Temperature sensitivity of the pyloric neuromuscular system and its modulation by dopamine

We report here the effects of temperature on the p1 neuromuscular system of the stomatogastric system of the lobster (Panulirus interruptus). Muscle force generation, in response to both the spontaneously rhythmic in vitro pyloric network neural activity and direct, controlled motor nerve stimulation, dramatically decreased as temperature increased, sufficiently that stomach movements would very unlikely be maintained at warm temperatures. However, animals fed in warm tanks showed statistically identical food digestion to those in cold tanks. Applying dopamine, a circulating hormone in crustacea, increased muscle force production at all temperatures and abolished neuromuscular system temperature dependence. Modulation may thus exist not only to increase the diversity of produced behaviors, but also to maintain individual behaviors when environmental conditions (such as temperature) vary

MonoAminergic Receptors in the Stomatogastric Nervous System: Characterization and Localization in Panulirus Interruptus

Neural circuit flexibility is fundamental to the production of adaptable behaviors. Invertebrate models offer relatively simple networks consisting of large, identifiable neurons that are useful for investigating the electrophysiological properties that contribute to circuit output. In particular, central pattern generating circuits within the crustacean stomatogastric nervous system have been well characterized with regard to their synaptic connectivities, cellular properties, and response to modulatory influences. Monoaminergic modulation is essential for the production of adaptable circuit output in most species. Monoamines, such as dopamine and serotonin, signal via metabotropic receptors, which activate intracellular signaling cascades. Many of the neuronal and network targets of monoaminergic modulation in the crustacean stomatogastric nervous system are known, but nothing is known of the signal transduction cascades that mediate the biophysical response. This work represents a thorough characterization of monoaminergic receptors in the crustacean stomatogastric nervous system. We took advantage of the close phylogenetic relationship between crustaceans and insects to clone monoaminergic receptors from the spiny lobster. Using a novel database mining strategy, we were able to identify several uncharacterized monoaminergic receptors in the Panulirus interruptus genome. We cloned one serotonin (5-HT2βPan) and three dopamine receptors (D1αPan, D1βPan, and D2αPan), and characterized them with regard to G protein coupling and signal transduction cascades. We used a heterologous expression system to show that G protein couplings and signaling properties of monoaminergic receptors are strongly conserved among vertebrate and invertebrate species. This work further shows that DAR-G protein couplings in the stomatogastric nervous system are unique for a given receptor subtype, and receptors can couple to multiple signaling pathways, similar to their mammalian homologs. Custom made antibodies were used to localize monoamine receptors in the stomatogastric ganglion, and in identified neurons. Pyloric neurons show unique receptor expression profiles, which supports the idea of receptor expression as an underlying mechanism for cell-type specific effects of a given modulator. Receptors are localized to the synaptic neuropil, but are not expressed in the membrane of large diameter processes or the soma. The localization of dopamine receptors in identified pyloric neurons suggests that they may respond to synaptic, paracrine or neurohormonal dopamine signals. This work also supports the idea that different types of signals can be generated by a single receptor

Identification and Functional Analysis of Crustacean Serotonin Receptors.

Constantly changing environments force animals to adapt by cycling through multiple physiological states. Plasticity in sensory, motor, and modulatory neural circuits is an essential part of these adaptive processes. Invertebrates with their accessible, identifiable neurons are excellent models for investigating the molecular and cellular mechanisms underlying state-dependent neural plasticity, and provide insight into similar processes in more complex systems. These properties have allowed highly detailed characterization of several crustacean circuits with respect to their connectivities, cellular properties, responses to various inputs, and outputs. Serotonin (5-HT) is an important neuromodulator in virtually every animal species. 5-HT signals are mediated primarily by a large family of metabotropic receptors on target cells that activate diverse intracellular signaling cascades. Although 5-HT’s effects on crustacean circuits have been studied in detail, the mediating receptors have been inaccessible until recently. Crustacean receptors had not been cloned and specific drugs for use in physiological experiments could therefore not be identified. Coupling properties of 5-HT receptor families are strongly conserved between phyla, but pharmacological profiles are not. The extent of pharmacological divergence among invertebrates is unclear, however, as no systematic functional profile of 5-HT receptors from related species has been determined. This work shows that orthologs of two 5-HT receptors, 5-HT2b and 5-HT1a, are highly conserved at the molecular, functional and pharmacological level between two distantly related decapod crustaceans, Panulirus interruptus and Procambarus clarkii. A suite of drugs was functionally characterized at Panulirus and Procambarus 5-HT2b and 5-HT1a receptors in cell culture, which were then used to investigate the roles of the receptors in pyloric cycle frequency modulation in the stomatogastric ganglion, a model central pattern generator. The two receptor subtypes were found to serve different roles in the circuit and their function depends on the initial state of the circuit. Finally, an antibody recognizing 5-HT1a was used to map the localization of this receptor within the crayfish nervous system. 5-HT1a is localized to somata and neuropil throughout the nerve cord, suggesting it may respond to synaptic, paracrine or neurohormonal 5-HT signals. The protein and mRNA expression levels are variable between individual animals, perhaps reflecting distinct physiological states

