Central nervous system projections to and from the commissural ganglion of the crab Cancer borealis

Higher-order inputs provide important regulatory control to motor circuits, but few cellular-level studies of such inputs have been performed. To begin studying higher-order neurons in an accessible model system, we have localized, in the supraesophageal ganglion (brain), neurons that are candidates for influencing the well-characterized motor circuits in the stomatogastric nervous system (STNS) of the crab Cancer borealis. The STNS is an extension of the central nervous system and includes four ganglia, within which are a set of motor circuits that regulate the ingestion and processing of food. These motor circuits are locally regulated by a set of modulatory neurons, most of which are located in the paired commissural ganglia (CoGs). These modulatory neurons are well-positioned to receive input from brain neurons because the circumesophageal commissures (CoCs) connect the brain with the CoGs. We have performed a series of CoC backfills to localize the brain neurons that are likely to innervate the CoGs and are, therefore, candidates for influencinng the STNS motor patterns. CoC backfill-labeled neuronal somata within the brain are clustered around a subset of anatomically defined neuropil regions. We have concomitantly localized many CoG neurons that project into the brain. This latter pathway presumably includes neurons that provide feedback regarding ongoing STNS activity. Interestingly, nearly all of these brain and CoG neurons project through the medial aspect of the CoC. This work provides an initial framework for future studies to determine the way that higher-order input regulates rhythmic motor patterns. This work was supported by a grant from the National Institute of Neurological Disorders and Strokes (NS42813 to M.P.N.) and a National Science Foundation Fellowship (DGE9616278 to M.S.K.).     

Premotor neurons in the feeding system of Aplysia californica

Central pattern generator (CPG) circuits control cyclic motor output underlying rhythmic behaviors. Although there have been extensive behavioral and cellular studies of food-induced feeding arousal as well as satiation in Aplysia, very little is known about the neuronal circuits controlling rhythmic consummatory feeding behavior. However, recent studies have identified premotor neurons that initiate and maintain buccal motor programs underlying ingestion and egestion in Aplysia. Other newly identified neurons receive synaptic input from feeding CPGs and in turn synapse with and control the output of buccal motor neurons. Some of these neurons and their effects within the buccal system are modulated by endogenous neuropeptides. With this information we can begin to understand how neuronal networks control buccal motor output and how their activity is modulated to produce flexibility in observed feeding behavior.     

Central pattern generators: some principles learned from invertebrate model systems.

1. Central pattern generators (CPGs) underlie a wide variety of rhythmic behaviours such as locomotion and respiration in most multi-cellular organisms. 2. The CPG's are capable of generating a patterned output without phasic sensory input. 3. The organization of the CPG is due to both intrinsic properties of the individual neurons and their network interactions. 4. To gain an understanding of the mechanisms which underlie rhythmicity a CPG has been reconstructed in culture. This will allow investigators to test directly the mechanisms underlying the generation of rhythmic output and will allow the direct testing of the mechanisms by which various modulators affect the CPG.     

Regulation of afferent transmission in the feeding circuitry of Aplysia

Although feeding in Aplysia is mediated by a central pattern generator (CPG), the activity of this CPG is modified by afferent input. To determine how afferent activity produces the widespread changes in motor programs that are necessary if behavior is to be modified, we have studied two classes of feeding sensory neurons. We have shown that afferent-induced changes in activity are widespread because sensory neurons make a number of synaptic connections. For example, sensory neurons make monosynaptic excitatory connections with feeding motor neurons. Sensori-motor transmission is, however, regulated so that changes in the periphery do not disrupt ongoing activity. This results from the fact that sensory neurons are also electrically coupled to feeding interneurons. During motor programs sensory neurons are, therefore, rhythmically depolarized via central input. These changes in membrane potential profoundly affect sensori-motor transmission. For example, changes in membrane potential alter spike propagation in sensory neurons so that spikes are only actively transmitted to particular output regions when it is behaviorally appropriate. To summarize, afferent activity alters motor output because sensory neurons make direct contact with motor neurons. Sensori-motor transmission is, however, centrally regulated so that changes in the periphery alter motor programs in a phase-dependent manner.     

