Functional consequences of neuropeptide and small-molecule co-transmission.

Colocalization of small-molecule and neuropeptide transmitters is common throughout the nervous system of all animals. The resulting co-transmission, which provides conjoint ionotropic ('classical') and metabotropic ('modulatory') actions, includes neuropeptide- specific aspects that are qualitatively different from those that result from metabotropic actions of small-molecule transmitter release. Here, we focus on the flexibility afforded to microcircuits by such co-transmission, using examples from various nervous systems. Insights from such studies indicate that co-transmission mediated even by a single neuron can configure microcircuit activity via an array of contributing mechanisms, operating on multiple timescales, to enhance both behavioural flexibility and robustness.

Mass spectrometry profiling and quantitation of changes in circulating hormones secreted over time in Cancer borealis hemolymph due to feeding behavior.

The crustacean stomatogastric ganglion (STG) is a valuable model for understanding circuit dynamics in neuroscience as it contains a small number of neurons, all easily distinguishable and most of which contribute to two complementary feeding-related neural circuits. These circuits are modulated by numerous neuropeptides, with many gaining access to the STG as hemolymph-transported hormones. Previous work characterized neuropeptides in the hemolymph of the crab Cancer borealis but was limited by low peptide abundance in the presence of a complex biological matrix and the propensity for rapid peptide degradation. To improve their detection, a data-independent acquisition (DIA) mass spectrometry (MS) method was implemented. This approach improved the number of neuropeptides detected by approximately twofold and showed greater reproducibility between experimental and biological replicates. This method was then used to profile neuropeptides at different stages of the feeding process, including hemolymph from crabs that were unfed, or 0 min, 15 min, 1 h, and 2 h post-feeding. The results show differences both in the presence and relative abundance of neuropeptides at the various time points. Additionally, 96 putative neuropeptide sequences were identified with de novo sequencing, indicating there may be more key modulators within this system than is currently known. These results suggest that a distinct cohort of neuropeptides provides modulation to the STG at different times in the feeding process, providing groundwork for targeted follow-up electrophysiological studies to better understand the functional role of circulating hormones in the neural basis of feeding behavior.

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.

Different microcircuit responses to comparable input from one versus both copies of an identified projection neuron.

Neuronal inputs to microcircuits are often present as multiple copies of apparently equivalent neurons. Thus far, however, little is known regarding the relative influence on microcircuit output of activating all or only some copies of such an input. We examine this issue in the crab (Cancer borealis) stomatogastric ganglion, where the gastric mill (chewing) microcircuit is activated by modulatory commissural neuron 1 (MCN1), a bilaterally paired modulatory projection neuron. Both MCN1s contain the same co-transmitters, influence the same gastric mill microcircuit neurons, can drive the biphasic gastric mill rhythm, and are co-activated by all identified MCN1-activating pathways. Here, we determine whether the gastric mill microcircuit response is equivalent when stimulating one or both MCN1s under conditions where the pair are matched to collectively fire at the same overall rate and pattern as single MCN1 stimulation. The dual MCN1 stimulations elicited more consistently coordinated rhythms, and these rhythms exhibited longer phases and cycle periods. These different outcomes from single and dual MCN1 stimulation may have resulted from the relatively modest, and equivalent, firing rate of the gastric mill neuron LG (lateral gastric) during each matched set of stimulations. The LG neuron-mediated, ionotropic inhibition of the MCN1 axon terminals is the trigger for the transition from the retraction to protraction phase. This LG neuron influence on MCN1 was more effective during the dual stimulations, where each MCN1 firing rate was half that occurring during the matched single stimulations. Thus, equivalent individual- and co-activation of a class of modulatory projection neurons does not necessarily drive equivalent microcircuit output.

Similarities and differences in circuit responses to applied Gly1-SIFamide and peptidergic (Gly1-SIFamide) neuron stimulation.

