Comodulation reduces interindividual variability of circuit output.

Ionic current levels of identified neurons vary substantially across individual animals. Yet, under similar conditions, neural circuit output can be remarkably similar, as evidenced in many motor systems. All neural circuits are influenced by multiple neuromodulators which provide flexibility to their output. These neuromodulators often overlap in their actions by modulating the same channel type or synapse, yet have neuron-specific actions resulting from distinct receptor expression. Because of this different receptor expression pattern, in the presence of multiple convergent neuromodulators, a common downstream target would be activated more uniformly in circuit neurons across individuals. We therefore propose that a baseline tonic (non-saturating) level of comodulation by convergent neuromodulators can reduce interindividual variability of circuit output. We tested this hypothesis in the pyloric circuit of the crab, Cancer borealis. Multiple excitatory neuropeptides converge to activate the same voltage-gated current in this circuit, but different subsets of pyloric neurons have receptors for each peptide. We quantified the interindividual variability of the unmodulated pyloric circuit output by measuring the activity phases, cycle frequency and intraburst spike number and frequency. We then examined the variability in the presence of different combinations and concentrations of three neuropeptides. We found that at mid-level concentration (30 nM) but not at near-threshold (1 nM) or saturating (1 µM) concentrations, comodulation by multiple neuropeptides reduced the circuit output variability. Notably, the interindividual variability of response properties of an isolated neuron was not reduced by comodulation, suggesting that the reduction of output variability may emerge as a network effect.

Synaptic Dynamics Convey Differential Sensitivity to Input Pattern Changes in Two Muscles Innervated by the Same Motor Neurons.

Postsynaptic responses depend on input patterns as well as short-term synaptic plasticity, summation, and postsynaptic membrane properties, but the interactions of those dynamics with realistic input patterns are not well understood. We recorded the responses of the two pyloric dilator (PD) muscles, cpv2a and cpv2b, that are innervated by and receive identical periodic bursting input from the same two motor neurons in the lobster Homarus americanusCpv2a and cpv2b showed quantitative differences in membrane nonlinearities and synaptic summation. At a short timescale, responses in both muscles were dominated by facilitation, albeit with different frequency and time dependence. Realistic burst stimulations revealed more substantial differences. Across bursts, cpv2a showed transient depression, whereas cpv2b showed transient facilitation. Steady-state responses to bursting input also differed substantially. Neither muscle had a monotonic dependence on frequency, but cpv2b showed particularly pronounced bandpass filtering. Cpv2a was sensitive to changes in both burst frequency and intra-burst spike frequency, whereas, despite its much slower responses, cpv2b was largely insensitive to changes in burst frequency. Cpv2a was sensitive to both burst duration and number of spikes per burst, whereas cpv2b was sensitive only to the former parameter. Neither muscle showed consistent sensitivity to changes in the overall spike interval structure, but cpv2b was surprisingly sensitive to changes in the first intervals in each burst, a parameter known to be regulated by dopamine (DA) modulation of spike propagation of the presynaptic axon. These findings highlight how seemingly minor circuit output changes mediated by neuromodulation could be read out differentially at the two synapses.

Mutual Suppression of Proximal and Distal Axonal Spike Initiation Determines the Output Patterns of a Motor Neuron.

Axonal spike initiation at sites far from somatodendritic integration occurs in a range of systems, but its contribution to neuronal output activity is not well understood. We studied the interactions of distal and proximal spike initiation in an unmyelinated motor axon of the stomatogastric nervous system in the lobster, Homarus americanus. The peripheral axons of the pyloric dilator (PD) neurons generate tonic spiking in response to dopamine application. Centrally generated bursting activity and peripheral spike initiation had mutually suppressive effects. The two PD neurons and the electrically coupled oscillatory anterior burster (AB) neuron form the pacemaker ensemble of the pyloric central pattern generator, and antidromic invasion of central compartments by peripherally generated spikes caused spikelets in AB. Antidromic spikes suppressed burst generation in an activity-dependent manner: slower rhythms were diminished or completely disrupted, while fast rhythmic activity remained robust. Suppression of bursting was based on interference with the underlying slow wave oscillations in AB and PD, rather than a direct effect on spike initiation. A simplified multi-compartment circuit model of the pacemaker ensemble replicated this behavior. Antidromic activity disrupted slow wave oscillations by resetting the inward and outward current trajectories in each spike interval. Centrally generated bursting activity in turn suppressed peripheral spike initiation in an activity-dependent manner. Fast bursting eliminated peripheral spike initiation, while slower bursting allowed peripheral spike initiation to continue during the intervals between bursts. The suppression of peripheral spike initiation was associated with a small after-hyperpolarization in the sub-millivolt range. A realistic model of the PD axon replicated this behavior and showed that a sub-millivolt cumulative after-hyperpolarization across bursts was sufficient to eliminate peripheral spike initiation. This effect was based on the dynamic interaction between slow activity-dependent hyperpolarization caused by the Na+/K+-pump and inward rectification through the hyperpolarization-activated inward current, I h . These results demonstrate that interactions between different spike initiation sites based on spike propagation can shift the relative contributions of different types of activity in an activity-dependent manner. Therefore, distal axonal spike initiation can play an important role in shaping neural output, conditional on the relative level of centrally generated activity.

