Search

Oscillatory networks underlying rhythmic motor behaviors, and sensory and complex neural processing, are flexible, even in their neuronal composition. Neuromodulatory inputs enable neurons to switch participation between networks or participate in multiple networks simultaneously. Neuromodulation of internetwork synapses can both recruit and coordinate a switching neuron in a second network. We previously identified an example in which a neuron is recruited into dual-network activity via peptidergic modulation of intrinsic properties. We now ask whether the same neuropeptide also modulates internetwork synapses for internetwork coordination. The crab (Cancer borealis) stomatogastric nervous system contains two well-defined feeding-related networks (pyloric, food filtering, ∼1 Hz; gastric mill, food chewing, ∼0.1 Hz). The projection neuron MCN5 uses the neuropeptide Gly1-SIFamide to recruit the pyloric-only lateral posterior gastric (LPG) neuron into dual pyloric- plus gastric mill-timed bursting via modulation of LPG's intrinsic properties. Descending input is not required for a coordinated rhythm, thus intranetwork synapses between LPG and its second network must underlie coordination among these neurons. However, synapses between LPG and gastric mill neurons have not been documented. Using two-electrode voltage-clamp recordings, we found that graded synaptic currents between LPG and gastric mill neurons (lateral gastric, inferior cardiac, and dorsal gastric) were primarily negligible in saline, but were enhanced by Gly1-SIFamide. Furthermore, LPG and gastric mill neurons entrain each other during Gly1-SIFamide application, indicating bidirectional, functional connectivity. Thus, a neuropeptide mediates neuronal switching through parallel actions, modulating intrinsic properties for recruitment into a second network and as shown here, also modulating bidirectional internetwork synapses for coordination. NEW & NOTEWORTHY: Neuromodulation can enable neurons to simultaneously coordinate with separate networks. Both recruitment into, and coordination with, a second network can occur via modulation of internetwork synapses. Alternatively, recruitment can occur via modulation of intrinsic ionic currents. We find that the same neuropeptide previously determined to modulate intrinsic currents also modulates bidirectional internetwork synapses that are typically ineffective. Thus, complementary modulatory peptide actions enable recruitment and coordination of a neuron into a second network. [ABSTRACT FROM AUTHOR]

Neuromodulation lends flexibility to neural circuit operation but the general notion that different neuromodulators sculpt neural circuit activity into distinct and characteristic patterns is complicated by interindividual variability. In addition, some neuromodulators converge onto the same signaling pathways, with similar effects on neurons and synapses. We compared the effects of three neuropeptides on the rhythmic pyloric circuit in the stomatogastric ganglion of male crabs, Cancer borealis. Proctolin (PROC), crustacean cardioactive peptide (CCAP), and red pigment concentrating hormone (RPCH) activate the same modulatory inward current, IMI, and have convergent actions on synapses. However, while PROC targets all four neuron types in the core pyloric circuit, CCAP and RPCH target the same subset of only two neurons. After removal of spontaneous neuromodulator release, none of the neuropeptides restored the control cycle frequency, but all restored the relative timing between neuron types. Consequently, differences between neuropeptide effects were mainly found in the spiking activity of different neuron types. We performed statistical comparisons using the Euclidean distance in the multidimensional space of normalized output attributes to obtain a single measure of difference between modulatory states. Across preparations, the circuit output in PROC was distinguishable from CCAP and RPCH, but CCAP and RPCH were not distinguishable from each other. However, we argue that even between PROC and the other two neuropeptides, population data overlapped enough to prevent reliable identification of individual output patterns as characteristic for a specific neuropeptide. We confirmed this notion by showing that blind classifications by machine learning algorithms were only moderately successful. [ABSTRACT FROM AUTHOR]

Robustness of neuronal activity is a property necessary for a neuronal network to withstand perturbations, which may otherwise disrupt or destroy the system. The robustness of complex systems has been shown to depend on a number of features of the system, including morphology and heterogeneity of the activity of the component neurons, size of the networks, synaptic connectivity, and neuromodulation. The activity of small networks, such as the pyloric network of the crustacean stomatogastric nervous system, appears to be robust despite some of the factors not being consistent with the expected properties of complex systems, e.g., small size and homogeneity of the synaptic connections. The activity of the pyloric network has been shown to be stable and robust in a neuromodulatory state-dependent manner. When neuromodulatory inputs are severed, activity is initially disrupted, losing both stability and robustness. Over the long term, however, stable activity homeostatically recovers without the restoration of neuromodulatory input. The question we address in this study is whether robustness can also be restored as the network reorganizes itself to compensate for the loss of neuromodulatory input and recovers the lost activity. Here, we use temperature changes as a perturbation to probe the robustness of the network’s activity. We develop a simple metric of robustness, i.e., the variances of the network phase relationships, and show that robustness is indeed restored simultaneously along with its stable network activity, indicating that, whatever the reorganization of the network entails, it is deep enough also to restore this important property.

Recently, activity has been proposed as a primary feedback mechanism used by continuously bursting neurons to coordinate ion channel mRNA relationships that underlie stable output. However, some neuron types only have intermittent periods of activity and so may require alternative mechanisms that induce and constrain the appropriate ion channel profile in different states of activity. To address this, we used the pyloric dilator (PD; constitutively active) and the lateral gastric (LG; periodically active) neurons of the stomatogastric ganglion (STG) of the crustacean Cancer borealis. We experimentally stimulated descending inputs to the STG to cause release of neuromodulators known to elicit the active state of LG neurons and quantified the mRNA abundances and pairwise relationships of 11 voltage-gated ion channels in active and silent LG neurons. The same stimulus does not significantly alter PD activity. Activation of LG upregulated ion channel mRNAs and lead to a greater number of positively correlated pairwise channel mRNA relationships. Conversely, this stimulus did not induce major changes in ion channel mRNA abundances and relationships of PD cells, suggesting their ongoing activity is sufficient to maintain channel mRNA relationships even under changing modulatory conditions. In addition, we found that ion channel mRNA correlations induced by the active state of LG are influenced by a combination of activity- and neuromodulator-dependent feedback mechanisms. Interestingly, some of these same correlations are maintained by distinct mechanisms in PD, suggesting that these motor networks use distinct feedback mechanisms to coordinate the same mRNA relationships across neuron types. NEW & NOTEWORTHY Neurons use various feedback mechanisms to adjust and maintain their output. Here, we demonstrate that different neurons within the same network can use distinct signaling mechanisms to regulate the same ion channel mRNA relationships. [ABSTRACT FROM AUTHOR]