Rhythmic behaviour and pattern-generating circuits in the locust: Key concepts and recent updates

There is growing recognition that rhythmic activity patterns are widespread in our brain and play an important role in all aspects of the functioning of our nervous system, from sensory integration to central processing and motor control. The study of the unique properties that enable central circuits to generate their rhythmic output in the absence of any patterned, sensory or descending, inputs, has been very rewarding in the relatively simple invertebrate preparations. The locust, specifically, is a remarkable example of an organism in which central pattern generator (CPG) networks have been suggested and studied in practically all aspects of their behaviour. Here we present an updated overview of the various rhythmic behaviours in the locust and aspects of their neural control. We focus on the fundamental concepts of multifunctional neuronal circuits, neural centre interactions and neuromodulation of CPG networks. We are certain that the very broad and solid knowledge base of locust rhythmic behaviour and pattern-generating circuits will continue to expand and further contribute to our understanding of the principles behind the functioning of the nervous system and, indeed, the brain.     

Anatomical Organization of Multiple Modulatory Inputs in a Rhythmic Motor System

In rhythmic motor systems, descending projection neuron inputs elicit distinct outputs from their target central pattern generator (CPG) circuits. Projection neuron activity is regulated by sensory inputs and inputs from other regions of the nervous system, relaying information about the current status of an organism. To gain insight into the organization of multiple inputs targeting a projection neuron, we used the identified neuron MCN1 in the stomatogastric nervous system of the crab, Cancer borealis. MCN1 originates in the commissural ganglion and projects to the stomatogastric ganglion (STG). MCN1 activity is differentially regulated by multiple inputs including neuroendocrine (POC) and proprioceptive (GPR) neurons, to elicit distinct outputs from CPG circuits in the STG. We asked whether these defined inputs are compact and spatially segregated or dispersed and overlapping relative to their target projection neuron. Immunocytochemical labeling, intracellular dye injection and three-dimensional (3D) confocal microscopy revealed overlap of MCN1 neurites and POC and GPR terminals. The POC neuron terminals form a defined neuroendocrine organ (anterior commissural organ: ACO) that utilizes peptidergic paracrine signaling to act on MCN1. The MCN1 arborization consistently coincided with the ACO structure, despite morphological variation between preparations. Contrary to a previous 2D study, our 3D analysis revealed that GPR axons did not terminate in a compact bundle, but arborized more extensively near MCN1, arguing against sparse connectivity of GPR onto MCN1. Consistent innervation patterns suggest that integration of the sensory GPR and peptidergic POC inputs occur through more distributed and more tightly constrained anatomical interactions with their common modulatory projection neuron target than anticipated.     

Neuropeptide modulation of microcircuits

Neuropeptides provide functional flexibility to microcircuits, their inputs and effectors by modulating presynaptic and postsynaptic properties and intrinsic currents. Recent studies have relied less on applied neuropeptide and more on their neural release. In rhythmically active microcircuits (central pattern generators, CPGs), recent studies show that neuropeptide modulation can enable particular activity patterns by organizing specific circuit motifs. Neuropeptides can also modify microcircuit output indirectly, by modulating circuit inputs. Recently elucidated consequences of neuropeptide modulation include changes in motor patterns and behavior, stabilization of rhythmic motor patterns and changes in CPG sensitivity to sensory input. One aspect of neuropeptide modulation that remains enigmatic is the presence of multiple peptide family members in the same nervous system and even the same neurons.     

