Circuit function is more robust to changes in synaptic than intrinsic conductances.

Circuit function results from both intrinsic conductances of network neurons and the synaptic conductances that connect them. In models of neural circuits, different combinations of maximal conductances can give rise to similar activity. We compared the robustness of a neural circuit to changes in their intrinsic versus synaptic conductances. To address this, we performed a sensitivity analysis on a population of conductance-based models of the pyloric network from the crustacean stomatogastric ganglion (STG). The model network consists of three neurons with nine currents: a sodium current (Na), three potassium currents (Kd, KCa, A-type), two calcium currents (CaS and CaT), a hyperpolarization-activated current (H), a non-voltage-gated leak current (leak), and a neuromodulatory current (MI). The model cells are connected by seven synapses of two types, glutamatergic and cholinergic. We produced one hundred models of the pyloric network that displayed similar activities with values of maximal conductances distributed over wide ranges. We evaluated the robustness of each model to changes in their maximal conductances. We found that individual models have different sensitivities to changes in their maximal conductances, both in their intrinsic and synaptic conductances. As expected the models become less robust as the extent of the changes increase. Despite quantitative differences in their robustness, we found that in all cases, the model networks are more sensitive to the perturbation of their intrinsic conductances than their synaptic conductances.

Alterations in network robustness upon simultaneous temperature and pH perturbations.

Nervous systems have evolved to function consistently in the face of the normal environmental fluctuations experienced by animals. The stomatogastric nervous system (STNS) of the crab, Cancer borealis, produces a motor output that has been studied for its remarkable robustness in response to single global perturbations. Changes in environments, however, are often complex and multifactorial. Therefore, we studied the robustness of the pyloric network in the stomatogastric ganglion (STG) in response to simultaneous perturbations of temperature and pH. We compared the effects of elevated temperatures on the pyloric rhythm at control, acid, or base pHs. In each pH recordings were made at 11°C, and then the temperature was increased until the rhythms became disorganized ("crashed"). Pyloric burst frequencies and phase relationships showed minor differences between pH groups until reaching close to the crash temperatures. However, the temperatures at which the rhythms were disrupted were lower in the two extreme pH conditions. This indicates that one environmental stress can make an animal less resilient to a second stressor.NEW & NOTEWORTHY Resilience to environmental fluctuations is important for all animals. It is common that animals encounter multiple stressful events at the same time, the cumulative impacts of which are largely unknown. This study examines the effects of temperature and pH on the nervous system of crabs that live in the fluctuating environments of the Northern Atlantic Ocean. The ranges of tolerance to one perturbation, temperature, are reduced under the influence of a second, pH.

Blue light responses in Cancer borealis stomatogastric ganglion neurons.

In many animals, the daily cycling of light is a key environmental cue, encoded in part by specialized light-sensitive neurons without visual functions. We serendipitously discovered innate light-responsiveness while imaging the extensively studied stomatogastric ganglion (STG) of the crab, Cancer borealis. The STG houses a motor circuit that controls the rhythmic contractions of the foregut, and the system has facilitated deep understanding of circuit function and neuromodulation. We illuminated the crab STG in vitro with different wavelengths and amplitudes of light and found a dose-dependent increase in neuronal activity upon exposure to blue light (λ460-500 nm). The response was elevated in the absence of neuromodulatory inputs to the STG. The pacemaker kernel that drives the network rhythm was responsive to light when synaptically isolated, and light shifted the threshold for slow wave and spike activity in the hyperpolarized direction, accounting for the increased activity patterns. Cryptochromes are evolutionarily conserved blue-light photoreceptors that are involved in circadian behaviors.1 Their activation by light can lead to enhanced neuronal activity.2 We identified cryptochrome sequences in the C. borealis transcriptome as potential mediators of this response and confirmed their expression in pyloric dilator (PD) neurons, which are part of the pacemaker kernel, by single-cell RNA-seq analysis.

Feeding state-dependent modulation of feeding-related motor patterns.

Studies elucidating modulation of microcircuit activity in isolated nervous systems have revealed numerous insights regarding neural circuit flexibility, but this approach limits the link between experimental results and behavioral context. To bridge this gap, we studied feeding behavior-linked modulation of microcircuit activity in the isolated stomatogastric nervous system (STNS) of male Cancer borealis crabs. Specifically, we removed hemolymph from a crab that was unfed for ≥24 h ("unfed" hemolymph) or fed 15 min to 2 h before hemolymph removal ("fed" hemolymph). After feeding, the first significant foregut emptying occurred >1 h later and complete emptying required ≥6 h. We applied the unfed or fed hemolymph to the stomatogastric ganglion (STG) in an isolated STNS preparation from a separate, unfed crab to determine its influence on the VCN (ventral cardiac neuron)-triggered gastric mill (chewing) and pyloric (filtering of chewed food) rhythms. Unfed hemolymph had little influence on these rhythms, but fed hemolymph from each examined time-point (15 min, 1 h, or 2 h after feeding) slowed one or both rhythms without weakening circuit neuron activity. There were also distinct parameter changes associated with each time-point. One change unique to the 1-h time-point (i.e., reduced activity of one circuit neuron during the transition from the gastric mill retraction to protraction phase) suggested that the fed hemolymph also enhanced the influence of a projection neuron that innervates the STG from a ganglion isolated from the applied hemolymph. Hemolymph thus provides a feeding state-dependent modulation of the two feeding-related motor patterns in the C. borealis STG.NEW & NOTEWORTHY Little is known about behavior-linked modulation of microcircuit activity. We show that the VCN-triggered gastric mill (chewing) and pyloric (food filtering) rhythms in the isolated crab Cancer borealis stomatogastric nervous system were changed by applying hemolymph from recently fed but not unfed crabs. This included some distinct parameter changes during each examined post-fed hemolymph time-point. These results suggest the presence of feeding-related changes in circulating hormones that regulate consummatory microcircuit activity.