Microcircuit modulation by peptides is well established, but the cellular/synaptic mechanisms whereby identified neurons with identified peptide transmitters modulate microcircuits remain unknown for most systems. Here, we describe the distribution of GYRKPPFNGSIFamide (Gly1-SIFamide) immunoreactivity (Gly1-SIFamide-IR) in the stomatogastric nervous system (STNS) of the crab Cancer borealis and the Gly1-SIFamide actions on the two feeding-related circuits in the stomatogastric ganglion (STG). Gly1-SIFamide-IR localized to somata in the paired commissural ganglia (CoGs), two axons in the nerves connecting each CoG with the STG, and the CoG and STG neuropil. We identified one Gly1-SIFamide-IR projection neuron innervating the STG as the previously identified modulatory commissural neuron 5 (MCN5). Brief (~10 s) MCN5 stimulation excites some pyloric circuit neurons. We now find that bath applying Gly1-SIFamide to the isolated STG also enhanced pyloric rhythm activity and activated an imperfectly coordinated gastric mill rhythm that included unusually prolonged bursts in two circuit neurons [inferior cardiac (IC), lateral posterior gastric (LPG)]. Furthermore, longer duration (>30 s) MCN5 stimulation activated a Gly1-SIFamide-like gastric mill rhythm, including prolonged IC and LPG bursting. The prolonged LPG bursting decreased the coincidence of its activity with neurons to which it is electrically coupled. We also identified local circuit feedback onto the MCN5 axon terminals, which may contribute to some distinctions between the responses to MCN5 stimulation and Gly1-SIFamide application. Thus, MCN5 adds to the few identified projection neurons that modulate a well-defined circuit at least partly via an identified neuropeptide transmitter and provides an opportunity to study peptide regulation of electrical coupled neurons in a functional context. NEW & NOTEWORTHY Limited insight exists regarding how identified peptidergic neurons modulate microcircuits. We show that the modulatory projection neuron modulatory commissural neuron 5 (MCN5) is peptidergic, containing Gly1-SIFamide. MCN5 and Gly1-SIFamide elicit similar output from two well-defined motor circuits. Their distinct actions may result partly from circuit feedback onto the MCN5 axon terminals. Their similar actions include eliciting divergent activity patterns in normally coactive, electrically coupled neurons, providing an opportunity to examine peptide modulation of electrically coupled neurons in a functional context.

State-dependent sensorimotor gating in a rhythmic motor system.

Sensory feedback influences motor circuits and/or their projection neuron inputs to adjust ongoing motor activity, but its efficacy varies. Currently, less is known about regulation of sensory feedback onto projection neurons that control downstream motor circuits than about sensory regulation of the motor circuit neurons themselves. In this study, we tested whether sensory feedback onto projection neurons is sensitive only to activation of a motor system, or also to the modulatory state underlying that activation, using the crab Cancer borealis stomatogastric nervous system. We examined how proprioceptor neurons (gastropyloric receptors, GPRs) influence the gastric mill (chewing) circuit neurons and the projection neurons (MCN1, CPN2) that drive the gastric mill rhythm. During gastric mill rhythms triggered by the mechanosensory ventral cardiac neurons (VCNs), GPR was shown previously to influence gastric mill circuit neurons, but its excitation of MCN1/CPN2 was absent. In this study, we tested whether GPR effects on MCN1/CPN2 are also absent during gastric mill rhythms triggered by the peptidergic postoesophageal commissure (POC) neurons. The VCN and POC pathways both trigger lasting MCN1/CPN2 activation, but their distinct influence on circuit feedback to these neurons produces different gastric mill motor patterns. We show that GPR excites MCN1 and CPN2 during the POC-gastric mill rhythm, altering their firing rates and activity patterns. This action changes both phases of the POC-gastric mill rhythm, whereas GPR only alters one phase of the VCN-gastric mill rhythm. Thus sensory feedback to projection neurons can be gated as a function of the modulatory state of an active motor system, not simply switched on/off with the onset of motor activity.NEW & NOTEWORTHY Sensory feedback influences motor systems (i.e., motor circuits and their projection neuron inputs). However, whether regulation of sensory feedback to these projection neurons is consistent across different versions of the same motor pattern driven by the same motor system was not known. We found that gating of sensory feedback to projection neurons is determined by the modulatory state of the motor system, and not simply by whether the system is active or inactive.

