A newly identified extrinsic input triggers a distinct gastric mill rhythm via activation of modulatory projection neurons.

Neuronal network flexibility enables animals to respond appropriately to changes in their internal and external states. We are using the isolated crab stomatogastric nervous system to determine how extrinsic inputs contribute to network flexibility. The stomatogastric system includes the well-characterized gastric mill (chewing) and pyloric (filtering of chewed food) motor circuits in the stomatogastric ganglion. Projection neurons with somata in the commissural ganglia (CoGs) regulate these rhythms. Previous work characterized a unique gastric mill rhythm that occurred spontaneously in some preparations, but whose origin remained undetermined. This rhythm includes a distinct protractor phase activity pattern, during which a key gastric mill circuit neuron (LG neuron) and the projection neurons MCN1 and CPN2 fire in a pyloric rhythm-timed activity pattern instead of the tonic firing pattern exhibited by these neurons during previously studied gastric mill rhythms. Here we identify a new extrinsic input, the post-oesophageal commissure (POC) neurons, relatively brief stimulation (30 s) of which triggers a long-lasting (tens of minutes) activation of this novel gastric mill rhythm at least in part via its lasting activation of MCN1 and CPN2. Immunocytochemical and electrophysiological data suggest that the POC neurons excite MCN1 and CPN2 by release of the neuropeptide Cancer borealis tachykinin-related peptide Ia (CabTRP Ia). These data further suggest that the CoG arborization of the POC neurons comprises the previously identified anterior commissural organ (ACO), a CabTRP Ia-containing neurohemal organ. This endocrine organ thus appears to also have paracrine actions, including activation of a novel and lasting gastric mill rhythm.

Convergent motor patterns from divergent circuits.

Neuromodulation changes the cellular and synaptic properties of neurons, thereby enabling individual neuronal circuits to generate multiple activity patterns. However, distinct modulatory inputs could conceivably also cona different motor circuits to generate similar activity patterns. Using the isolated stomatogastric ganglion (STG) of the crab Cancer borealis, we showed previously that pyrokinin (PK) peptides activate the gastric mill (chewing) rhythm without the participation of the projection neuron modulatory commissural neuron 1 (MCN1). MCN1, which does not contain the PK peptide, also activates the gastric mill rhythm and, at these times, is a gastric mill central pattern generator (CPG) neuron. Here, we show that the gastric mill rhythms elicited by PK superfusion and MCN1 stimulation in the isolated STG are comparable, in contrast to the distinct gastric mill rhythms elicited by other input pathways. We also identified several cellular and synaptic mechanisms underlying the PK- and MCN1-elicited gastric mill rhythms that are distinct, including additional differences in their core CPG neurons. For example, the presence of the inhibitory synapse from the pyloric pacemaker neuron anterior burster onto the gastric mill CPG was necessary only for generation of the PK-elicited gastric mill rhythm. Similarly, the dorsal gastric motor neuron regulated only the PK rhythm, apparently because of PK-mediated enhancement of its synaptic actions. Thus, we demonstrate that different modulatory inputs can elicit comparable, as well as distinct activity patterns from the same neuronal ensemble. Moreover, these comparable rhythms can result from distinct CPGs using overlapping, but distinct sets of cellular and synaptic mechanisms.

Modulation of rhythmic motor activity by pyrokinin peptides.