Neurons in oscillatory networks often exhibit membrane potential resonance, a peak impedance at a non-zero input frequency. In electrically coupled oscillatory networks, the coupling coefficient (the ratio of post- and prejunctional voltage responses) could also show resonance. Such coupling resonance may emerge from the interaction between the coupling current and resonance properties of the coupled neurons, but this relationship has not been clearly described. Additionally, it is unknown if the gap-junction mediated electrical coupling conductance may have frequency dependence. We examined these questions by recording a pair of electrically coupled neurons in the oscillatory pyloric network of the crab Cancer borealis. We performed dual current- and voltageclamp recordings and quantified the frequency preference of the coupled neurons, the coupling coefficient, the electrical conductance, and the postjunctional neuronal response. We found that all components exhibit frequency selectivity, but with distinct preferred frequencies. Mathematical and computational analysis showed that membrane potential resonance of the postjunctional neuron was sufficient to give rise to resonance properties of the coupling coefficient, but not the coupling conductance. A distinct coupling conductance resonance frequency therefore emerges either from other circuit components or from the gating properties of the gap junctions. Finally, to explore the functional effect of the resonance of the coupling conductance, we examined its role in synchronizing neuronal the activities of electrically coupled bursting model neurons. Together, our findings elucidate factors that produce electrical coupling resonance and the function of this resonance in oscillatory networks. [ABSTRACT FROM AUTHOR]

Neural network flexibility includes changes in neuronal participation between networks, such as the switching of neurons between single- and dual-network activity. We previously identified a neuron that is recruited to burst in time with an additional network via modulation of its intrinsic membrane properties, instead of being recruited synaptically into the second network. However, the modulated intrinsic properties were not determined. Here, we use small networks in the Jonah crab (Cancer borealis) stomatogastric nervous system (STNS) to examine modulation of intrinsic properties underlying neuropeptide (Gly1-SIFamide)- elicited neuronal switching. The lateral posterior gastric neuron (LPG) switches from exclusive participation in the fast pyloric (~1 Hz) network, due to electrical coupling, to dual-network activity that includes periodic escapes from the fast rhythm via intrinsically generated oscillations at the slower gastric mill network frequency (~0.1 Hz). We isolated LPG from both networks by pharmacology and hyperpolarizing current injection. Gly¹-SIFamide increased LPG intrinsic excitability and rebound from inhibition and decreased spike frequency adaptation, which can all contribute to intrinsic bursting. Using ion substitution and channel blockers, we found that a hyperpolarization-activated current, a persistent sodium current, and calcium or calcium-related current(s) appear to be primary contributors to Gly1-SIFamide-elicited LPG intrinsic bursting. However, this intrinsic bursting was more sensitive to blocking currents when LPG received rhythmic electrical coupling input from the fast network than in the isolated condition. Overall, a switch from single- to dual-network activity can involve modulation of multiple intrinsic properties, while synaptic input from a second network can shape the contributions of these properties. [ABSTRACT FROM AUTHOR]

Relationships among membrane currents allow central pattern generator (CPG) neurons to reliably drive motor programs. We hypothesize that continually active CPG neurons utilize activity-dependent feedback to correlate expression of ion channel genes to balance essential membrane currents. However, episodically activated neurons experience absences of activity-dependent feedback and, thus, presumably employ other strategies to coregulate the balance of ionic currents necessary to generate appropriate output after periods of quiescence. To investigate this, we compared continually active pyloric dilator (PD) neurons with episodically active lateral gastric (LG) CPG neurons of the stomatogastric ganglion (STG) in male Cancer borealis crabs. After experimentally activating LG for 8 h, we measured three potassium currents and abundances of their corresponding channel mRNAs. We found that ionic current relationships were correlated in LG's silent state, but ion channel mRNA relationships were correlated in the active state. In continuously active PD neurons, ion channel mRNAs and ionic currents are simultaneously correlated. Therefore, two distinct relationships exist between channel mRNA abundance and the ionic current encoded in these cells: in PD, a direct correlation exists between Shal channel mRNA levels and the A-type potassium current it carries. Conversely, such channel mRNA-current relationships are not detected and appear to be temporally uncoupled in LG neurons. Our results suggest that ongoing feedback maintains membrane current and channel mRNA relationships in continually active PD neurons, while in LG neurons, episodic activity serves to establish channel mRNA relationships necessary to produce the ionic current profile necessary for the next bout of activity., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Viteri et al.)

Like their chemical counterparts, electrical synapses show complex dynamics such as rectification and voltage dependence that interact with other electrical processes in neurons. The consequences arising from these interactions for the electrical behavior of the synapse, and the dynamics they create, remain largely unexplored. Using a voltage-dependent electrical synapse between a descending modulatory projection neuron (MCN1) and a motor neuron (LG) in the crustacean stomatogastric ganglion, we find that the influence of the hyperpolarization-activated inward current (Ih) is critical to the function of the electrical synapse. When we blocked Ih with CsCl, the apparent voltage dependence of the electrical synapse shifted by 18.7 mV to more hyperpolarized voltages, placing the dynamic range of the electrical synapse outside of the range of voltages used by the LG motor neuron (-60.2 mV to -44.9 mV). With dual electrode current- and voltage-clamp recordings, we demonstrate that this voltage shift is not due to a change in the properties of the gap junction itself, but is a result of a sustained effect of Ih on the presynaptic MCN1 axon terminal membrane potential. Ih-induced depolarization of the axon terminal membrane potential increased the electrical postsynaptic potentials and currents. With Ih present, the axon terminal resting membrane potential is depolarized, shifting the dynamic range of the electrical synapse toward the functional range of the motor neuron. We thus demonstrate that the function of an electrical synapse is critically influenced by a voltage-dependent ionic current (Ih). [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Schneider et al.)

Peptide neuromodulation has been implicated to shield neuronal activity from acute temperature changes that can otherwise lead to loss of motor control or failure of vital behaviors. However, the cellular actions neuropeptides elicit to support temperature- robust activity remain unknown. Here, we find that peptide neuromodulation restores rhythmic bursting in temperature-compromised central pattern generator (CPG) neurons by counteracting membrane shunt and increasing dendritic electrical spread. We show that acutely rising temperatures reduced spike generation and interrupted ongoing rhythmic motor activity in the crustacean gastric mill CPG. Neuronal release and extrinsic application of Cancer borealis tachykinin-related peptide Ia (CabTRP Ia), a substance-P-related peptide, restored rhythmic activity. Warming led to a significant decrease in membrane resistance and a shunting of the dendritic signals in the main gastric mill CPG neuron. Using a combination of fluorescent calcium imaging and electrophysiology, we observed that postsynaptic potentials and antidromic action potentials propagated less far within the dendritic neuropil as the system warmed. In the presence of CabTRP Ia, membrane shunt decreased and both postsynaptic potentials and antidromic action potentials propagated farther. At elevated temperatures, CabTRP Ia restored dendritic electrical spread or extended it beyond that at cold temperatures. Selective introduction of the CabTRP Ia conductance using a dynamic clamp demonstrated that the CabTRP Ia voltage-dependent conductance was sufficient to restore rhythmic bursting. Our findings demonstrate that a substance-P-related neuropeptide can boost dendritic electrical spread to maintain neuronal activity when perturbed and reveals key neurophysiological components of neuropeptide actions that support pattern generation in temperature-compromised conditions. [ABSTRACT FROM AUTHOR]

In oscillatory systems, neuronal activity phase is often independent of network frequency. Such phase maintenance requires adjustment of synaptic input with network frequency, a relationship that we explored using the crab, Cancer borealis, pyloric network. The burst phase of pyloric neurons is relatively constant despite a > two fold variation in network frequency. We used noise input to characterize how input shape influences burst delay of a pyloric neuron, and then used dynamic clamp to examine how burst phase depends on the period, amplitude, duration, and shape of rhythmic synaptic input. Phase constancy across a range of periods required a proportional increase of synaptic duration with period. However, phase maintenance was also promoted by an increase of amplitude and peak phase of synaptic input with period. Mathematical analysis shows how short-term synaptic plasticity can coordinately change amplitude and peak phase to maximize the range of periods over which phase constancy is achieved.

