Neuromodulation by peptides and amines is a primary source of plasticity in the nervous system as it adapts the animal to an ever-changing environment. The crustacean stomatogastric nervous system is one of the premier systems to study neuromodulation and its effects on motor pattern generation at the cellular level. It contains the extensively modulated central pattern generators that drive the gastric mill (chewing) and pyloric (food filtering) rhythms. Neuromodulators affect all stages of neuronal processing in this system, from membrane currents and synaptic transmission in network neurons to the properties of the effector muscles. The ease with which distinct neurons are identified and their activity is recorded in this system has provided considerable insight into the mechanisms by which neuromodulators affect their target cells and modulatory neuron function. Recent evidence suggests that neuromodulators are involved in homeostatic processes and that the modulatory system itself is under modulatory control, a fascinating topic whose surface has been barely scratched. Future challenges include exploring the behavioral conditions under which these systems are activated and how their effects are regulated.

The marbled crayfish Procambarus virginalis is a unique freshwater crayfish characterized by very recent speciation and parthenogenetic reproduction. Marbled crayfish also represent an emerging invasive species and have formed wild populations in diverse freshwater habitats. However, our understanding of marbled crayfish biology, evolution and invasive spread has been hampered by the lack of freshwater crayfish genome sequences. We have now established a de novo draft assembly of the marbled crayfish genome. We determined the genome size at approximately 3.5 gigabase pairs and identified >21,000 genes. Further analysis confirmed the close relationship to the genome of the slough crayfish, Procambarusfallax, and also established a triploid AA’B genotype with a high level of heterozygosity. Systematic fieldwork and genotyping demonstrated the rapid expansion of marbled crayfish on Madagascar and established the marbled crayfish as a potent invader of freshwater ecosystems. Furthermore, comparative whole-genome sequencing demonstrated the clonality of the population and their genetic identity with the oldest known stock from the German aquarium trade. Our study closes an important gap in the phylogenetic analysis of animal genomes and uncovers the unique evolutionary history of an emerging invasive species.

Specificity in the actions of different modulatory neurons is often attributed to their having distinct cotransmitter complements. We are assessing the validity of this hypothesis with the stomatogastric nervous system of the crab Cancer borealis. In this nervous system, the stomatogastric ganglion (STG) contains a multifunctional network that generates the gastric mill and pyloric rhythms. Two identified projection neurons [modulatory proctolin neuron (MPN) and modulatory commissural neuron 1 (MCN1)] that innervate the STG and modulate these rhythms contain GABA and the pentapeptide proctolin, but only MCN1 contains Cancer borealis tachykinin-related peptide (CabTRP Ia). Selective activation of each projection neuron elicits different rhythms from the STG. MPN elicits only a pyloric rhythm, whereas MCN1 elicits a distinct pyloric rhythm as well as a gastric mill rhythm. We tested the degree to which CabTRP Ia distinguishes the actions of MCN1 and MPN. To this end, we used the tachykinin receptor antagonist Spantide I to eliminate the actions of CabTRP Ia. With Spantide I present, MCN1 no longer elicited the gastric mill rhythm and the resulting pyloric rhythm was changed. Although this rhythm was more similar to the MPN-elicited pyloric rhythm, these rhythms remained different. Thus, CabTRP Ia partially confers the differences in rhythm generation resulting from MPN versus MCN1 activation. This result suggests that different projection neurons may use the same cotransmitters differently to elicit distinct pyloric rhythms. It also supports the hypothesis that different projection neurons use a combination of strategies, including using distinct cotransmitter complements, to elicit different outputs from the same neuronal network.

Co-transmission is a common means of neuronal communication, but its consequences for neuronal signaling within a defined neuronal circuit remain unknown in most systems. We are addressing this issue in the crab stomatogastric nervous system by characterizing how the identified modulatory commissural neuron (MCN)1 uses its co-transmitters to activate the gastric mill (chewing) rhythm in the stomatogastric ganglion (STG). MCN1 contains gamma-aminobutyric acid (GABA) plus the peptides proctolin and Cancer borealis tachykinin-related peptide Ia (CabTRP Ia), which it co-releases during the retractor phase of the gastric mill rhythm to influence both retractor and protractor neurons. By focally applying each MCN1 co-transmitter and pharmacologically manipulating each co-transmitter action during MCN1 stimulation, we found that MCN1 has divergent co-transmitter actions on the gastric mill central pattern generator (CPG), which includes the neurons lateral gastric (LG) and interneuron 1 (Int1), plus the STG terminals of MCN1 (MCN1(STG)). MCN1 used only CabTRP Ia to influence LG, while it used only GABA to influence Int1 and the contralateral MCN1(STG). These MCN1 actions caused a slow excitation of LG, a fast excitation of Int1 and a fast inhibition of MCN1(STG). MCN1-released proctolin had no direct influence on the gastric mill CPG, although it likely indirectly regulates this CPG via its influence on the pyloric rhythm. MCN1 appeared to have no ionotropic actions on the gastric mill follower motor neurons, but it did use proctolin and/or CabTRP Ia to excite them. Thus, a modulatory projection neuron can elicit rhythmic motor activity by using distinct co-transmitters, with different time courses of action, to simultaneously influence different CPG neurons.