Partly shared spinal cord networks for locomotion and scratching

Animals produce a variety of behaviors using a limited number of muscles and motor neurons. Rhythmic behaviors are often generated in basic form by networks of neurons within the central nervous system, or central pattern generators (CPGs). It is known from several invertebrates that different rhythmic behaviors involving the same muscles and motor neurons can be generated by a single CPG, multiple separate CPGs, or partly overlapping CPGs. Much less is known about how vertebrates generate multiple, rhythmic behaviors involving the same muscles. The spinal cord of limbed vertebrates contains CPGs for locomotion and multiple forms of scratching. We investigated the extent of sharing of CPGs for hind limb locomotion and for scratching. We used the spinal cord of adult red-eared turtles. Animals were immobilized to remove movement-related sensory feedback and were spinally transected to remove input from the brain. We took two approaches. First, we monitored individual spinal cord interneurons (i.e., neurons that are in between sensory neurons and motor neurons) during generation of each kind of rhythmic output of motor neurons (i.e., each motor pattern). Many spinal cord interneurons were rhythmically activated during the motor patterns for forward swimming and all three forms of scratching. Some of these scratch/swim interneurons had physiological and morphological properties consistent with their playing a role in the generation of motor patterns for all of these rhythmic behaviors. Other spinal cord interneurons, however, were rhythmically activated during scratching motor patterns but inhibited during swimming motor patterns. Thus, locomotion and scratching may be generated by partly shared spinal cord CPGs. Second, we delivered swim-evoking and scratch-evoking stimuli simultaneously and monitored the resulting motor patterns. Simultaneous stimulation could cause interactions of scratch inputs with subthreshold swim inputs to produce normal swimming, acceleration of the swimming rhythm, scratch-swim hybrid cycles, or complete cessation of the rhythm. The type of effect obtained depended on the level of swim-evoking stimulation. These effects suggest that swim-evoking and scratch-evoking inputs can interact strongly in the spinal cord to modify the rhythm and pattern of motor output. Collectively, the single-neuron recordings and the results of simultaneous stimulation suggest that important elements of the generation of rhythms and patterns are shared between locomotion and scratching in limbed vertebrates.     

Sprouting and connectivity of embryonic leech heart excitor (HE) motor neurons in the absence of their peripheral target

The rhythmic pumping of the hearts in the medicinal leech,Hirudo medicinalis, is neurogenic and mediated by a defined circuit involving identified interneurons in a central pattern generator (CPG) and segmentally iterated motor neurons that drive the heart muscle. During early embryogenesis, presumptive heart excitor (HE) motor neurons extend many axon branches into the body wall; they later innervate the heart while retracting the supernumerary peripheral axons, and only much later in development receive synaptic input from the central pattern generator (Jellies, Kopp and Bledsoe (1992)J. Exp. Biol., 170, 71–92.)- In this study, HE motor neurons were deprived of an early interaction with the heart by surgical ablation of a circumscribed portion of body wall including the heart primordium. Anatomical and electrophysiological data were obtained using intracellular techniques to examine the hypothesis that peripheral interactions with the developing heart provide instructive cues for the final differentiation of these neurons. Target-deprived HE motor neurons continued to extend multiple axons in ventral, lateral and dorsal body wall throughout late embryonic and into postembryonic stages and they extended anomalous axons within the CNS. This resembles the early embryonic growth of HE motor neurons before heart tube differentiation. Furthermore, HE motor neurons deprived of heart contact exhibited tonic activity similar to the situation during early development before they are contacted by the CPG interneurons. In contrast, sham-operated and contralateral HE motor neurons oscillated normally. These results suggest that heart tube contact is specifically required for at least some aspects of HE development and provide a framework in which to identify cell-cell interactions that are involved in matching neurons and targets to generate behaviorally relevant neural circuits.     

Associative neural network model for the generation of temporal patterns. Theory and application to central pattern generators.

Cyclic patterns of motor neuron activity are involved in the production of many rhythmic movements, such as walking, swimming, and scratching. These movements are controlled by neural circuits referred to as central pattern generators (CPGs). Some of these circuits function in the absence of both internal pacemakers and external feedback. We describe an associative neural network model whose dynamic behavior is similar to that of CPGs. The theory predicts the strength of all possible connections between pairs of neurons on the basis of the outputs of the CPG. It also allows the mean operating levels of the neurons to be deduced from the measured synaptic strengths between the pairs of neurons. We apply our theory to the CPG controlling escape swimming in the mollusk Tritonia diomedea. The basic rhythmic behavior is shown to be consistent with a simplified model that approximates neurons as threshold units and slow synaptic responses as elementary time delays. The model we describe may have relevance to other fixed action behaviors, as well as to the learning, recall, and recognition of temporally ordered information.     