Understanding circuit organization depends on identification of cell types. Recent advances in transcriptional profiling methods have enabled classification of cell types by their gene expression. While exceptionally powerful and high throughput, the ground-truth validation of these methods is difficult: If cell type is unknown, how does one assess whether a given analysis accurately captures neuronal identity? To shed light on the capabilities and limitations of solely using transcriptional profiling for cell-type classification, we performed 2 forms of transcriptional profiling--RNA-seq and quantitative RT-PCR, in single, unambiguously identified neurons from 2 small crustacean neuronal networks: The stomatogastric and cardiac ganglia. We then combined our knowledge of cell type with unbiased clustering analyses and supervised machine learning to determine how accurately functionally defined neuron types can be classified by expression profile alone. The results demonstrate that expression profile is able to capture neuronal identity most accurately when combined with multimodal information that allows for post hoc grouping, so analysis can proceed from a supervised perspective. Solely unsupervised clustering can lead to misidentification and an inability to distinguish between 2 or more cell types. Therefore, this study supports the general utility of cell identification by transcriptional profiling, but adds a caution: It is difficult or impossible to know under what conditions transcriptional profiling alone is capable of assigning cell identity. Only by combining multiple modalities of information such as physiology, morphology, or innervation target can neuronal identity be unambiguously determined. [ABSTRACT FROM AUTHOR]

Many neurons receive synchronous input from heterogeneous presynaptic neurons with distinct properties. An instructive example is the crustacean stomatogastric pyloric circuit pacemaker group, consisting of the anterior burster (AB) and pyloric dilator (PD) neurons, which are active synchronously and exert a combined synaptic action on most pyloric follower neurons. Previous studies in lobster have indicated that AB is glutamatergic, whereas PD is cholinergic. However, although the stomatogastric system of the crab Cancer borealis has become a preferred system for exploration of cellular and synaptic basis of circuit dynamics, the pacemaker synaptic output has not been carefully analyzed in this species. We examined the synaptic properties of these neurons using a combination of single-cell mRNA analysis, electrophysiology, and pharmacology. The crab PD neuron expresses high levels of choline acetyltransferase and the vesicular acetylcholine transporter mRNAs, hallmarks of cholinergic neurons. In contrast, the AB neuron expresses neither cholinergic marker but expresses high levels of vesicular glutamate transporter mRNA, consistent with a glutamatergic phenotype. Notably, in the combined synapses to follower neurons, 70-75% of the total current was blocked by putative glutamatergic blockers, but short-term synaptic plasticity remained unchanged, and although the total pacemaker current in two follower neuron types was different, this difference did not contribute to the phasing of the follower neurons. These findings provide a guide for similar explorations of heterogeneous synaptic connections in other systems and a baseline in this system for the exploration of the differential influence of neuromodulators.NEW & NOTEWORTHY The pacemaker-driven pyloric circuit of the Jonah crab stomatogastric nervous system is a well-studied model system for exploring circuit dynamics and neuromodulation, yet the understanding of the synaptic properties of the two pacemaker neuron types is based on older analyses in other species. We use single-cell PCR and electrophysiology to explore the neurotransmitters used by the pacemaker neurons and their distinct contribution to the combined synaptic potentials. [ABSTRACT FROM AUTHOR]

Neurons operate within defined activity limits, and feedback control mechanisms dynamically tune ionic currents to maintain this optimal range. This study describes a novel, rapid feedback mechanism that uses SUMOylation to continuously adjust ionic current densities according to changes in activity. Small ubiquitin-like modifier (SUMO) is a peptide that can be post-translationally conjugated to ion channels to influence their surface expression and biophysical properties. Neuronal activity can regulate the extent of protein SUMOylation. This study on the single, unambiguously identifiable lateral pyloric neuron (LP), a component of the pyloric network in the stomatogastric nervous system of male and female spiny lobsters (Panulirus in te rru p ts), focused on dynamic SUMOylation in the context of activity homeostasis. There were four major findings: First, neuronal activity adjusted the balance between SUMO conjugation and deconjugation to continuously and bidirectionally fine-tune the densities of two opposing conductances: the hyperpolarization activated current (Ih) and the transient potassium current (IA). Second, tonic 5 nM dopamine (DA) gated activity-dependent SUMOylation to permit and prevent activity-dependent regulation of Ih and IA, respectively. Third, DA-gated, activity-dependent SUMOylation contributed to a feedback mechanism that restored the timing and duration of LP activity during prolonged modulation by 5 ptM DA, which initially altered these and other activity features. Fourth, DA modulatory and metamoduatory (gating) effects were tailored to simultaneously alter and stabilize neuronal output. Our findings suggest that modulatory tone may select a subset of rapid activitydependent mechanisms from a larger menu to achieve homeostasis under varying conditions. [ABSTRACT FROM AUTHOR]

Recent genomic/transcriptomic studies have identified a novel peptide family whose members share the carboxyl terminal sequence -GSEFLamide. However, the presence/identity of the predicted isoforms of this peptide group have yet to be confirmed biochemically, and no physiological function has yet been ascribed to any member of this peptide family. To determine the extent to which GSEFLamides are conserved within the Arthropoda, we searched publicly accessible databases for genomic/transcriptomic evidence of their presence. GSEFLamides appear to be highly conserved within the Arthropoda, with the possible exception of the Insecta, in which sequence evidence was limited to the more basal orders. One crustacean in which GSEFLamides have been predicted using transcriptomics is the lobster, Homarus americanus. Expression of the previously published transcriptome-derived sequences was confirmed by reverse transcription (RT)-PCR of brain and eyestalk ganglia cDNAs; mass spectral analyses confirmed the presence of all six of the predicted GSEFLamide isoforms - IGSEFLamide, MGSEFLamide, AMGSEFLamide, VMGSEFLamide, ALGSEFLamide and AVGSEFLamide - in H. americanus brain extracts. AMGSEFLamide, of which there are multiple copies in the cloned transcripts, was the most abundant isoform detected in the brain. Because the GSEFLamides are present in the lobster nervous system, we hypothesized that they might function as neuromodulators, as is common for neuropeptides. We thus asked whether AMGSEFLamide modulates the rhythmic outputs of the cardiac ganglion and the stomatogastric ganglion. Physiological recordings showed that AMGSEFLamide potently modulates the motor patterns produced by both ganglia, suggesting that the GSEFLamides may serve as important and conserved modulators of rhythmic motor activity in arthropods. [ABSTRACT FROM AUTHOR]