The complexity of small circuits: the stomatogastric nervous system.

The crustacean stomatogastric nervous system is a long-standing test bed for studies of circuit dynamics and neuromodulation. We give a brief update on the most recent work on this system, with an emphasis on the broader implications for understanding neural circuits. In particular, we focus on new findings underlining that different levels of dynamics taking place at different time scales all interact in multiple ways. Dynamics due to synaptic and intrinsic neuronal properties, neuromodulation, and long-term gene expression-dependent regulation are not independent, but influence each other. Extensive research on the stomatogastric system shows that these dynamic interactions convey robustness to circuit operation, while facilitating the flexibility of producing multiple circuit outputs.

Neuropeptide receptor transcript expression levels and magnitude of ionic current responses show cell type-specific differences in a small motor circuit.

We studied the relationship between neuropeptide receptor transcript expression and current responses in the stomatogastric ganglion (STG) of the crab, Cancer borealis. We identified a transcript with high sequence similarity to crustacean cardioactive peptide (CCAP) receptors in insects and mammalian neuropeptide S receptors. This transcript was expressed throughout the nervous system, consistent with the role of CCAP in a range of different behaviors. In the STG, single-cell qPCR showed expression in only a subset of neurons. This subset had previously been shown to respond to CCAP with the activation of a modulator-activated inward current (IMI), with one exception. In the one cell type that showed expression but no IMI responses, we found CCAP modulation of synaptic currents. Expression levels within STG neuron types were fairly variable, but significantly different between some neuron types. We tested the magnitude and concentration dependence of IMI responses to CCAP application in two identified neurons, the lateral pyloric (LP) and the inferior cardiac (IC) neurons. LP had several-fold higher expression and showed larger current responses. It also was more sensitive to low CCAP concentrations and showed saturation at lower concentrations, as sigmoid fits showed smaller EC50 values and steeper slopes. In addition, occlusion experiments with proctolin, a different neuropeptide converging onto IMI, showed that saturating concentrations of CCAP activated all available IMI in LP, but only approximately two-thirds in IC, the neuron with lower receptor transcript expression. The implications of these findings for comodulation are discussed.

Short-term synaptic plasticity compensates for variability in number of motor neurons at a neuromuscular junction.

We studied how similar postsynaptic responses are maintained in the face of interindividual variability in the number of presynaptic neurons. In the stomatogastric ganglion of the lobster, Homarus americanus, the pyloric (PY) neurons exist in variable numbers across animals. We show that each individual fiber of the stomach muscles innervated by PY neurons received synaptic input from all neurons present. We performed intracellular recordings of excitatory junction potentials (EJPs) in the muscle fibers to determine the consequences of differences in the number of motor neurons. Despite the variability in neuron number, the compound electrical response of muscle fibers to natural bursting input was similar across individuals. The similarity of total synaptic activation was not due to differences in the spiking activity of individual motor neurons across animals with different numbers of PY neurons. The amplitude of a unitary EJP in response to a single spike in a single motor neuron also did not depend on the number of PY neurons present. Consequently, the compound EJP in response to a single stimulus that activated all motor axons present was larger in individuals with more PY neurons. However, when axons were stimulated with trains of pulses mimicking bursting activity, EJPs facilitated more in individuals with fewer PY neurons. After a few stimuli, this resulted in depolarizations similar to the ones in individuals with more PY neurons. We interpret our findings as evidence that compensatory or homeostatic regulatory mechanisms can act on short-term synaptic dynamics instead of absolute synaptic strength.

