Reciprocally inhibitory circuits operating with distinct mechanisms are differently robust to perturbation and modulation
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Evaluation Summary:
Morozova et al. describe potential mechanisms contributing to the flexibility of burst patterns and dynamic responses to perturbations within an isolated reciprocally inhibitory circuit derived from the stomatogastric ganglion of the crab. The authors use the dynamic clamp approach to study the interactions between pharmacologically isolated, intrinsically silent gastric mill neurons, an approach pioneered by Andrew Sharp in the 1990s. The authors demonstrate that the mechanisms of switching between components of the reciprocally organized half-center network are not fixed and may shift to favor a release or escape mechanism depending on factors such as the synaptic threshold, Ih conductance, and synaptic conductance. This is a fundamentally important study because reciprocally organized networks are ubiquitous and found virtually in every organism. It is assumed that this half-center-type network organization governs rhythmic activity with a wide range of functions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewers #1, #2 and #3 agreed to share their names with the authors.)
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Abstract
Reciprocal inhibition is a building block in many sensory and motor circuits. We studied the features that underly robustness in reciprocally inhibitory two neuron circuits. We used the dynamic clamp to create reciprocally inhibitory circuits from pharmacologically isolated neurons of the crab stomatogastric ganglion by injecting artificial graded synaptic (I Syn ) and hyperpolarization-activated inward (I H ) currents. There is a continuum of mechanisms in circuits that generate antiphase oscillations, with ‘release’ and ‘escape’ mechanisms at the extremes, and mixed mode oscillations between these extremes. In release, the active neuron primarily controls the off/on transitions. In escape, the inhibited neuron controls the transitions. We characterized the robustness of escape and release circuits to alterations in circuit parameters, temperature, and neuromodulation. We found that escape circuits rely on tight correlations between synaptic and H conductances to generate bursting but are resilient to temperature increase. Release circuits are robust to variations in synaptic and H conductances but fragile to temperature increase. The modulatory current (I MI ) restores oscillations in release circuits but has little effect in escape circuits. Perturbations can alter the balance of escape and release mechanisms and can create mixed mode oscillations. We conclude that the same perturbation can have dramatically different effects depending on the circuits’ mechanism of operation that may not be observable from basal circuit activity.
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Evaluation Summary:
Morozova et al. describe potential mechanisms contributing to the flexibility of burst patterns and dynamic responses to perturbations within an isolated reciprocally inhibitory circuit derived from the stomatogastric ganglion of the crab. The authors use the dynamic clamp approach to study the interactions between pharmacologically isolated, intrinsically silent gastric mill neurons, an approach pioneered by Andrew Sharp in the 1990s. The authors demonstrate that the mechanisms of switching between components of the reciprocally organized half-center network are not fixed and may shift to favor a release or escape mechanism depending on factors such as the synaptic threshold, Ih conductance, and synaptic conductance. This is a fundamentally important study because reciprocally organized networks are ubiquitous and …
Evaluation Summary:
Morozova et al. describe potential mechanisms contributing to the flexibility of burst patterns and dynamic responses to perturbations within an isolated reciprocally inhibitory circuit derived from the stomatogastric ganglion of the crab. The authors use the dynamic clamp approach to study the interactions between pharmacologically isolated, intrinsically silent gastric mill neurons, an approach pioneered by Andrew Sharp in the 1990s. The authors demonstrate that the mechanisms of switching between components of the reciprocally organized half-center network are not fixed and may shift to favor a release or escape mechanism depending on factors such as the synaptic threshold, Ih conductance, and synaptic conductance. This is a fundamentally important study because reciprocally organized networks are ubiquitous and found virtually in every organism. It is assumed that this half-center-type network organization governs rhythmic activity with a wide range of functions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewers #1, #2 and #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