Convergent neuromodulation onto a network neuron can have divergent effects at the network level.

Different neuromodulators often target the same ion channel. When such modulators act on different neuron types, this convergent action can enable a rhythmic network to produce distinct outputs. Less clear are the functional consequences when two neuromodulators influence the same ion channel in the same neuron. We examine the consequences of this seeming redundancy using a mathematical model of the crab gastric mill (chewing) network. This network is activated in vitro by the projection neuron MCN1, which elicits a half-center bursting oscillation between the reciprocally-inhibitory neurons LG and Int1. We focus on two neuropeptides which modulate this network, including a MCN1 neurotransmitter and the hormone crustacean cardioactive peptide (CCAP). Both activate the same voltage-gated current (I MI ) in the LG neuron. However, I MI-MCN1 , resulting from MCN1 released neuropeptide, has phasic dynamics in its maximal conductance due to LG presynaptic inhibition of MCN1, while I MI-CCAP retains the same maximal conductance in both phases of the gastric mill rhythm. Separation of time scales allows us to produce a 2D model from which phase plane analysis shows that, as in the biological system, I MI-MCN1 and I MI-CCAP primarily influence the durations of opposing phases of this rhythm. Furthermore, I MI-MCN1 influences the rhythmic output in a manner similar to the Int1-to-LG synapse, whereas I MI-CCAP has an influence similar to the LG-to-Int1 synapse. These results show that distinct neuromodulators which target the same voltage-gated ion channel in the same network neuron can nevertheless produce distinct effects at the network level, providing divergent neuromodulator actions on network activity.

Motor circuit-specific burst patterns drive different muscle and behavior patterns.

In the isolated CNS, different modulatory inputs can enable one motor network to generate multiple output patterns. Thus far, however, few studies have established whether different modulatory inputs also enable a defined network to drive distinct muscle and movement patterns in vivo, much as they enable these distinctions in behavioral studies. This possibility is not a foregone conclusion, because additional influences present in vivo (e.g., sensory feedback, hormonal modulation) could alter the motor patterns. Additionally, rhythmic neuronal activity can be transformed into sustained muscle contractions, particularly in systems with slow muscle dynamics, as in the crab (Cancer borealis) stomatogastric system used here. We assessed whether two different versions of the biphasic (protraction, retraction) gastric mill (chewing) rhythm, triggered in the isolated stomatogastric system by the modulatory ventral cardiac neurons (VCNs) and postoesophageal commissure (POC) neurons, drive different muscle and movement patterns. One distinction between these rhythms is that the lateral gastric (LG) protractor motor neuron generates tonic bursts during the VCN rhythm, whereas its POC-rhythm bursts are divided into fast, rhythmic burstlets. Intracellular muscle fiber recordings and tension measurements show that the LG-innervated muscles retain the distinct VCN-LG and POC-LG neuron burst structures. Moreover, endoscope video recordings in vivo, during VCN-triggered and POC-triggered chewing, show that the lateral teeth protraction movements exhibit the same, distinct protraction patterns generated by LG in the isolated nervous system. Thus, the multifunctional nature of an identified motor network in the isolated CNS can be preserved in vivo, where it drives different muscle activity and movement patterns.

Convergent rhythm generation from divergent cellular mechanisms.

