two rhythms remains more or less intact across temperature variations. It is a nice descriptive finding, but rather disappointing in that there is so much more that could have been done rather easily that would have given much more depth to this finding. Most obviously, because it is known that the source of the coupling is the inhibitory synapse from the pyloric pacemaker to the gastric mill half-center, it is quite important to know how the strength of this synapse affects the interaction at different temperatures. That is, to expand what Bartos et al 1999 did across a range of temperatures. Short of that, it would have been nice at least to perturb the cycle period of the pyloric rhythm and see whether the interaction would remain robust across temperature despite changes in cycle period.

    While the study convinces the reader that integer coupling between pyloric and evoked gastric rhythms is robust to temperature changes, it does not attempt to to explore the origin of this robustness, e.g. by using different methods to activate the gastric rhythm or testing if integer coupling is present with spontaneous gastric rhythms.

  3. eLife

    ###Reviewer #2:

    In the present paper, Powell and colleagues investigated how coupled oscillatory circuits maintain their coordination over a wide range of temperature. To do so they used the stomatogastric system of the crab Cancer borealis that contains the fast (1Hz) pyloric network and the slow (0.1 Hz) gastric mill network. The two generated rhythms are coordinated such that there are an integer number of pyloric cycles per gastric cycle. Both rhythms exhibit temperature-induced frequency changes, but their coordination is well maintained even at high temperature. Therefore, this study shows that the relative coordination between rhythmic circuits can be maintained as temperature changes, thus ensuring appropriate physiological functions even under global perturbations.

    This study, that uses a fantastic model for investigating neural networks in general, addresses an important physiological question. However, I have a few concerns that could be probably clarified with some additional explanations in the text:

    -While the intrinsic temperature sensitivity of the pyloric rhythm has been nicely investigated in some previous excellent publications (most done by the authors), that of the gastric rhythm is less well known. Stadele has shown that increasing the temperature leads to a breakdown of the gastric rhythm that can be rescued by modulatory afferences. What do we know about the temperature sensitivity of the afferent neurons that are stimulated to trigger the gastric rhythm here? Is there the possibility that what is observed also includes an effect of the temperature changes on these neurons (MCN1 function for example) or that the gastric temperature sensitivity described here reflects in fact that of the afferences?

    -All experiments were performed in conditions in which the gastric rhythm is triggered by stimulation of the two dorsal posterior esophageal nerves (dpons) that contain axons of modulatory afferent neurons. However stimulating these nerves also modulates the pyloric network that is also a target of those afferences (as stipulated in the text line 583-584). Isn't this a bias in the experiments and their interpretations? Also, because as schematically represented in Fig 1, the pyloric pacemaker neuron AB has direct connections with Int1 gastric neuron that is itself connected to the LG gastric neuron, the simplest interpretation of the experiments would be that this connection is preserved and remains efficient even under high temperature. Is it finally one of the conclusions of the paper?

    -In the same vain, the sensitivity to temperature changes of the gastric rhythm has been studied here but with the pyloric network, being itself intrinsically sensitive to temperature changes, still active (Fig 3 and related text). What do we know about the intrinsic temperature sensitivity of the gastric rhythm when elicited by dpons stimulation but isolated from the pyloric network (AB neuron killed for example)?

    -Data presented here show that coordination between PD and LG neurons is preserved after temperature increase, but that this is not the case between PD and DG neuron that shows no phase-coupling at high temperature (Fig 6). The PD neurons are used here as an indicator of the pyloric rhythm while the LG neurons indicate the gastric rhythm. Then what would be the conclusions of the authors if the DG neuron would have been used as the gastric rhythm indicator? How do you conciliate everything together?

  4. eLife

    ###Reviewer #1:

    Powell and colleagues measured coordination robustness between pyloric and gastric rhythms in in vitro preparations of Cancer borealis exposed to temperature variations (7-23C degrees). Using extracellular recordings, they first show that spontaneous rhythms are not stable, likely resulting from multiple physiological processes that are difficult to monitor. Therefore, they rather used bouts of activity reproducibly evoked by stimulation of a neuromodulatory pathway. As expected, cold temperatures slowed down rhythms, warm temperatures accelerated rhythms in a similar manner. Despite this variation in rhythm frequency across temperatures, coordination between pyloric and gastric rhythms was stable . This suggested that the activity of rhythmogenic neurons is coordinated across temperatures. Powell and colleagues also found that the gastric Lateral Gastric motor neuron (LG) was phase-locked with the Pyloric Dilatator neuron (PD), suggesting they may be involved in coordination robustness.

    The originality of the study is that the authors focused on the coordination of pyloric (1 Hz) and gastric (0.1 Hz) networks. A large quantity of raw data is beautifully illustrated. Data analysis is sophisticated and convincingly supports the interpretations on the authors. The text is exquisitely written in a clear style and pleasant to read. In my view, the study contains the first experiments of a potentially exceptionally interesting study, once more mechanistic insights are added. To further strengthen the relevance of the study, I would suggest pursuing one of the three options below to further uncover the mechanisms underlying the effects described. 1.) Could the authors design causality-based experiments to identify which neuron is responsible for the coordination of the rhythms at different temperatures? There are many interconnected neurons in Figure 1C. Even if LG is phase locked to PD, is it possible that another neuron drives PD and LG? If PD controls LG, would it be relevant if the authors reversibly switched off PD (e.g. with tonic hyperpolarisation) and see the effect on gastric rhythm frequency at various temperatures?

    1. Could the authors identify using pharmacological tools whether distinct neuromodulatory substances influence coordination robustness over specific ranges of temperature, but not in others? It seems that Stadele et al. 2015 PLoS Biol 13(9):e1002265 used a different way to evoke the rhythm, and their gastric rhythm crashed at lower temperatures (13C degrees) than in the present study (27C degrees). Do the authors think that the different stimulation approaches used in the two studies could involve different neuromodulatory substances, which would result in different robustness profiles?

    2. Do the same intrinsic properties or synaptic connections underlie coordination robustness across temperatures? Modeling suggests that different conductances are involved in a temperature-dependent manner (Alonso and Marder 2020 Elife 9:e55470.2020). Is it possible for the authors to experimentally deactivate specific conductances using dynamic clamp in LG or PD or with pharmacological tools and determine whether this would reversibly disrupt the coordination between pyloric and gastric networks in some specific temperature ranges but not in others?

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    This study addresses an important question about the physiology of coupled oscillatory neuronal networks operating under a wide range of temperatures. The stomatogastric system of the crab Cancer borealis contains the fast (~1Hz) pyloric network and the slow (~0.1 Hz) gastric mill network. The two generated rhythms are coordinated so that there is a given number of pyloric cycles per gastric cycle. Powell and colleagues show that upon stimulation of a neuromodulatory pathway, these coupled oscillatory circuits exhibit reproducible bouts of activity and maintain their coordination, and that this coordination is maintained over a wide range of temperatures, thus ensuring appropriate physiological functions even under global perturbations.The authors show that the gastric Lateral Gastric motor neuron (LG) is phase-locked with the Pyloric Dilatator neuron (PD), suggesting these neurons may be involved in coordination robustness.

  6. Version published to 10.1101/2020.06.26.173427 on bioRxiv