Coping with variability in small neuronal networks

Experimental and corresponding modeling studies indicate that there is a 2- to 5-fold variation of intrinsic and synaptic parameters across animals while functional output is maintained. Here, we review experiments, using the heartbeat central pattern generator (CPG) in medicinal leeches, which explore the consequences of animal-to-animal variation in synaptic strength for coordinated motor output. We focus on a set of segmental heart motor neurons that all receive inhibitory synaptic input from the same four premotor interneurons. These four premotor inputs fire in a phase progression and the motor neurons also fire in a phase progression because of differences in synaptic strength profiles of the four inputs among segments. Our work tested the hypothesis that functional output is maintained in the face of animal-to-animal variation in the absolute strength of connections because relative strengths of the four inputs onto particular motor neurons is maintained across animals. Our experiments showed that relative strength is not strictly maintained across animals even as functional output is maintained, and animal-to-animal variations in strength of particular inputs do not correlate strongly with output phase. Further experiments measured the precise temporal pattern of the premotor inputs, the segmental synaptic strength profiles of their connections onto motor neurons, and the temporal pattern (phase progression) of those motor neurons all in the same animal for a series of 12 animals. The analysis of input and output in this sample of 12 individuals suggests that the number (four) of inputs to each motor neuron and the variability of the temporal pattern of input from the CPG across individuals weaken the influence of the strength of individual inputs. Moreover, the temporal pattern of the output varies as much across individuals as that of the input. Essentially, each animal arrives at a unique solution for how the network produces functional output.     

Neuropilar Projections of the Anterior Gastric Receptor Neuron in the Stomatogastric Ganglion of the Jonah Crab,Cancer Borealis

Sensory neurons provide important feedback to pattern-generating motor systems. In the crustacean stomatogastric nervous system (STNS), feedback from the anterior gastric receptor (AGR), a muscle receptor neuron, shapes the activity of motor circuits in the stomatogastric ganglion (STG) via polysynaptic pathways involving anterior ganglia. The AGR soma is located in the dorsal ventricular nerve posterior to the STG and it has been thought that its axon passes through the STG without making contacts. Using high-resolution confocal microscopy with dye-filled neurons, we show here that AGR from the crab Cancer borealis also has local projections within the STG and that these projections form candidate contact sites with STG motor neurons or with descending input fibers from other ganglia. We develop and exploit a new masking method that allows us to potentially separate presynaptic and postsynaptic staining of synaptic markers. The AGR processes in the STG show diversity in shape, number of branches and branching structure. The number of AGR projections in the STG ranges from one to three simple to multiply branched processes. The projections come in close contact with gastric motor neurons and descending neurons and may also be electrically coupled to other neurons of the STNS. Thus, in addition to well described long-loop pathways, it is possible that AGR is involved in integration and pattern regulation directly in the STG.     

Computer simulation study on central pattern generator: from biology to engineering

Central pattern generator (CPG) is a neuronal circuit in the nervous system that can generate oscillatory patterns for the rhythmic movements. Its simplified format, neural oscillator, is wildly adopted in engineering application. This paper explores the CPG from an integral view that combines biology and engineering together. Biological CPG and simplified CPG are both studied. Computer simulation reveals the mechanism of CPG. Some properties, such as effect of tonic input and sensory feedback, stable oscillation, robustness, entrainment etc., are further studied. The promising results provide foundation for the potential engineering application in future.     

Systems-level modeling of neuronal circuits for leech swimming

This paper describes a mathematical model of the neuronal central pattern generator (CPG) that controls the rhythmic body motion of the swimming leech. The systems approach is employed to capture the neuronal dynamics essential for generating coordinated oscillations of cell membrane potentials by a simple CPG architecture with a minimal number of parameters. Based on input/output data from physiological experiments, dynamical components (neurons and synaptic interactions) are first modeled individually and then integrated into a chain of nonlinear oscillators to form a CPG. We show through numerical simulations that the values of a few parameters can be estimated within physiologically reasonable ranges to achieve good fit of the data with respect to the phase, amplitude, and period. This parameter estimation leads to predictions regarding the synaptic coupling strength and intrinsic period gradient along the nerve cord, the latter of which agrees qualitatively with experimental observations.     