Different modulatory inputs commonly elicit distinct rhythmic motor patterns from a central pattern generator (CPG), but they can instead elicit the same pattern. We are determining the rhythm-generating mechanisms in this latter situation, using the gastric mill (chewing) CPG in the crab (Cancer borealis) stomatogastric ganglion, where stimulating the projection neuron MCN1 (modulatory commissural neuron 1) or bath applying CabPK (C. borealis pyrokinin) peptide elicits the same gastric mill motor pattern, despite configuring different gastric mill circuits. In both cases, the core rhythm generator includes the same reciprocally inhibitory neurons LG (lateral gastric) and Int1 (interneuron 1), but the pyloric (food-filtering) circuit pacemaker neuron AB (anterior burster) is additionally necessary only for CabPK rhythm generation. MCN1 drives this rhythm generator by activating in the LG neuron the modulator-activated inward current (IMI), which waxes and wanes periodically due to phasic feedback inhibition of MCN1 transmitter release. Each buildup of IMI enables the LG neuron to generate a self-terminating burst and thereby alternate with Int1 activity. Here we establish that CabPK drives gastric mill rhythm generation by activating in the LG neuron IMI plus a slowly activating transient, low-threshold inward current (ITrans-LTS) that is voltage, time, and Ca(2+) dependent. Unlike MCN1, CabPK maintains a steady IMI activation, causing a subthreshold depolarization in LG that facilitates a periodic postinhibitory rebound burst caused by the regular buildup and decay of the availability of ITrans-LTS. Thus, different modulatory inputs can use different rhythm-generating mechanisms to drive the same neuronal rhythm. Additionally, the same ionic current (IMI) can play different roles under these different conditions, while different currents (IMI, ITrans-LTS) can play the same role.

Modulation of circuit feedback specifies motor circuit output.

Bidirectional communication (i.e., feedforward and feedback pathways) between functional levels is common in neural systems, but in most systems little is known regarding the function and modifiability of the feedback pathway. We are exploring this issue in the crab (Cancer borealis) stomatogastric nervous system by examining bidirectional communication between projection neurons and their target central pattern generator (CPG) circuit neurons. Specifically, we addressed the question of whether the peptidergic post-oesophageal commissure (POC) neurons trigger a specific gastric mill (chewing) motor pattern in the stomatogastric ganglion solely by activating projection neurons, or by additionally altering the strength of CPG feedback to these projection neurons. The POC-triggered gastric mill rhythm is shaped by feedback inhibition onto projection neurons from a CPG neuron. Here, we establish that POC stimulation triggers a long-lasting enhancement of feedback-mediated IPSC/Ps in the projection neurons, which persists for the same duration as POC-gastric mill rhythms. This strengthened CPG feedback appears to result from presynaptic modulation, because it also occurs in other projection neurons whose activity does not change after POC stimulation. To determine the function of this strengthened feedback synapse, we compared the influence of dynamic-clamp-injected feedback IPSPs of pre- and post-POC amplitude into a pivotal projection neuron after POC stimulation. Only the post-POC amplitude IPSPs elicited the POC-triggered activity pattern in this projection neuron and enabled full expression of the POC-gastric mill rhythm. Thus, the strength of CPG feedback to projection neurons is modifiable and can be instrumental to motor pattern selection.

The same core rhythm generator underlies different rhythmic motor patterns.

Rhythmically active motor circuits can generate different activity patterns in response to different inputs. In most systems, however, it is not known whether the same neurons generate the underlying rhythm for each different pattern. Thus far, information regarding the degree of conservation of rhythm generator neurons is limited to a few pacemaker-driven circuits, in most of which the core rhythm generator is unchanged across different output patterns. We are addressing this issue in the network-driven, gastric mill (chewing) circuit in the crab stomatogastric nervous system. We first establish that distinct gastric mill motor patterns are triggered by separate stimulation of two extrinsic input pathways, the ventral cardiac neurons (VCNs) and postoesophageal commissure (POC) neurons. A prominent feature that distinguishes these gastric mill motor patterns is the LG (lateral gastric) protractor motor neuron activity pattern, which is tonic during the VCN rhythm and exhibits fast rhythmic bursting during the POC rhythm. These two motor patterns also differed in their cycle period and some motor neuron phase relationships, duty cycles, and burst durations. Despite the POC and VCN motor patterns being distinct, rhythm generation during each motor pattern required the activity of the same two, reciprocally inhibitory gastric mill neurons [LG, Int1 (interneuron 1)]. Specifically, reversibly hyperpolarizing LG or Int1, but no other gastric mill neuron, delayed the start of the next gastric mill cycle until after the imposed hyperpolarization. Thus, the same circuit neurons can comprise the core rhythm generator during different versions of a network-driven rhythmic motor pattern.