Robustness of neuronal activity is a property necessary for a neuronal network to withstand perturbations, which may otherwise disrupt or destroy the system. The robustness of complex systems has been shown to depend on a number of features of the system, including morphology and heterogeneity of the activity of the component neurons, size of the networks, synaptic connectivity, and neuromodulation. The activity of small networks, such as the pyloric network of the crustacean stomatogastric nervous system, appears to be robust despite some of the factors not being consistent with the expected properties of complex systems, e.g., small size and homogeneity of the synaptic connections. The activity of the pyloric network has been shown to be stable and robust in a neuromodulatory state-dependent manner. When neuromodulatory inputs are severed, activity is initially disrupted, losing both stability and robustness. Over the long term, however, stable activity homeostatically recovers without the restoration of neuromodulatory input. The question we address in this study is whether robustness can also be restored as the network reorganizes itself to compensate for the loss of neuromodulatory input and recovers the lost activity. Here, we use temperature changes as a perturbation to probe the robustness of the network's activity. We develop a simple metric of robustness, i.e., the variances of the network phase relationships, and show that robustness is indeed restored simultaneously along with its stable network activity, indicating that, whatever the reorganization of the network entails, it is deep enough also to restore this important property., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 More-Potdar and Golowasch.)

Choline chloride is often, and Nmethyl-D-glucamine (NMDG) sometimes, used to replace sodium chloride in studies of sodium-activated potassium channels. Given the high concentrations used in sodium replacement protocols, it is essential to test that it is not the replacement substances themselves, as opposed to the lack of sodium, that cause any observed effects. We therefore compared, in lobster stomatogastric neurons and leech Retzius cells, the effects of applying salines in which choline chloride replaced sodium chloride, and in which choline hydroxide or sucrose was added to normal saline. We also tested, in stomatogastric neurons, the effect of adding NMDG to normal saline. These protocols allowed us to measure the direct effects (i.e., effects not due to changes in sodium concentration or saline osmolarity or ionic strength) of choline on stomatogastric and leech currents, and of NMDG on stomatogastric currents. Choline directly reduced transient and sustained depolarization-activated outward currents in both species, and NMDG directly reduced transient depolarization-activated outward currents in stomatogastric neurons. Experiments with lower choline concentrations showed that adding as little as 150 mM (stomatogastric) or 5 mM (leech) choline reduced at least some depolarization-activated outward currents. Reductions in outward current with choline chloride or NMDG replacement alone are thus not evidence of sodium-activated potassium currents. NEW & NOTEWORTHY We show that choline or N-methyl-Dglucamine (NMDG) directly (i.e., not due to changes in extracellular sodium) decrease outward currents. Prior work studying sodium-activated potassium channels in which sodium was replaced with choline or NMDG without an addition control may therefore be artifactual. [ABSTRACT FROM AUTHOR]

Multiple neuromodulators act in concert to shape the properties of neural circuits. Different neuromodulators usually activate distinct receptors but can have overlapping targets. Therefore, circuit output depends on neuromodulator interactions at shared targets, a poorly understood process.Weexplored quantitative rules of co-modulation of two principal targets of neuromodulation: synapses and voltagegated ionic currents. In the stomatogastric ganglion of the male crab Cancer borealis, the neuropeptides proctolin (Proc) and the crustacean cardioactive peptide (CCAP) modulate synapses of the pyloric circuit and activate a voltage-gated current (IMI) in multiple neurons. We examined the validity of a simple dose-dependent quantitative rule, that co-modulation by Proc and CCAP is predicted by the linear sum of the individual effects of each modulator up to saturation.Wefound that this rule is valid for co-modulation of synapses, but not for the activation of IMI, in which co-modulation was sublinear. The predictions for the co-modulation of IMI activation were greatly improved if we assumed that the intracellular pathways activated by two peptide receptors inhibit one another. These findings suggest that the pathways activated by two neuromodulators could have distinct interactions, leading to distinct co-modulation rules for different targets even in the same neuron. Given the evolutionary conservation of neuromodulator receptors and signaling pathways, such distinct rules for co-modulation of different targets are likely to be common across neuronal circuits. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The crustacean stomatogastric nervous system is a classic model for understanding the effects of modulating ionic currents and synapses at both the cell and network levels. The stomatogastric ganglion in this system contains two distinct central pattern generators: a slow gastric mill network that generates flexible rhythmic outputs (8-20 s) and is often silent, and a fast pyloric network that generates more consistent rhythmic outputs (0.5-2 s) and is always active in vitro. Different ionic conductances contribute to the properties of individual neurons and therefore to the overall dynamics of the pyloric and gastric mill networks. However, the contributions of ionic currents to different dynamics between the pyloric and gastric mill networks are not well understood. The goal of this study is to evaluate how changes in outward potassium current ( I ) in the stomatogastric ganglion affect the dynamics of the pyloric and gastric mill rhythms by interfering with normal I activity. We bath-applied the specific I blocker 4-aminopyridine to reduce I 's effect in the stomatogastric ganglion in vitro and evaluated quantitatively the changes in both rhythms. We found that blocking I in the stomatogastric ganglion alters the synchronization between pyloric neurons, and consistently activates the gastric mill rhythm in quiescent preparations. [ABSTRACT FROM AUTHOR]

Motor neurons of the crustacean cardiac ganglion generate virtually identical, synchronized output despite the fact that each neuron uses distinct conductance magnitudes. As a result of this variability, manipulations that target ionic conductances have distinct effects on neurons within the same ganglion, disrupting synchronized motor neuron output that is necessary for proper cardiac function. We hypothesized that robustness in network output is accomplished via plasticity that counters such destabilizing influences. By blocking high-threshold K+ conductances in motor neurons within the ongoing cardiac network, we discovered that compensation both resynchronized the network and helped restore excitability. Using model findings to guide experimentation, we determined that compensatory increases of both GA and electrical coupling restored function in the network. This is one of the first direct demonstrations of the physiological regulation of coupling conductance in a compensatory context, and of synergistic plasticity across cell- and network-level mechanisms in the restoration of output.