The stomatogastric nervous system as a model for studying sensorimotor interactions in real-time closed-loop conditions.

The perception of proprioceptive signals that report the internal state of the body is one of the essential tasks of the nervous system and helps to continuously adapt body movements to changing circumstances. Despite the impact of proprioceptive feedback on motor activity it has rarely been studied in conditions in which motor output and sensory activity interact as they do in behaving animals, i.e., in closed-loop conditions. The interaction of motor and sensory activities, however, can create emergent properties that may govern the functional characteristics of the system. We here demonstrate a method to use a well-characterized model system for central pattern generation, the stomatogastric nervous system, for studying these properties in vitro. We created a real-time computer model of a single-cell muscle tendon organ in the gastric mill of the crab foregut that uses intracellular current injections to control the activity of the biological proprioceptor. The resulting motor output of a gastric mill motor neuron is then recorded intracellularly and fed into a simple muscle model consisting of a series of low-pass filters. The muscle output is used to activate a one-dimensional Hodgkin-Huxley type model of the muscle tendon organ in real-time, allowing closed-loop conditions. Model properties were either hand tuned to achieve the best match with data from semi-intact muscle preparations, or an exhaustive search was performed to determine the best set of parameters. We report the real-time capabilities of our models, its performance and its interaction with the biological motor system.

Regulation of motor patterns by the central spike-initiation zone of a sensory neuron.

Sensory feedback from muscles and peripheral sensors acts to initiate, tune or reshape motor activity according to the state of the body. Yet, sensory neurons often show low levels of activity even in the absence of sensory input. Here we examine the functional role of spontaneous low-frequency activity of such a sensory neuron. The anterior gastric receptor (AGR) is a muscle-tendon organ in the crab stomatogastric nervous system whose phasic activity shapes the well-characterized gastric mill (chewing) and pyloric (filtering) motor rhythms. Phasic activity is driven by a spike-initiation zone near the innervated muscle. We demonstrate that AGR possesses a second spike-initiation zone, which is located spatially distant from the innervated muscle in a central section of the axon. This initiation zone generates tonic activity and is responsible for the spontaneous activity of AGR in vivo, but does not code sensory information. Rather, it is sensitive to the neuromodulator octopamine. A computational model indicates that the activity at this initiation zone is not caused by excitatory input from another neuron, but generated intrinsically. This tonic activity is functionally relevant, because it modifies the activity state of the gastric mill motor circuit and changes the pyloric rhythm. The sensory function of AGR is not impaired as phasic activity suppresses spiking at the central initiation zone. Our results thus demonstrate that sensory neurons are not mere reporters of sensory signals. Neuromodulators can elicit non-sensory coding activity in these neurons that shapes the state of the motor system.

Regulation of motor pattern frequency by reversals in proprioceptive feedback.

Proprioceptive sensory feedback has important functions for motor pattern generation in which phasic negative and positive feedback is used to coordinate neural and musculoskeletal dynamics. Whether and how feedback sign regulates the motor patterns in behaviorally relevant closed-loop conditions has not been fully elucidated. We characterized the feedback provided by the anterior gastric receptor (AGR), a muscle tendon organ in the stomatogastric nervous system of the crab Cancer pagurus, to the gastric mill motor pattern in intact animals. AGR innervates the protractor muscles and was activated either during the protraction or retraction phase of the rhythm. Experiments with neuromuscular preparations imply that this was due to isometric contractions of the protractor muscles and their passive stretch by the antagonistic retractor muscles. As AGR excited the protractors and inhibited the retractors independently of the timing of its activation, the timing switch changed AGR feedback from positive to negative. We tested the effects of this change in feedback sign on the motor pattern in the isolated nervous system by activating AGR at the corresponding phases of the rhythm, using intracellular current injection. When AGR was activated during the protractor phase and provided positive feedback, it prolonged the burst activities of protractor and retractor neurons and slowed ongoing rhythms. When activated during the retraction phase and thus provided negative feedback, burst durations decreased and the rhythm cycle frequency increased. Our study thus shows that the cycle frequency of centrally generated activity patterns can be regulated by switching the sign of phasic proprioceptive feedback.