Distribution of Ih Channels and their Function in the Stomatogastric Ganglion

Generation of rhythmic patterns in the absence of descending commands is an essential and powerful trait of many motor networks. Cyclic rhythmic discharges of motoneurons in repeated motor activities like locomotion, mastication and respiration require underlying circuits of neurons, which are called central pattern generators (CPG). This study examined the possible roles of Ih cation channels in the pyloric network of the stomatogastric nervous system, a rhythmically active network of motoneurons that controls movements of the lobster foregut. Of specific interest were the H-current�s involvement in maintaining firing properties, the distribution of Ih channels within the stomatogastric ganglion, and a potential role for Ih in regulation of synaptic strength. I was able to confirm a homeostatic interaction of Ih with A-type potassium channels, where the over-expression of the IA shal gene after RNA injection evoked a compensatory increase of Ih in different motoneuron types. I observed an additional, non-Ih component of the hyperpolarization activated current, which was more likely to occur in shal-RNA and gfp-RNA injected neurons, compared to untreated neurons. Further, I showed that the homeostatic response of Ih increase is unidirectional; overexpression of the Ih protein PIIH did not lead to an increase of IA. In an immunocytochemical study, I found high concentrations of Ih protein localized in the fine neuropil of the stomatogastric ganglion, an area which is rich in synaptic contacts. Finally, I demonstrate a potential role for Ih in regulating synaptic transmission, for which I found evidence in electrophysiological experiments, where the amplitude of inhibitory postsynaptic potentials decreased with increasing activation of Ih

Precise Temperature Compensation of Phase in a Rhythmic Motor Pattern

Computational modeling and experimentation in a model system for network dynamics reveal how network phase relationships are temperature-compensated in terms of their underlying synaptic and intrinsic membrane currents

Ion Channel SUMOylation: a Novel Role for SUMO in Homeostatic Regulation of Multiple Ionic Conductances

Neurons can adjust their ionic currents to maintain a stable output. The homeostatic mechanisms that produce compensatory changes in ionic currents operate over multiple time scales. The rapid mechanisms that act over minutes are mostly unknown. We have been characterizing a fast homeostatic mechanism that stabilizes activity phase in the rhythmically active lateral pyloric neuron (LP) of the crustacean stomatogastric ganglion. LP activity phase is invariant. It is determined, in part, by the balance between the hyperpolarization-activated current (Ih) and the transient potassium current (IA). When LP IA is experimentally decreased, activity phase is initially disrupted, but then it recovers over minutes. This is because the decrease in IA modifies LP activity, which in turn alters cytosolic Ca2+ levels. Ca-dependent enzymes then mediate a reduction in LP Ih to restore the balance between the two conductances. We have been studying the molecular mechanisms that correlate LP IA and Ih in an activity- dependent fashion. We have found that neuronal activity adjusts the level of ion channel post- translational modification by Small Ubiquitin-like Modifier (SUMO), a peptide which when conjugated to target proteins alters their protein-protein interactions. Using a heterologous expression system, we showed that enhancing SUMOylation of HCN or Kv4 ion channels that mediate Ih and IA, respectively, produced opposite effects on the amplitudes of Ih and IA. We also demonstrated that a given change in activity produced the opposite effect on SUMOylation levels associated with each current. Thus, activity-dependent regulation of ion channel SUMOylation specified a positive correlation between the two currents. We have also demonstrated that activity-dependent regulation of ion channel SUMOylation is conditional; it only occurs in the presence of the appropriate modulatory tone. We showed this is because modulators, like dopamine, specify the targets of the SUMOylation machinery. In sum, we have discovered a novel mechanism that acts over minutes to correlate ionic conductances and thereby stabilize neuronal output

Robust dynamical invariants in sequential neural activity

By studying different sources of temporal variability in central pattern generator (CPG) circuits, we unveil fundamental aspects of the instantaneous balance between flexibility and robustness in sequential dynamics -a property that characterizes many systems that display neural rhythms. Our analysis of the triphasic rhythm of the pyloric CPG (Carcinus maenas) shows strong robustness of transient dynamics in keeping not only the activation sequences but also specific cycle-by-cycle temporal relationships in the form of strong linear correlations between pivotal time intervals, i.e. dynamical invariants. The level of variability and coordination was characterized using intrinsic time references and intervals in long recordings of both regular and irregular rhythms. Out of the many possible combinations of time intervals studied, only two cycle-by-cycle dynamical invariants were identified, existing even outside steady states. While executing a neural sequence, dynamical invariants reflect constraints to optimize functionality by shaping the actual intervals in which activity emerges to build the sequence. Our results indicate that such boundaries to the adaptability arise from the interaction between the rich dynamics of neurons and connections. We suggest that invariant temporal sequence relationships could be present in other networks, including those shaping sequences of functional brain rhythms, and underlie rhythm programming and functionalityThis work has been supported by Spanish grants MINECO DPI2015-65833-P, TIN2017-84452-R, PGC2018-095895-B-I00 (http:// www.mineco.gob.es/), and ONRG grant N62909-14-1-N279

