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 (ISyn) and hyperpolarization-activated inward (IH) 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 in 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 (IMI) 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.

What features are important for circuit robustness? Reciprocal inhibition is a building block in many circuits. We used dynamic clamp to create reciprocally inhibitory circuits from GM neurons of the crab stomatogastric ganglion by injecting artificial synaptic and hyperpolarization-activated inward (H) currents. In "release", the active neuron 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. 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 (IMI) restores oscillations in release circuits but has little effect in escape. Thus, the same perturbation can have dramatically different effects depending on the circuits' mechanism of operation that may not be observable from circuit output.

Changes in ambient temperature affect all biological processes. However, these effects are process specific and often vary non-linearly. It is thus a non-trivial problem for neuronal circuits to maintain coordinated, functional output across a range of temperatures. The cardiac nervous systems in two species of decapod crustaceans, Homarus americanus and Cancer borealis, can maintain function across a wide but physiologically relevant temperature range. However, the processes that underlie temperature resilience in neuronal circuits and muscle systems are not fully understood. Here, we demonstrate that the non-isolated cardiac nervous system (i.e., the whole heart: neurons, effector organs, intrinsic feedback systems) in the American lobster, H. americanus, is more sensitive to warm temperatures than the isolated cardiac ganglion (CG) that controls the heartbeat. This was surprising as modulatory processes known to stabilize the output from the CG are absent when the ganglion is isolated. One source of inhibitory feedback in the intact cardiac neuromuscular system is nitric oxide (NO), which is released in response to heart contractions. We hypothesized that the greater temperature tolerance observed in the isolated CG is due to the absence of NO feedback. Here, we demonstrate that applying an NO donor to the isolated CG reduces its temperature tolerance. Similarly, we show that the NO synthase inhibitor L-nitroarginine (LNA) increases the temperature tolerance of the non-isolated nervous system. This is sufficient to explain differences in temperature tolerance between the isolated CG and the whole heart. However, in an intact lobster, the heart and CG are modulated by an array of endogenous peptides and hormones, many of which are positive regulators of the heartbeat. Many studies have demonstrated that excitatory modulators increase temperature resilience. However, this neuromuscular system is regulated by both excitatory and inhibitory peptide modulators. Perfusing SGRNFLRFamide, a FLRFamide-like peptide, through the heart increases the non-isolated nervous system’s tolerance to high temperatures. In contrast, perfusing myosuppressin, a peptide that negatively regulates the heartbeat frequency, decreases the temperature tolerance. Our data suggest that, in this nervous system, positive regulators of neural output increase temperature tolerance of the neuromuscular system, while modulators that decrease neural output decrease temperature tolerance.

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