This is a very careful and systematic hybrid system analysis of the mechanism underlying alternating bursting in mutually inhibitory neurons or half-center oscillators (HCOs). By clever use of dynamic clamp, the authors create HCOs between non-connected living neurons (from the crab stomatogastric ganglion) of the same type by adding artificial synapses and h-current. This hybrid system allows them to manipulate synaptic threshold as a control variable to engage different oscillatory mechanisms escape and release, which are based on a theoretical understanding of HCO operation. They also have control of synaptic and h-current conductance and dynamics (activation-deactivation) and manipulate these variables or as proxies for changes in temperature of circuit operation. Using the synaptic threshold control …
Reviewer #1 (Public Review):
This is a very careful and systematic hybrid system analysis of the mechanism underlying alternating bursting in mutually inhibitory neurons or half-center oscillators (HCOs). By clever use of dynamic clamp, the authors create HCOs between non-connected living neurons (from the crab stomatogastric ganglion) of the same type by adding artificial synapses and h-current. This hybrid system allows them to manipulate synaptic threshold as a control variable to engage different oscillatory mechanisms escape and release, which are based on a theoretical understanding of HCO operation. They also have control of synaptic and h-current conductance and dynamics (activation-deactivation) and manipulate these variables or as proxies for changes in temperature of circuit operation. Using the synaptic threshold control variable to set escape or release mode they discern difference of these manipulations on burst characteristics in escape vs release modes. In separate experiments, they also add a modulatory current (similar to a persistent Na current) in dynamic clamp and explore it effects on HCOs in escape and release modes. The end result is a thorough analysis of how oscillator mechanism in an HCO, a basic circuit building block, affects circuit responses to perturbation and modulation.
The experiments are well performed, and a deep and rich data set is generated that is appropriately analyzed. The findings are significant for all interested in oscillatory network function and its resilience to perturbation and modulation.Concerns:
Robustness is often mentioned but is not precisely defined. Operationally robustness seems in this paper to stand for robustness to 1) activity regime change under parameter variation, 2) stability of burst characteristics with parameter variation, and 3) slow-wave amplitude, spiking strength (spike frequency), and symmetry of bursting. These are three very different things and should be clearly differentiated in the text so that when robustness is mentioned, the type of robustness is made clear. Perhaps robustness should be limited to the first, activity regime, and some other terms used for the other two.
On several occasion in the text the authors refer to irregularity in bursting of the hybrid HCOs, but this is not quantified beyond displaying exemplars that seem to have irregular bursting. Pooled data should be analyzed in the different modes and manipulations and analyzed for statistical difference in the CoV of cycle frequency (or period) and burst duration. Similarly, the authors cite changes in symmetry in bursting in exemplars but do not present pooled quantitative data in support of the claim, just visual inspection of exemplars.
In the stomatogastric networks, synaptic transmission is largely graded (based on release mediated by the slow wave of oscillation) and not so much spike-mediated, so it is reasonable that synaptic threshold should be a control variable in this system. Moreover, spikes, recorded in the cell bodies are not reflective of their amplitude at the SIZ. In other system transmission can be largely mediated by spikes. At the beginning of the paper (Figure 1), it is clear that release mode in their hybrid HCOs depends on spike-mediated transmission because synaptic threshold is above the slow-wave depolarization, thus spike frequency is a key feature determining the mechanism of oscillation. However, in escape mode the transmission is purely graded because synaptic threshold is so low that transmission is saturated by the slow-wave depolarization and spikes contribute little if anything, thus spike frequency is immaterial to the mechanism of oscillation. This situation should be addressed at the beginning of the paper in reference to Figure 1. How this spike-mediated vs. graded balance plays out in the mixed mechanism modes remains to be explored.
In Figure 1C, the authors show convincingly that there is a vast landscape where their hybrid HCO operate in a mixed mechanistic mode somewhere between escape and release corresponding to synaptic thresholds in the middle range. This mixed mode is addressed only with a single exemplar in Figure 8B as a case for how modulation affects mixed mode circuits. The Discussion should reflect plainly that this mixed mode is likely common in biological circuits and may go hand-in-hand with significant reliance on spike-mediated transmission.