Monoaminergic Tone Supports Conductance Correlations and Stabilizes Activity Features in Pattern Generating Neurons of the Lobster, Panulirus interruptus

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

Structural and Kinetic Studies of Drug-Resistant Mutants of HIV-1 Protease

The employment of HIV-1 protease (PR) inhibitors (PIs) in antiviral therapy has been successful in reducing mortality of HIV/AIDS patients. However, the long-term efficacy of PIs is challenged by the rapid emergence of drug-resistant mutants of PR. To understand the underlying mechanism of drug resistance, structures and activities of HIV-1 PR and its drug resistant mutants have been extensively studied. Here, PR mutants PRR8Q, PRD30N, PRI47V, PRI50V, PRI54M, PRV82A, and PRN88D/S bearing single substitutions have been investigated by crystallography and kinetics. GRL-0519 is a potent new antiviral inhibitor of HIV-1 PR that possesses tris-tetrahydrofuran (tris-THF) as the P2 ligand. The crystal structures of GRL-0519 were determined at resolutions of 1.06-1.49 Å in complex with the mutants PRR8Q, PRD30N, PRI50V, PRI54M, and PRV82A. I50V lost its interaction with inhibitor while V82Aand I54M compensated for the mutation through the main chain shift and flexibility of 80’s loop (residues 78-82), respectively. The structural changes may account for the worst inhibition of GRL-0519 for PRI50V (60-fold decrease relative to wild-type enzyme)and moderate inhibition for PRI54M and PRV82A (6-7-fold decrease). The large tris-THF group at P2 provides a good fit in the S2 subsite and may be effective against resistant virus with mutations of residues in this subsite. SQV and DRV are two clinical inhibitors that were designed to target the wild type PR and its drug resistant mutants, respectively. The crystal structures of PR mutants PRI47V, PRN88D/s in complex with DRV and mutants PRI47V and PRN88D in complex with SQV with resolutions of 1.13-1.72 Å were also analyzed. Mutation I47V gained more hydrophobic interactions with DRV and SQV. Interestingly, the structural changes did not affect the inhibition of both inhibitors for PRI47V (relative Ki is 0.7 and 1 for DRV and SQV, respectively). DRV and SQV showed 8-fold increase in Ki for PRN88D and only very subtle local changes have been observed on the structures. DRV induced 0.3 fold reduction in Ki for PRN88S and the distal structural changes have been transferred to the active site. This study provided fundamental information for understanding drug resistance and future design of potential antiviral drugs

The pyloric neural circuit of the herbivorous crab Pugettia producta shows limited sensitivity to several neuromodulators that elicit robust effects in more opportunistically feeding decapods

Modulation of neural circuits in the crustacean stomatogastric nervous system (STNS) allows flexibility in the movements of the foregut musculature. The extensive repertoire of such resulting motor patterns in dietary generalists is hypothesized to permit these animals to process varied foods. The foregut and STNS of Pugettia producta are similar to those of other decapods, but its diet is more uniform, consisting primarily of kelp. We investigated the distribution of highly conserved neuromodulators in the stomatogastric ganglion (STG) and neuroendocrine organs of Pugettia, and documented their effects on its pyloric rhythm. Using immunohistochemistry, we found that the distributions of Cancer borealis tachykinin-related peptide I (CabTRP I), crustacean cardioactive peptide (CCAP), proctolin, red pigment concentrating hormone (RPCH) and tyrosine hydroxylase (dopamine) were similar to those of other decapods. For all peptides except proctolin, the isoforms responsible for the immunoreactivity were confirmed by mass spectrometry to be the authentic peptides. Only two modulators had physiological effects on the pyloric circuit similar to those seen in other species. In non-rhythmic preparations, proctolin and the muscarinic acetylcholine agonist oxotremorine consistently initiated a full pyloric rhythm. Dopamine usually activated a pyloric rhythm, but this pattern was highly variable. In only about 25% of preparations, RPCH activated a pyloric rhythm similar to that seen in other species. CCAP and CabTRP I had no effect on the pyloric rhythm. Thus, whereas Pugettia possesses all the neuromodulators investigated, its pyloric rhythm, when compared with other decapods, appears less sensitive to many of them, perhaps because of its limited diet