Hormonal modulation of sensorimotor integration.

Neuronal circuits commonly receive simultaneous inputs from descending, ascending, and hormonal systems. Thus far, however, most such inputs have been studied individually to determine their influence on a given circuit. Here, we examine the integrated action of the hormone crustacean cardioactive peptide (CCAP) and the gastropyloric receptor (GPR) proprioceptor neuron on the biphasic gastric mill (chewing) rhythm driven by the projection neuron modulatory commissural neuron 1 (MCN1) in the isolated crab stomatogastric ganglion. In control saline, GPR stimulation selectively prolongs the gastric mill retractor phase, via presynaptic inhibition of MCN1. In the absence of GPR stimulation, CCAP does not alter retraction duration and modestly prolongs protraction. Here, we show, using computational modeling and dynamic-clamp manipulations, that the presence of CCAP weakens or eliminates the GPR effect on the gastric mill rhythm. This CCAP action results from its ability to activate the same modulator-activated conductance (G(MI)) as MCN1 in the gastric mill circuit neuron lateral gastric (LG). Because GPR prolongs retraction by weakening MCN1 activation of G(MI) in LG, the parallel G(MI) activation by CCAP reduces the impact of GPR regulation of this conductance. The CCAP-activated G(MI) thus counteracts the GPR-mediated decrease in the MCN1-activated G(MI) in LG and reduces the GPR ability to regulate the gastric mill rhythm. Consequently, although CCAP neither changes retraction duration nor alters GPR inhibition of MCN1, its activation of a modulator-activated conductance in a pivotal downstream circuit neuron enables CCAP to weaken or eliminate sensory regulation of motor circuit output.

Perturbation-specific responses by two neural circuits generating similar activity patterns.

A fundamental question in neuroscience is whether neuronal circuits with variable circuit parameters that produce similar outputs respond comparably to equivalent perturbations.1-4 Work on the pyloric rhythm of the crustacean stomatogastric ganglion (STG) showed that highly variable sets of intrinsic and synaptic conductances can generate similar circuit activity patterns.5-9 Importantly, in response to physiologically relevant perturbations, these disparate circuit solutions can respond robustly and reliably,10-12 but when exposed to extreme perturbations the underlying circuit parameter differences produce diverse patterns of disrupted activity.7,12,13 In this example, the pyloric circuit is unchanged; only the conductance values vary. In contrast, the gastric mill rhythm in the STG can be generated by distinct circuits when activated by different modulatory neurons and/or neuropeptides.14-21 Generally, these distinct circuits produce different gastric mill rhythms. However, the rhythms driven by stimulating modulatory commissural neuron 1 (MCN1) and bath-applying CabPK (Cancer borealis pyrokinin) peptide generate comparable output patterns, despite having distinct circuits that use separate cellular and synaptic mechanisms.22-25 Here, we use these two gastric mill circuits to determine whether such circuits respond comparably when challenged with persisting (hormonal: CCAP) or acute (sensory: GPR neuron) metabotropic influences. Surprisingly, the hormone-mediated action separates these two rhythms despite activating the same ionic current in the same circuit neuron during both rhythms, whereas the sensory neuron evokes comparable responses despite acting via different synapses during each rhythm. These results highlight the need for caution when inferring the circuit response to a perturbation when that circuit is not well defined physiologically.

Mass Spectrometry Quantification, Localization, and Discovery of Feeding-Related Neuropeptides in Cancer borealis.