The authors state "The modulatory current (IMI) restores oscillations in release circuits but has little effect in escape circuits." but this is supported by a single exemplar (Figure 8E) and no pooled data is presented. -
Reviewer #2 (Public Review):
This manuscript provides a very detailed and thorough examination of an important issue in neural circuit research, namely how the mechanisms underlying neural activity relate to robustness in the face of perturbations. It examines the simplest neural circuit possible, one involving just two neurons that reciprocally inhibit each other, which is capable of producing rhythmic alternating activity. The research shows that there is a continuum of mechanisms based on synaptic and membrane properties of the two neurons that can generate a robust output. At one end of the continuum, each neuron escapes from the inhibition of the other. At the other end, each neuron releases the other from inhibition. In the middle, both mechanisms contribute to generation of rhythmic activity. The effects that perturbations such …
Reviewer #2 (Public Review):
This manuscript provides a very detailed and thorough examination of an important issue in neural circuit research, namely how the mechanisms underlying neural activity relate to robustness in the face of perturbations. It examines the simplest neural circuit possible, one involving just two neurons that reciprocally inhibit each other, which is capable of producing rhythmic alternating activity. The research shows that there is a continuum of mechanisms based on synaptic and membrane properties of the two neurons that can generate a robust output. At one end of the continuum, each neuron escapes from the inhibition of the other. At the other end, each neuron releases the other from inhibition. In the middle, both mechanisms contribute to generation of rhythmic activity. The effects that perturbations such as temperature and neuromodulators have on the circuit depend upon where the mechanism of oscillation lies along this continuum.
This paper has several important strengths:
It uses dynamic clamp technique to artificially couple two real neurons and provide them with a membrane conductance that they don't normally have. This is a powerful technique that merges experimental and theoretical neuroscience because the researchers are able to systematically alter parameter values such as synaptic strength and ionic conductance that are not feasible to modify biologically. Yet they are also monitoring the activity of real neurons.
The manuscript thoroughly represents the results and convincingly demonstrates how release and escape mechanisms are differentially affected by perturbations. The method of data visualization is very effect at summarizing complex results.
An important conclusion drawn from the results is that half-center oscillators using a release mechanism are more robust to variations in synaptic and membrane conductance.
Another important conclusion is that the same circuit can produce a similar output using different mechanisms and that it is not possible to know which mechanism is used without looking at the effect of perturbation.
The weaknesses in the manuscript are minor and are merely aspects related to the presentation of the work, not its substance.
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Reviewer #3 (Public Review):
The authors demonstrate that the mechanisms of switching between components of the reciprocally organized half-center network are not fixed and may shift to favor a release or escape mechanism depending on factors such as the synaptic threshold, Ih conductance, and synaptic conductance. This is a fundamentally important study because reciprocally organized networks are ubiquitous and found virtually in every organism.
This study leads to the important conclusion that a given rhythmic output alone does not reveal the underlying rhythmogenic mechanisms. A rhythmic output is not based on one "fixed" mechanism, but on the interplay between different rhythmogenic modes. Moreover, because of this interplay it is impossible to predict how this network will respond to perturbations.The study is an important …
Reviewer #3 (Public Review):
The authors demonstrate that the mechanisms of switching between components of the reciprocally organized half-center network are not fixed and may shift to favor a release or escape mechanism depending on factors such as the synaptic threshold, Ih conductance, and synaptic conductance. This is a fundamentally important study because reciprocally organized networks are ubiquitous and found virtually in every organism.
This study leads to the important conclusion that a given rhythmic output alone does not reveal the underlying rhythmogenic mechanisms. A rhythmic output is not based on one "fixed" mechanism, but on the interplay between different rhythmogenic modes. Moreover, because of this interplay it is impossible to predict how this network will respond to perturbations.The study is an important reminder that even a small two neuron network with a well defined, extremely simple "connectome" is strikingly flexible and complex: an important lessons for those aspiring to obtain complete connectomes in mammals in the hope to reveal the secrets of the brain.
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