Neuromodulation for behavior in the locust frontal ganglion

Neuromodulators orchestrate complex behavioral routines by their multiple and combined effects on the nervous system. In the desert locust, Schistocerca gregaria, frontal ganglion neurons innervate foregut dilator muscles and play a key role in the control of foregut motor patterns. To further investigate the role of the frontal ganglion in locust behavior, we currently focus on the frontal ganglion central pattern generator as a target for neuromodulation. Application of octopamine, a well-studied insect neuromodulator, generated reversible disruption of frontal ganglion rhythmic activity. The threshold for the modulatory effects of octopamine was 10^–6 mol l^–1, and 10^–4 mol l^–1 always abolished the ongoing rhythm. In contrast to this straightforward modulation, allatostatin, previously reported to be a myoinhibitor of insect gut muscles, showed complex, tri-modal, dose-dependent effects on frontal ganglion rhythmic pattern. Using a novel cross-correlation analysis technique, we show that different allatostatin concentrations have very different effects not only on cycle period but also on temporal characteristics of the rhythmic bursts of action potentials. Allatostatin also altered the frontal ganglion rhythm in vivo. The analysis technique we introduce may be instrumental in the study of not fully characterized neural circuits and their modulation. The physiological significance of our results and the role of the modulators in locust behavior are discussed.Abbreviation CPG central pattern generator - FG frontal ganglion - JH juvenile hormone - STNS stomatogastric nervous system     

The cortex as a central pattern generator

Vertebrate spinal cord and brainstem central pattern generator (CPG) circuits share profound similarities with neocortical circuits. CPGs can produce meaningful functional output in the absence of sensory inputs. Neocortical circuits could be considered analogous to CPGs as they have rich spontaneous dynamics that, similar to CPGs, are powerfully modulated or engaged by sensory inputs, but can also generate output in their absence. We find compelling evidence for this argument at the anatomical, biophysical, developmental, dynamic and pathological levels of analysis. Although it is possible that cortical circuits are particularly plastic types of CPG ('learning CPGs'), we argue that present knowledge about CPGs is likely to foretell the basic principles of the organization and dynamic function of cortical circuits.     

Functional organization of a neural network for aversive olfactory learning in Caenorhabditis elegans

Many animals use their olfactory systems to learn to avoid dangers, but how neural circuits encode naive and learned olfactory preferences, and switch between those preferences, is poorly understood. Here, we map an olfactory network, from sensory input to motor output, which regulates the learned olfactory aversion of Caenorhabditis elegans for the smell of pathogenic bacteria. Naive animals prefer smells of pathogens but animals trained with pathogens lose this attraction. We find that two different neural circuits subserve these preferences, with one required for the naive preference and the other specifically for the learned preference. Calcium imaging and behavioral analysis reveal that the naive preference reflects the direct transduction of the activity of olfactory sensory neurons into motor response, whereas the learned preference involves modulations to signal transduction to downstream neurons to alter motor response. Thus, two different neural circuits regulate a behavioral switch between naive and learned olfactory preferences.     

Patterns of spinal sensory-motor connectivity prescribed by a dorsoventral positional template

Sensory-motor circuits in the spinal cord are constructed with a fine specificity that coordinates motor behavior, but the mechanisms that direct sensory connections with their motor neuron partners remain unclear. The dorsoventral settling position of motor pools in the spinal cord is known to match the distal-to-proximal position of their muscle targets in the limb, but the significance of invariant motor neuron positioning is unknown. An analysis of sensory-motor connectivity patterns in FoxP1 mutant mice, where motor neuron position has been scrambled, shows that the final pattern of sensory-motor connections is initiated by the projection of sensory axons to discrete dorsoventral domains of the spinal cord without regard for motor neuron subtype or, indeed, the presence of motor neurons. By implication, the clustering and dorsoventral settling position of motor neuron pools serve as a determinant of the pattern of sensory input specificity and thus motor coordination.     

Multi-neuronal refractory period adapts centrally generated behaviour to reward

Oscillating neuronal circuits, known as central pattern generators (CPGs), are responsible for generating rhythmic behaviours such as walking, breathing and chewing. The CPG model alone however does not account for the ability of animals to adapt their future behaviour to changes in the sensory environment that signal reward. Here, using multi-electrode array (MEA) recording in an established experimental model of centrally generated rhythmic behaviour we show that the feeding CPG of Lymnaea stagnalis is itself associated with another, and hitherto unidentified, oscillating neuronal population. This extra-CPG oscillator is characterised by high population-wide activity alternating with population-wide quiescence. During the quiescent periods the CPG is refractory to activation by food-associated stimuli. Furthermore, the duration of the refractory period predicts the timing of the next activation of the CPG, which may be minutes into the future. Rewarding food stimuli and dopamine accelerate the frequency of the extra-CPG oscillator and reduce the duration of its quiescent periods. These findings indicate that dopamine adapts future feeding behaviour to the availability of food by significantly reducing the refractory period of the brain's feeding circuitry.