Background: Crustaceans have been studied extensively as model systems for nervous system function from single neuron properties to behavior. However, lack of molecular sequence information and tools have slowed the adoption of these physiological systems as molecular model systems. In this study, we sequenced and performed de novo assembly for the nervous system transcriptomes of two decapod crustaceans: the Jonah crab (Cancer borealis) and the American lobster (Homarus americanus). Results: Forty-two thousand, seven hundred sixty-six and sixty thousand, two hundred seventy-three contigs were assembled from C. borealis and H. americanus respectively, representing 9,489 and 11,061 unique coding sequences. From these transcripts, genes associated with neural function were identified and manually curated to produce a characterization of multiple gene families important for nervous system function. This included genes for 34 distinct ion channel types, 17 biogenic amine and 5 GABA receptors, 28 major transmitter receptor subtypes including glutamate and acetylcholine receptors, and 6 gap junction proteins - the Innexins. Conclusion: With this resource, crustacean model systems are better poised for incorporation of modern genomic and molecular biology technologies to further enhance the interrogation of fundamentals of nervous system function. [ABSTRACT FROM AUTHOR]

Oscillatory networks often include neurons with membrane potential resonance, exhibiting a peak in the voltage amplitude as a function of current input at a nonzero (resonance) frequency (fres). Although fres has been correlated to the network frequency (fnet) in a variety of systems, a causal relationship between the two has not been established. We examine the hypothesis that combinations of biophysical parameters that shift fres, without changing other attributes of the impedance profile, also shift fnet in the same direction. We test this hypothesis, computationally and experimentally, in an electrically coupled network consisting of intrinsic oscillator (O) and resonator (R) neurons. We use a two-cell model of such a network to show that increasing fres of R directly increases fnet and that this effect becomes more prominent if the amplitude of resonance is increased. Notably, the effect of fres on fnet is independent of the parameters that define the oscillator or the combination of parameters in R that produce the shift in fres, as long as this combination produces the same impedance vs. frequency relationship. We use the dynamic clamp technique to experimentally verify the model predictions by connecting a model resonator to the pacemaker pyloric dilator neurons of the crab Cancer borealis pyloric network using electrical synapses and show that the pyloric network frequency can be shifted by changing fres in the resonator. Our results provide compelling evidence that fres and resonance amplitude strongly influence fnet, and therefore, modulators may target these attributes to modify rhythmic activity. [ABSTRACT FROM AUTHOR]

Essential to understanding the process of neuronal signal integration is the knowledge of where within a neuron action potentials (APs) are generated. Recent studies support the idea that the precise location where APs are initiated and the properties of spike initiation zones define the cell's information processing capabilities. Notably, the location of spike initiation can be modified homeostatically within neurons to adjust neuronal activity. Here we show that this potential mechanism for neuronal plasticity can also be exploited in a rapid and dynamic fashion. We tested whether dislocation of the spike initiation zone affects signal integration by studying ectopic spike initiation in the anterior gastric receptor neuron (AGR) of the stomatogastric nervous system of Cancer borealis. Like many other vertebrate and invertebrate neurons, AGR can generate ectopic APs in regions distinct from the axon initial segment. Using voltagesensitive dyes and electrophysiology, we determined that AGR's ectopic spike activity was consistently initiated in the neuropil region of the stomatogastric ganglion motor circuits. At least one neurite branched off the AGR axon in this area; and indeed, we found that AGR's ectopic spike activity was influenced by local motor neurons. This sensorimotor interaction was state-dependent in that focal axon modulation with the biogenic amine octopamine, abolished signal integration at the primary spike initiation zone by dislocating spike initiation to a distant region of the axon. We demonstrate that the site of ectopic spike initiation is important for signal integration and that axonal neuromodulation allows for a dynamic adjustment of signal integration. [ABSTRACT FROM AUTHOR]

Long-term intrinsic and synaptic plasticity must be coordinated to ensure stability and flexibility in neuronal circuits. Coordination might be achieved through shared transduction components. Dopamine (DA) is a well-established participant in many forms of long-term synaptic plasticity. Recent work indicates that DA is also involved in both activity-dependent and -independent forms of long-term intrinsic plasticity. We previously examined DA-enabled long-term intrinsic plasticity in a single identified neuron. The lateral pyloric (LP) neuron is a component of the pyloric network in the crustacean stomatogastric nervous system (STNS). LP expresses type 1 DA receptors (D1Rs). A 1hr bath application of 5nM DA followed by washout produced a significant increase in the maximal conductance (Gmax) of the LP transient potassium current (IA) that peaked ~4hr after the start of DA application; furthermore, if a change in neuronal activity accompanied the DA application, then a persistent increase in the LP hyperpolarization activated current (Ih) was also observed. Here, we repeated these experiments with pharmacological and peptide inhibitors to determine the cellular processes and signaling proteins involved. We discovered that the persistent, DA-induced activity-independent (IA) and activity-dependent (Ih) changes in ionic conductances depended upon many of the same elements that enable long-term synaptic plasticity, including: the D1R-protein kinase A (PKA) axis, RNA polymerase II transcription, RNA interference (RNAi), and mechanistic target of rapamycin (mTOR)-dependent translation. We interpret the data to mean that increasing the tonic DA concentration enhances expression of a microRNA(s) (miRs), resulting in increased cap-dependent translation of an unidentified protein(s).

Experimental and computational studies demonstrate that different sets of intrinsic and synaptic conductances can give rise to equivalent activity patterns. This is because the balance of conductances, not their absolute values, defines a given activity feature. Activity-dependent feedback mechanisms maintain neuronal conductance correlations and their corresponding activity features. This study demonstrates that tonic nM concentrations of monoamines enable slow, activity-dependent processes that can maintain a correlation between the transient potassium current (IA) and the hyperpolarization activated current (Ih) over the long-term (i.e., regulatory change persists for hours after removal of modulator). Tonic 5 nM DA acted through an RNA interference silencing complex (RISC)- and RNA polymerase II-dependent mechanism to maintain a long-term positive correlation between IA and Ih in the lateral pyloric neuron (LP) but not in the pyloric dilator neuron (PD). In contrast, tonic 5 nM 5HT maintained a RISC- dependent positive correlation between IA and Ih in PD but not LP over the long-term. Tonic 5 nM OCT maintained a long-term negative correlation between IA and Ih in PD but not LP; however, it was only revealed when RISC was inhibited. This study also demonstrated that monoaminergic tone can also preserve activity features over the long-term: the timing of LP activity, LP duty cycle and LP spike number per burst were maintained by tonic 5 nM DA. The data suggest that low-level monoaminergic tone acts through multiple slow processes to permit cell-specific, activity-dependent regulation of ionic conductances to maintain conductance correlations and their corresponding activity features over the long-term. [ABSTRACT FROM AUTHOR]