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

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.

Two central pattern generators from the crab, Cancer borealis, respond robustly and differentially to extreme extracellular pH.

The activity of neuronal circuits depends on the properties of the constituent neurons and their underlying synaptic and intrinsic currents. We describe the effects of extreme changes in extracellular pH - from pH 5.5 to 10.4 - on two central pattern generating networks, the stomatogastric and cardiac ganglia of the crab, Cancer borealis. Given that the physiological properties of ion channels are known to be sensitive to pH within the range tested, it is surprising that these rhythms generally remained robust from pH 6.1 to pH 8.8. The pH sensitivity of these rhythms was highly variable between animals and, unexpectedly, between ganglia. Animal-to-animal variability was likely a consequence of similar network performance arising from variable sets of underlying conductances. Together, these results illustrate the potential difficulty in generalizing the effects of environmental perturbation across circuits, even within the same animal.

Circuit Robustness to Temperature Perturbation Is Altered by Neuromodulators.

In the ocean, the crab Cancer borealis is subject to daily and seasonal temperature changes. Previous work, done in the presence of descending modulatory inputs, had shown that the pyloric rhythm of the crab increases in frequency as temperature increases but maintains its characteristic phase relationships until it "crashes" at extremely high temperatures. To study the interaction between neuromodulators and temperature perturbations, we studied the effects of temperature on preparations from which the descending modulatory inputs were removed. Under these conditions, the pyloric rhythm was destabilized. We then studied the effects of temperature on preparations in the presence of oxotremorine, proctolin, and serotonin. Oxotremorine and proctolin enhanced the robustness of the pyloric rhythm, whereas serotonin made the rhythm less robust. These experiments reveal considerable animal-to-animal diversity in their crash stability, consistent with the interpretation that cryptic differences in many cell and network parameters are revealed by extreme perturbations.

Phase maintenance in a rhythmic motor pattern during temperature changes in vivo.

Central-pattern-generating neural circuits function reliably throughout an animal's life, despite constant molecular turnover and environmental perturbations. Fluctuations in temperature pose a problem to the nervous systems of poikilotherms because their body temperature follows the ambient temperature, thus affecting the temperature-dependent dynamics of various subcellular components that constitute neuronal circuits. In the crustacean stomatogastric nervous system, the pyloric circuit produces a triphasic rhythm comprising the output of the pyloric dilator, lateral pyloric, and pyloric constrictor neurons. In vitro, the phase relationships of these neurons are maintained over a fourfold change in pyloric frequency as temperature increases from 7°C to 23°C. To determine whether these temperature effects are also found in intact crabs, in the presence of sensory feedback and neuromodulator-rich environments, we measured the temperature dependence of the pyloric frequency and phases in vivo by implanting extracellular electrodes into Cancer borealis and Cancer pagurus and shifting tank water temperature from 11°C to 26°C. Pyloric frequency in the intact crab increased significantly with temperature (Q10 = 2-2.5), while pyloric phases were generally conserved. For a subset of the C. borealis experiments, animals were subsequently dissected and the stomatogastric ganglion subjected to a similar temperature ramp in vitro. We found that the maximal frequency attained at high temperatures in vivo is lower than it is under in vitro conditions. Our results demonstrate that, over a wide temperature range, the phases of the pyloric rhythm in vivo are generally preserved, but that the frequency range is more restricted than it is in vitro.

Pacemaker neuron and network oscillations depend on a neuromodulator-regulated linear current.

Linear leak currents have been implicated in the regulation of neuronal excitability, generation of neuronal and network oscillations, and network state transitions. Yet, few studies have directly tested the dependence of network oscillations on leak currents or explored the role of leak currents on network activity. In the oscillatory pyloric network of decapod crustaceans neuromodulatory inputs are necessary for pacemaker activity. A large subset of neuromodulators is known to activate a single voltage-gated inward current I(MI), which has been shown to regulate the rhythmic activity of the network and its pacemaker neurons. Using the dynamic clamp technique, we show that the crucial component of I(MI) for the generation of oscillatory activity is only a close-to-linear portion of the current-voltage relationship. The nature of this conductance is such that the presence or the absence of neuromodulators effectively regulates the amount of leak current and the input resistance in the pacemaker neurons. When deprived of neuromodulatory inputs, pyloric oscillations are disrupted; yet, a linear reduction of the total conductance in a single neuron within the pacemaker group recovers not only the pacemaker activity in that neuron, but also leads to a recovery of oscillations in the entire pyloric network. The recovered activity produces proper frequency and phasing that is similar to that induced by neuromodulators. These results show that the passive properties of pacemaker neurons can significantly affect their capacity to generate and regulate the oscillatory activity of an entire network, and that this feature is exploited by neuromodulatory inputs.