Pyrokinin (PK) peptides localize to the central and peripheral nervous systems of arthropods, but their actions in the CNS have yet to be studied in any species. Here, we identify PK peptide family members in the crab Cancer borealis and characterize their actions on the gastric mill (chewing) and pyloric (filtering) motor circuits in the stomatogastric ganglion (STG). We identified PK-like immunolabeling in the STG neuropil, in projection neuron inputs to this ganglion, and in the neuroendocrine pericardial organs. By combining MALDI mass spectrometry (MS) and ESI tandem MS techniques, we identified the amino acid sequences of two C. borealis pyrokinins (CabPK-I, CabPK-II). Both CabPKs contain the PK family-specific carboxy-terminal amino acid sequence (FXPRLamide). PK superfusion to the isolated STG had little influence on the pyloric rhythm but excited many gastric mill neurons and consistently activated the gastric mill rhythm. Both CabPKs had comparable actions in the STG and these actions were equivalent to those of Pevpyrokinin (shrimp) and Leucopyrokinin (cockroach). The PK-elicited gastric mill rhythm usually occurred without activation of the projection neuron MCN1. MCN1, which does not contain CabPKs, effectively drives the gastric mill rhythm and at such times is also a gastric mill central pattern generator (CPG) neuron. Because the PK-elicited gastric mill rhythm is independent of MCN1, the underlying core CPG of this rhythm is different from the one responsible for the MCN1-elicited rhythm. Thus neuromodulation, which commonly alters motor circuit output without changing the core CPG, can also change the composition of this core circuit.

Actions of kinin peptides in the stomatogastric ganglion of the crab Cancer borealis.

To fully understand neuronal network operation, the influence of all inputs onto that network must be characterized. As in most systems, many neuronal and hormonal pathways influence the multifunctional motor circuits of the crustacean stomatogastric ganglion (STG), but the actions of only some of them are known. Therefore, we characterized the influence of the kinin peptide family on the gastric mill (chewing) and pyloric (filtering of chewed food) motor circuits in the STG of the crab Cancer borealis. The kinins are myoactive in arthropods and they occur within the arthropod central nervous system (CNS), but their CNS actions are not well characterized in any species. The pevkinins were first identified in the shrimp Penaeus vannamei, but they have yet to be studied in the STG of any species. We identified kinin-like immunolabeling (KLI) in the pericardial organs (POs) in C. borealis, but there was no KLI within the STG. The POs are a major source of hormonal influence on the STG. Pevkinin peptides activated the pyloric circuit and they caused a modest increase in the speed of ongoing pyloric rhythms. This modest influence on cycle speed resulted in part from pevkinin excitation of the lateral pyloric neuron, whose strengthened inhibitory synapse onto the pyloric pacemaker neurons limited the pevkinin-mediated increase in cycle speed. The pevkinin excitation of the pyloric rhythm was not strong enough to interfere with the previously documented, gastric mill rhythm-mediated weakening of the pyloric rhythm. Pevkinin also had little influence on the gastric mill rhythm. These results indicate that the kinin peptides have distinct and selective modulatory actions on the pyloric rhythm.

Proprioceptor regulation of motor circuit activity by presynaptic inhibition of a modulatory projection neuron.

Phasically active sensory systems commonly influence rhythmic motor activity via synaptic actions on the relevant circuit and/or motor neurons. Using the crab stomatogastric nervous system (STNS), we identified a distinct synaptic action by which an identified proprioceptor, the gastropyloric muscle stretch receptor (GPR) neuron, regulates the gastric mill (chewing) motor rhythm. Previous work showed that rhythmically stimulating GPR in a gastric mill-like pattern, in the isolated STNS, elicits the gastric mill rhythm via its activation of two identified projection neurons, modulatory commissural neuron 1 (MCN1) and commissural projection neuron 2, in the commissural ganglia. Here, we determine how activation of GPR with a behaviorally appropriate pattern (active during each gastric mill retractor phase) influences an ongoing gastric mill rhythm via actions in the stomato gastric ganglion, where the gastric mill circuit is located. Stimulating GPR during each retractor phase selectively prolongs that phase and thereby slows the ongoing rhythm. This selective action on the retractor phase results from two distinct GPR actions. First, GPR presynaptically inhibits the axon terminals of MCN1, reducing MCN1 excitation of all gastric mill neurons. Second, GPR directly excites the retractor phase neurons. Because MCN1 transmitter release occurs during each retractor phase, these parallel GPR actions selectively reduce the buildup of excitatory drive to the protractor phase neurons, delaying each protractor burst. Thus, rhythmic proprioceptor feedback to a motor circuit can result from a global reduction in excitatory drive to that circuit, via presynaptic inhibition, coupled with a phase-specific excitatory input that prolongs the excited phase by delaying the onset of the subsequent phase.