We recorded from lobster and leech neurons with two sharp electrodes filled with solutions often used with these preparations (lobster: 0.6 M K2SO4 or 2.5 M KAc; leech: 4 M KAc), with solutions approximately matching neuron cytoplasm ion concentrations, and with 6.5 M KAc (lobster, leech) and 0.6 M KAc (lobster). We measured membrane potential, input resistance, and transient and sustained depolarization-activated outward current amplitudes in leech and these neuron properties and hyperpolarization-activated current time constant in lobster, every 10 min for 60 min after electrode penetration. Neuron properties varied with electrode fill. For fills with molarities ≥2.5 M, neuron properties also varied strongly with time after electrode penetration. Depending on the property being examined, these variations could be large. In leech, cell size also increased with noncytoplasmic fills. The changes in neuron properties could be due to the ions being injected from the electrodes during current injection. We tested this possibility in lobster with the 2.5 M KAc electrode fill by making measurements only 10 and 60 min after penetration. Neuron properties still changed, although the changes were less extreme. Making measurements every 2 min showed that the time-dependent variations in neuron properties occurred in concert with each other. Neuron property changes with high molarity electrode-fill solutions were great enough to decrease neuron firing strongly. An experiment with 14C-glucose electrode fill confirmed earlier work showing substantial leak from sharp electrodes. Sharp electrode work should thus be performed with cytoplasm- matched electrode fills. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A variety of neurons and synapses shows a maximal response at a preferred frequency, generally considered to be important in shaping network activity. We are interested in whether all neurons and synapses in a recurrent oscillatory network can have preferred frequencies and, if so, whether these frequencies are the same or correlated, and whether they influence the network activity. We address this question using identified neurons in the pyloric network of the crab Cancer borealis. Previous work has shown that the pyloric pacemaker neurons exhibit membrane potential resonance whose resonance frequency is correlated with the network frequency. The follower lateral pyloric (LP) neuron makes reciprocally inhibitory synapses with the pacemakers. We find that LP shows resonance at a higher frequency than the pacemakers and the network frequency falls between the two. We also find that the reciprocal synapses between the pacemakers and LP have preferred frequencies but at significantly lower values. The preferred frequency of the LP to pacemaker synapse is correlated with the presynaptic preferred frequency, which is most pronounced when the peak voltage of the LP waveform is within the dynamic range of the synaptic activation curve and a shift in the activation curve by the modulatory neuropeptide proctolin shifts the frequency preference. Proctolin also changes the power of the LP neuron resonance without significantly changing the resonance frequency. These results indicate that different neuron types and synapses in a network may have distinct preferred frequencies, which are subject to neuromodulation and may interact to shape network oscillations. [ABSTRACT FROM AUTHOR]

Rhythmic activity which underlies motor output is often initiated and controlled by descending modulatory projection pathways onto central pattern generator (CPG) networks. In turn, these descending pathways receive synaptic feedback from their target CPG network, which can influence the CPG output. However, the mechanisms underlying such bidirectional synaptic interactions are mostly unexplored. We develop a reduced mathematical model, including both feed-forward and feedback circuitry, to examine how the synaptic interactions involving two projection neurons, MCN1 and CPN2, can produce and shape the activity of the gastric mill CPG in the crab stomatogastric nervous system. We use simplifying assumptions that are based on the behavior of the biological system to reduce this model down to 2 dimensions, which allows for phase plane analysis of the model output. The model shows a distinct activity for the gastric mill rhythm that is elicited when MCN1 and CPN2 are coactive compared to the rhythm elicited by MCN1 activity alone. Furthermore, the presence of feedback to the projection neuron CPN2 provides a distinct locus of pattern generation in the model which does not require reciprocally inhibitory interactions between the gastric mill CPG neurons, but is instead based on a half-center oscillator that occurs through a trisynaptic pathway that includes CPN2. Our modeling results show that feedback to projection pathways may provide additional mechanisms for the generation of motor activity. These mechanisms can have distinct dependence on network parameters and may therefore provide additional flexibility for the rhythmic motor output. [ABSTRACT FROM AUTHOR]

The lobster gastric mill CPG is located in the stomatogastric ganglion and consists of eleven neurons whose circuitry is well known. Because all of the neurons are identifiable and accessible, it can serve as a prime experimental model for analyzing how microcircuits generate multiphase oscillatory spatiotemporal patterns. The neurons that comprise the gastric mill CPG consist of one interneuron, five burster neurons and six tonically firing neurons. The single interneuron (Int 1) is shared by the medial tooth subcircuit (containing the AM, DG and GMs) and the lateral teeth subcircuit (LG, MG and LPGs). By surveying cell-to-cell connections and the cooperative dynamics of the neurons we find that the medial subcircuit is essentially a feed forward system of oscillators. The Int 1 neuron entrains the DG and AM cells by delayed excitation and this pair then periodically inhibits the tonically firing GMs causing them to burst. The lateral subcircuit consists of two negative feedback loops of reciprocal inhibition from Int 1 to the LG/MG pair and from the LG/MG to the LPGs. Following a fast inhibition from Int 1, the LG/MG neurons receive a slowly developing excitatory input similar to that which Int 1 puts onto DG/AM. Thus Int 1 plays a key role in synchronizing both subcircuits. This coordinating role is assisted by additional, weaker connections between the two subsets but those are not sufficient to synchronize them in the absence of Int 1. In addition to the experiments, we developed a conductance-based model of a slightly simplified gastric circuit. The mathematical model can reproduce the fundamental rhythm and many of the experimentally induced perturbations. Our findings shed light on the functional role of every cell and synapse in this small circuit providing a detailed understanding of the rhythm generation and pattern formation in the gastric mill network.

Neuromodulators alter network output and have the potential to destabilize a circuit. The mechanisms maintaining stability in the face of neuromodulation are not well described. Using the pyloric network in the crustacean stomatogastric nervous system, we show that dopamine (DA) does not simply alter circuit output, but activates a closed loop in which DA-induced alterations in circuit output consequently drive a change in an ionic conductance to preserve a conductance ratio and its activity correlate. DA acted at low affinity type 1 receptors (D1Rs) to induce an immediate modulatory decrease in the transient potassium current (IA) of a pyloric neuron. This, in turn, advanced the activity phase of that component neuron, which disrupted its network function and thereby destabilized the circuit. DA simultaneously acted at high affinity D1Rs on the same neuron to confer activity-dependence upon the hyperpolarization activated current (Ih) such that the DA induced changes in activity subsequently reduced Ih. This DA-enabled, activity-dependent, intrinsic plasticity exactly compensated for the modulatory decrease in IA to restore the IA:Ih ratio and neuronal activity phase, thereby closing an open loop created by the modulator. Activation of closed loops to preserve conductance ratios may represent a fundamental operating principle neuromodulatory systems use to ensure stability in their target networks. [ABSTRACT FROM AUTHOR]