The crab Cancer borealis nervous system is an important model for understanding neural circuit dynamics and modulation, but the identity of neuromodulatory substances and their influence on circuit dynamics in this system remains incomplete, particularly with respect to behavioral state-dependent modulation. Therefore, we used a multifaceted mass spectrometry (MS) method to identify neuropeptides that differentiate the unfed and fed states. Duplex stable isotope labeling revealed that the abundance of 80 of 278 identified neuropeptides was distinct in ganglia and/or neurohemal tissue from fed vs unfed animals. MS imaging revealed that an additional 7 and 11 neuropeptides exhibited altered spatial distributions in the brain and the neuroendocrine pericardial organs (POs), respectively, during these two feeding states. Furthermore, de novo sequencing yielded 69 newly identified putative neuropeptides that may influence feeding state-related neuromodulation. Two of these latter neuropeptides were determined to be upregulated in PO tissue from fed crabs, and one of these two peptides influenced heartbeat in ex vivo preparations. Overall, the results presented here identify a cohort of neuropeptides that are poised to influence feeding-related behaviors, providing valuable opportunities for future functional studies.

Parallel regulation of a modulator-activated current via distinct dynamics underlies comodulation of motor circuit output.

The cellular mechanisms underlying comodulation of neuronal networks are not elucidated in most systems. We are addressing this issue by determining the mechanism by which a peptide hormone, crustacean cardioactive peptide (CCAP), modulates the biphasic (protraction/retraction) gastric mill (chewing) rhythm driven by the projection neuron MCN1 in the crab stomatogastric ganglion. MCN1 activates this rhythm by slow peptidergic (CabTRP Ia) and fast GABAergic excitation of the reciprocally inhibitory central pattern generator neurons LG (protraction) and Int1 (retraction), respectively. MCN1 synaptic transmission is limited to the retraction phase, because LG inhibits MCN1 during protraction. Bath-applied CCAP also excites both LG and Int1, but selectively prolongs protraction. Here, we use computational modeling and dynamic-clamp manipulations to establish that CCAP prolongs the gastric mill protractor (LG) phase and maintains the retractor (Int1) phase duration by activating the same modulator-activated inward current (I(MI)) in LG as MCN1-released CabTRP Ia. However, the CCAP-activated current (I(MI-CCAP)) and MCN1-activated current (I(MI-MCN1)) exhibit distinct time courses in LG during protraction. This distinction results from I(MI-CCAP) being regulated only by postsynaptic voltage, whereas I(MI-MCN1) is also regulated by LG presynaptic inhibition of MCN1. Hence, without CCAP, retraction and protraction duration are determined by the time course of I(MI-MCN1) buildup and feedback inhibition-mediated decay, respectively, in LG. With I(MI-CCAP) continually present, the impact of the feedback inhibition is reduced, prolonging protraction and maintaining retraction duration. Thus, comodulation of rhythmic motor activity can result from convergent activation, via distinct dynamics, of a single voltage-dependent current.

State-dependent presynaptic inhibition regulates central pattern generator feedback to descending inputs.

Central pattern generators (CPGs) provide feedback to their projection neuron inputs. However, it is unknown whether this feedback is regulated and how that might shape CPG output. We are studying feedback from the pyloric CPG to identified projection neurons that regulate the gastric mill CPG, in the crab stomatogastric nervous system. Both CPGs are located in the stomatogastric ganglion (STG) and are influenced by projection neurons originating in the paired commissural ganglia (CoGs). Two extrinsic inputs [ventral cardiac neurons (VCNs) and postoesophageal commissure (POC) neurons] trigger distinct gastric mill rhythms despite acting via the same projection neurons [modulatory commissural neuron 1 (MCN1); commissural projection neuron 2 (CPN2)]. These projection neurons receive feedback inhibition from the pyloric CPG interneuron anterior burster (AB), resulting in their exhibiting pyloric-timed activity during the retraction phase of the VCN- and POC-triggered gastric mill rhythms. However, during the gastric mill protraction phase, MCN1/CPN2 exhibit pyloric-timed activity during the POC-triggered rhythm but fire tonically during the VCN-triggered rhythm. Here, we show that the latter, tonic activity pattern results from the elimination of AB inhibition of MCN1/CPN2, despite persistent AB actions within the STG and AB action potentials still propagating into each CoG. This loss of pyloric-timed AB input likely results from presynaptic inhibition of AB in each CoG because, when a secondary rhythmic AB burst initiation zone in the CoG is activated, the associated action potentials are selectively suppressed during the VCN protraction phase. Thus, rhythmic CPG feedback can be locally regulated, in a state-dependent manner, enabling the same projection neurons to drive multiple motor patterns from the same neuronal circuit.