Histamine is a neurotransmitter with actions throughout the nervous system of vertebrates and invertebrates. Nevertheless, the actions of only a few identified histamine-containing neurons have been characterized. Here, we present the actions of a histaminergic projection neuron on the rhythmically active pyloric and gastric mill circuits within the stomatogastric ganglion (STG) of the crab Cancer borealis. An antiserum generated against histamine labeled profiles throughout the C. borealis stomatogastric nervous system. Labeling occurred in several somata and neuropil within the paired commissural ganglia as well as in neuropil within the STG and at the junction of the superior oesophageal and stomatogastric nerves. The source of all histamine-like immunolabeling in the STG neuropil was one pair of neuronal somata, the previously identified inferior ventricular (IV) neurons, located in the supraoesophageal ganglion. These neurons also exhibited FLRFamide-like immunoreactivity. Activation of the IV neurons in the crab inhibited some pyloric and gastric mill neurons and, with inputs from the commissural ganglia eliminated, terminated both rhythms. Focal application of histamine had comparable effects. The actions of both applied histamine and IV neuron stimulation were blocked, reversibly, by the histamine type-2 receptor antagonist cimetidine. With the commissural ganglia connected to the STG, IV neuron stimulation elicited a longer-latency activation of commissural projection neurons which in turn modified the pyloric rhythm and activated the gastric mill rhythm. These results support the hypothesis that the histaminergic/peptidergic IV neurons are projection neurons with direct and indirect actions on the STG circuits of the crab C. borealis.

Central-pattern-generating neural circuits function reliably throughout an animal's life, despite constant molecular turnover and environmental perturbations. Fluctuations in temperature pose a problem to the nervous systems of poikilotherms because their body temperature follows the ambient temperature, thus affecting the temperature-dependent dynamics of various subcellular components that constitute neuronal circuits. In the crustacean stomatogastric nervous system, the pyloric circuit produces a triphasic rhythm comprising the output of the pyloric dilator, lateral pyloric, and pyloric constrictor neurons. In vitro, the phase relationships of these neurons are maintained over a fourfold change in pyloric frequency as temperature increases from 7°C to 23°C. To determine whether these temperature effects are also found in intact crabs, in the presence of sensory feedback and neuromodulator-rich environments, we measured the temperature dependence of the pyloric frequency and phases in vivo by implanting extracellular electrodes into Cancer borealis and Cancer pagurus and shifting tank water temperature from 11°C to 26°C. Pyloric frequency in the intact crab increased significantly with temperature (Q10 = 2-2.5), while pyloric phases were generally conserved. For a subset of the C. borealis experiments, animals were subsequently dissected and the stomatogastric ganglion subjected to a similar temperature ramp in vitro. We found that the maximal frequency attained at high temperatures in vivo is lower than it is under in vitro conditions. Our results demonstrate that, over a wide temperature range, the phases of the pyloric rhythm in vivo are generally preserved, but that the frequency range is more restricted than it is in vitro.

Stable rhythmic neural activity depends on the well-coordinated interplay of synaptic and cell-intrinsic conductances. Since all biophysical processes are temperature dependent, this interplay is challenged during temperature fluctuations. How the nervous system remains functional during temperature perturbations remains mostly unknown. We present a hitherto unknown mechanism of how temperature-induced changes in neural networks are compensated by changing their neuromodulatory state: activation of neuromodulatory pathways establishes a dynamic coregulation of synaptic and intrinsic conductances with opposing effects on neuronal activity when temperature changes, hence rescuing neuronal activity. Using the well-studied gastric mill pattern generator of the crab, we show that modest temperature increase can abolish rhythmic activity in isolated neural circuits due to increased leak currents in rhythm-generating neurons. Dynamic clamp-mediated addition of leak currents was sufficient to stop neuronal oscillations at low temperatures, and subtraction of additional leak currents at elevated temperatures was sufficient to rescue the rhythm. Despite the apparent sensitivity of the isolated nervous system to temperature fluctuations, the rhythm could be stabilized by activating extrinsic neuromodulatory inputs from descending projection neurons, a strategy that we indeed found to be implemented in intact animals. In the isolated nervous system, temperature compensation was achieved by stronger extrinsic neuromodulatory input from projection neurons or by augmenting projection neuron influence via bath application of the peptide cotransmitter Cancer borealis tachykinin-related peptide Ia (CabTRP Ia). CabTRP Ia activates the modulator-induced current IMI (a nonlinear voltage-gated inward current) that effectively acted as a negative leak current and counterbalanced the temperature-induced leak to rescue neuronal oscillations. Computational modelling revealed the ability of IMI to reduce detrimental leak-current influences on neuronal networks over a broad conductance range and indicated that leak and IMI are closely coregulated in the biological system to enable stable motor patterns. In conclusion, these results show that temperature compensation does not need to be implemented within the network itself but can be conditionally provided by extrinsic neuromodulatory input that counterbalances temperature-induced modifications of circuit-intrinsic properties.

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 LGinnervated 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.