This article reviews the mechanical processes associated with digestion in decapod crustaceans. The decapod crustacean gut is essentially an internal tube that is divided into three functional areas, the foregut, midgut, and hindgut. The foregut houses the gastric mill apparatus which functions in mastication (cutting and grinding) of the ingested food. The processed food passes into the pyloric region of the foregut which controls movement of digesta into the midgut region and hepatopancreas where intracellular digestion takes place. The movements of the foregut muscles and gastric mill are controlled via nerves from the stomatogastric ganglion. Contraction rates of the gastric mill and foregut muscles can be influenced by environmental factors such as salinity, temperature, and oxygen levels. Gut contraction rates depend on the magnitude of the environmental perturbation and the physiological ability of each species. The subsequent transit of the digesta from the foregut into the midgut and through the hindgut has been followed in a wide variety of crustaceans. Transit rates are commonly used as a measure of food processing rates and are keys in understanding strategies of adaptation to trophic conditions. Transit times vary from as little as 30 min in small copepods to over 150 h in larger lobsters. Transit times can be influenced by the size and the type of the meal, the size and activity level of an animal and changes in environmental temperature, salinity and oxygen tension. Ultimately, changes in transit times influence digestive efficiency (the amount of nutrients absorbed across the gut wall). Digestive efficiencies tend to be high for carnivorous crustaceans, but somewhat lower for those that consume plant material. A slowing of the transit rate allows more time for nutrient absorption but this may be confounded by changes in the environment, which may reduce the energy available for active transport processes. Given the large number of articles already published on the stomatogastric ganglion and its control mechanisms, this area will continue to be of interest to scientists. There is also a push towards studying animals in a more natural environment or even in the field and investigation of the energetic costs of the components of digestion under varying biotic and environmental conditions will undoubtedly be an area that expands in the future. [ABSTRACT FROM AUTHOR]

The levels of voltage-gated and synaptic currents in the same neuron type can vary substantially across individuals. Yet, the phase relationships between neurons in oscillatory circuits are often maintained, even in the face of varying oscillation frequencies. We examined whether synaptic and intrinsic currents are matched to maintain constant activity phases across preparations, using the lateral pyloric (LP) neuron of the stomatogastric ganglion (STG) of the crab, Cancer borealis LP produces stable oscillatory bursts on release from inhibition, with an onset phase that is independent of oscillation frequency. We quantified the parameters that define the shape of the synaptic current inputs across preparations and found no linear correlations with voltage-gated currents. However, several synaptic parameters were correlated with oscillation period and burst onset phase, suggesting they may play a role in phase maintenance. We used dynamic clamp to apply artificial synaptic inputs and found that those synaptic parameters correlated with phase and period were ineffective in influencing burst onset. Instead, parameters that showed the least variability across preparations had the greatest influence. Thus, parameters that influence circuit phasing are constrained across individuals, while those that have little effect simply co-vary with phase and frequency., (Copyright © 2022 Anwar et al.)

In similar states, neural circuits produce similar outputs across individuals despite substantial interindividual variability in neuronal ionic conductances and synapses. Circuit states are largely shaped by neuromodulators that tune ionic conductances. It is therefore possible that, in addition to producing flexible circuit output, neuromodulators also contribute to output similarity despite varying ion channel expression. We studied whether neuromodulation at saturating concentrations can increase the output similarity of a single identified neuron across individual animals. Using the LP neuron of the crab stomatogastric ganglion (STG), we compared the variability of f-I curves and rebound properties in the presence of neuropeptides. The two neuropeptides we used converge to activate the same target current, which increases neuronal excitability. Output variability was lower in the presence of the neuropeptides, regardless of whether the neuropeptides significantly changed the mean of the corresponding parameter or not. However, the addition of the second neuropeptide did not add further to the reduction of variability. With a family of computational LP-like models, we explored how increased excitability and target variability contribute to output similarity and found two mechanisms: Saturation of the responses and a differential increase in baseline activity. Saturation alone can reduce the interindividual variability only if the population shares a similar ceiling for the responses. In contrast, reduction of variability due to the increase in baseline activity is independent of ceiling effects. Significance Statement The activity of single neurons and neural circuits can be very similar across individuals even though the ionic currents underlying activity are variable. The mechanisms that compensate for the underlying variability and promote output similarity are poorly understood but may involve neuromodulation. Using an identified neuron, we show that neuropeptide modulation of excitability can reduce interindividual variability of response properties at a single-neuron level in two ways. First, the neuropeptide increases baseline excitability in a differential manner, resulting in similar response thresholds. Second, the neuropeptide increases excitability towards a shared saturation level, promoting similar maximal firing rates across individuals. Such tuning of neuronal excitability could be an important mechanism compensating for interindividual variability of ion channel expression., Competing Interests: The authors report no conflict of interest, (Copyright © 2022 Schneider et al.)

Abstract: CbNav and CbIH encode channels that carry voltage-gated sodium and hyperpolarization activated cation currents respectively in the crab, Cancer borealis. We cloned and sequenced full length cDNAs for both CbNav and CbIH and found nine different regions of alternative splicing for the CbNav gene and four regions of alternative splicing for CbIH. We used RT-PCR to determine tissue-specific differences in splicing of both channel genes among cardiac muscle, skeletal muscle, brain, and stomatogastric ganglion (STG) tissue. We then examined the splice variant isoforms present in single, unambiguously identified neurons of the STG. We found cell-type specific patterns of alternative splicing for CbNav , indicating unique cell-specific pattern of post-transcriptional modification. Furthermore, we detected possible differences in cellular localization of alternatively spliced CbNav transcripts; distinct mRNA isoforms are present between the cell somata and the axons of the neurons. In contrast, we found no qualitative differences among different cell types for CbIH variants present, although this analysis did not represent the full spectrum of all possible CbIH variants. CbIH mRNA was not detected in axon samples. Finally, although cell-type specific patterns of splicing were detected for CbNav , the same cell type within and between animals also displayed variability in which splice forms were detected. These results indicate that channel splicing is differentially regulated at the level of single neurons of the same neural network, providing yet another mechanism by which cell-specific neuronal output can be achieved. [Copyright &y& Elsevier]

The pyloric network of decapod crustaceans has been intensively studied electrophysiologically in the infraorders Astacidea, Brachyura, and Palinura. The morphology of some or all pyloric neurons has been well described in Astacidea and Brachyura, but less so in Palinura. Given the large evolutionary distance between these three groups, and the large amount of electrophysiology that has been performed in palinuroid species, it is important to fill this gap. We describe here the gross morphology of all six pyloric neuron types in a palinuroid, P. interruptus. All pyloric neurons had complicated, extended dendritic trees that filled the majority of the neuropil, with most small diameter processes present in a shell near the surface of the ganglion. Certain neuron types showed modest preferences for somata location in the ganglion, but these differences were too weak to use as identifying characteristics. Quantitative measurements of secondary branch number, maximum branch order, total process length, and neuron somata diameter were also, in general, insufficient to distinguish among the neurons, although AB and LP neuron somata diameters differed from those of the other types. One neuron type (VD) had a distinctive neurite branching pattern consisting of a small initial branch followed shortly by a bifurcation of the main neurite. The processes arising from these two branches occupied largely non-overlapping neuropil. Electrophysiological recordings showed that each major branch had its own spike initiation zone and that, although the zones fired correlated spikes, they generated spikes independently. VD neurons in the other infraorders have similar morphologies, suggesting that having two arbors is important for the function of this neuron. These data are similar to those previously obtained in Brachyura and Astacidea. It thus appears that, despite their long evolutionary separation, neuron morphology in these three infraorders has not greatly diverged. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