Presynaptic inhibition selectively weakens peptidergic cotransmission in a small motor system.

The presence and influence of neurons containing multiple neurotransmitters is well established, including the ability of coreleased transmitters to influence the same or different postsynaptic targets. Little is known, however, regarding whether presynaptic regulation of multitransmitter neurons influences all transmission from these neurons. Using the identified neurons and motor networks in the crab stomatogastric ganglion, we document the ability of presynaptic inhibition to selectively inhibit peptidergic cotransmission. Specifically, we determine that the gastropyloric receptor (GPR) proprioceptor neuron uses presynaptic inhibition to selectively regulate peptidergic cotransmission from the axon terminals of MCN1, a projection neuron that drives the biphasic (retraction, protraction) gastric mill (chewing) rhythm. MCN1 drives this rhythm via fast GABAergic excitation of the retraction neuron Int1 and slow peptidergic excitation of the lateral gastric (LG) protraction neuron. We first demonstrate that GPR inhibition of the MCN1 axon terminals is serotonergic and then establish that this serotonergic inhibition weakens MCN1 peptidergic excitation of LG without altering MCN1 GABAergic excitation of Int1. At the circuit level, we show that this selective regulation of MCN1 peptidergic cotransmission is necessary for the normal GPR regulation of the gastric mill rhythm. This is the first demonstration, at the level of individual identified neurons, that a presynaptic input can selectively regulate a subset of coreleased transmitters. This selective regulation changes the balance of cotransmitter actions by the target multitransmitter neuron, thereby enabling this neuron to have state-dependent actions on its target network. This finding reveals additional flexibility afforded by the ability of neurons to corelease multiple neurotransmitters.

Mechanosensory gating of proprioceptor input to modulatory projection neurons.

Sensorimotor gating commonly occurs at sensory neuron synapses onto motor circuit neurons and motor neurons. Here, using the crab stomatogastric nervous system, we show that sensorimotor gating also occurs at the level of the projection neurons that activate motor circuits. We compared the influence of the gastro-pyloric receptor (GPR) muscle stretch-sensitive neuron on two projection neurons, modulatory commissural neuron 1 (MCN1) and commissural projection neuron 2 (CPN2), with and without a preceding activation of the mechanosensory ventral cardiac neurons (VCNs). MCN1 and CPN2 project from the paired commissural ganglia (CoGs) to the stomatogastric ganglion (STG), where they activate the gastric mill (chewing) motor circuit. When stimulated separately, the GPR and VCN neurons each elicit the gastric mill rhythm by coactivating MCN1 and CPN2. When GPR is instead stimulated during the VCN-gastric mill rhythm, it slows this rhythm. This effect results from a second GPR synapse onto MCN1 that presynaptically inhibits its STG terminals. Here, we show that, during the VCN-triggered rhythm, the GPR excitation of MCN1 and CPN2 in the CoGs is gated out, leaving only its influence in the STG. This gating effect appears to occur within the CoG and does not result from a ceiling effect on projection neuron firing frequency. Additionally, this gating action enables GPR to either activate rhythmic motor activity or act as a phasic sensorimotor feedback system. These results also indicate that the site of sensorimotor gating can occur at the level of the projection neurons that activate a motor circuit.

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.