A possible role of the insect stomatogastric nervous system (STNS) in ecdysis was first implied in early studies reporting on internal air pressure build-up in the digestive tract and air swallowing during ecdysis. The frontal ganglion, a major component of the insect STNS, was suggested to play an important part in this behaviour. Recent neurophysiological studies have confirmed the critical role of the STNS in the successful completion of both larval and adult moults in insects. In aquatic arthropods, though much less studied, the STNS plays an equally important and probably very similar role in water swallowing. Water uptake is instrumental in splitting the crustacean cuticle and allowing successful ecdysis. Current data are presented in a comparative view that contributes to our understanding of the role of the STNS in arthropod behaviour. It also sheds light on the question of homology of the STNS among the different arthropod groups. New insights into the neurohormonal control of ecdysis, related to the STNS in both insects and crustaceans, are also presented and comparatively discussed. [ABSTRACT FROM AUTHOR]

The rhythmic pyloric network of the lobster stomatogastric system approximately maintains phase (that is, the burst durations and durations between the bursts of its neurons change proportionally) when network cycle period is altered by current injection into the network pacemaker (Hooper, 1997a,b). When isolated from the network and driven by rhythmic hyperpolarizing current pulses, the delay to firing after each pulse of at least one network neuron type [pyloric (PY)] varies in a phase-maintaining manner when cycle period is varied (Hooper, 1998). These variations require PY neurons to have intrinsic mechanisms that respond to changes in neuron activity on time scales at least as long as 2 s. Slowly activating and deactivating conductances could provide such a mechanism. We tested this possibility by building models containing various slow conductances. This work showed that such conductances could indeed support intrinsic phase maintenance, and we show here results for one such conductance, a slow potassium conductance. These conductances supported phase maintenance because their mean activation level changed, hence altering neuron postinhibition firing delay, when the rhythmic input to the neuron changed. Switching the sign of the dependence of slow-conductance activation and deactivation on membrane potential resulted in neuron delays switching to change in an anti-phase-maintaining manner. These data suggest that slow conductances or similar slow processes such as changes in intracellular Ca2+ concentration could underlie phase maintenance in pyloric network neurons. [ABSTRACT FROM AUTHOR]

Emerging experimental evidence suggests that both networks and their component neurons respond to similar inputs differently, depending on the state of network activity. The network state is determined by the intrinsic dynamical structure of the network and may change as a function of neuromodulation, the balance or stochasticity of synaptic inputs to the network, and the history of network activity. Much of the knowledge on state-dependent effects comes from comparisons of awake and sleep states of the mammalian brain. Yet, the mechanisms underlying these states are difficult to unravel. Several vertebrate and invertebrate studies have elucidated cellular and synaptic mechanisms of state dependence resulting from neuromodulation, sensory input, and experience. Recent studies have combined modeling and experiments to examine the computational principles that emerge when network state is taken into account; these studies are highlighted in this article. We discuss these principles in a variety of systems (mammalian, crustacean, and mollusk) to demonstrate the unifying theme of state dependence of network output. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The transient potassium A-current is present in most neurons and plays an important role in determining the timing of action potentials. We examine the role of the A-current in the activity phase of a follower neuron in a rhythmic feed-forward inhibitory network with a reduced three-variable model and conduct experiments to verify the usefulness of our model. Using geometric analysis of dynamical systems, we explore the factors that determine the onset of activity in a follower neuron following release from inhibition. We first analyze the behavior of the follower neuron in a single cycle and find that the phase plane structure of the model can be used to predict the potential behaviors of the follower neuron following release from inhibition. We show that, depending on the relative scales of the inactivation time constant of the A-current and the time constant of the recovery variable, the follower neuron may or may not reach its active state following inhibition. Our simple model is used to derive a recursive set of equations to predict the contribution of the A-current parameters in determining the activity phase of a follower neuron as a function of the duration and frequency of the inhibitory input it receives. These equations can be used to demonstrate the dependence of activity phase on the period and duty cycle of the periodic inhibition, as seen by comparing the predictions of the model with the activity of the pyloric constrictor (PY) neurons in the crustacean pyloric network. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Electrical activity in identical neurons across individuals is often remarkably similar and stable over long periods. However, the ionic currents that determine the electrical activity of these neurons show wide animal-to-animal amplitude variability. This seemingly random variability of individual current amplitudes may obscure mechanisms that globally reduce variability and that contribute to the generation of similar neuronal output. One such mechanism could be the coordinated regulation of ionic current expression. Studying identified neurons of the Cancer borealis pyloric network, we discovered that the removal of neuromodulatory input to this network (decentralization) was accompanied by the loss of the coordinated regulation of ionic current levels. Additionally, decentralization induced large changes in the levels of several ionic currents. The loss of coregulation and the changes in current levels were prevented by continuous exogenous application of proctolin, an endogenous neuromodulatory peptide, to the pyloric network. This peptide does not exert fast regulatory actions on any of the currents affected by decentralization. We conclude that neuromodulatory inputs to the pyloric network have a novel role in the regulation of ionic current expression. They can control, over the long term, the coordinated expression of multiple voltage-gated ionic currents that they do not acutely modulate. Our results suggest that current coregulation places constraints on neuronal intrinsic plasticity and the ability of a network to respond to perturbations. The loss of conductance coregulation may be a mechanism to facilitate the recovery of function. [ABSTRACT FROM AUTHOR]

The postdevelopmental basis of cellular identity and the unique cellular output of a particular neuron type are of particular interest in the nervous system because a detailed understanding of circuits responsible for complex processes in the brain is impeded by the often ambiguous classification of neurons in these circuits. Neurons have been classified by morphological, electrophysiological, and neurochemical techniques. More recently, molecular approaches, particularly microarray, have been applied to the question of neuronal identity. With the realization that proteins expressed exclusively in only one type of neuron are rare, expression profiles obtained from neuronal subtypes are analyzed to search for diagnostic patterns of gene expression. However, this expression profiling hinges on one critical and implicit assumption: that neurons of the same type in different animals achieve their conserved functional output via conserved levels and quantitative relationships of gene expression. Here we exploit the unambiguously identifiable neurons in the crab stomatogastric ganglion to investigate the precise quantitative expression profiling of neurons at the level of single-cell ion channel expression. By measuring absolute mRNA levels of six different channels in the same individually identified neurons, we demonstrate that not only do individual cell types possess highly variable levels of channel expression but that this variability is constrained by unique patterns of correlated channel expression. [ABSTRACT FROM AUTHOR]