Nitric oxide synthesis and cGMP production is important for neurite growth and synapse remodeling after axotomy.

Nitric oxide (NO) is an important signaling molecule with a variety of functions in the CNS, including a potential role in modulating neuronal growth and synapse formation. In the present study, we used tractable, identified neurons in the CNS of the pond snail Lymnaea stagnalis to study the role of endogenous NO signaling in neuronal growth and synaptic remodeling after nerve injury. Axonal damage of L. stagnalis neurons B1 and B2 induces extensive central growth of neurites that is accompanied by changes in existing electrical connections, the transient formation of novel electrical connections, and the formation of a novel excitatory chemical synapse from B2 to B1 neurons. Partial chronic inhibition of endogenous NO synthesis reduces neurite growth in NO-synthase-expressing B2, but has only minor effects on NOS-negative B1 neurons. Chronic application of an NO donor while inhibiting endogenous NO synthesis rescues neurite extension in B2 neurons and boosts growth of B1 neurons. Blocking soluble guanylate cyclase activity completely suppresses neurite extension and synaptic remodeling after nerve crush, demonstrating the importance of cGMP in these processes. Interestingly, inhibition of cGMP-dependent protein kinase only suppresses chemical synapse formation without effects on neuronal growth and electrical synapse remodeling. We conclude that NO signaling via cGMP is an important modulator of both neurite growth and synaptic remodeling after nerve crush. However, differential effects of cGMP-dependent protein kinase inhibition on neurite growth and synaptic remodeling suggest that these effects are mediated by separate signaling pathways.

Cloning and characterization of a P2X receptor expressed in the central nervous system of Lymnaea stagnalis.

P2X receptors are membrane ion channels gated by extracellular ATP. Mammals possess seven distinct P2X subtypes (P2X1-7) that have important functions in a wide array of physiological processes including roles in the central nervous system (CNS) where they have been linked to modulation of neurotransmitter release. We report here the cloning and functional characterization of a P2X receptor from the mollusc Lymnaea stagnalis. This model organism has a relatively simple CNS consisting of large readily identifiable neurones, a feature which together with a well characterized neuronal circuitry for important physiological processes such as feeding and respiration makes it an attractive potential model to examine P2X function. Using CODEHOP PCR we identified a single P2X receptor (LymP2X) in Lymnaea CNS which was subsequently cloned by RT-PCR. When heterologously expressed in Xenopus oocytes, LymP2X exhibited ATP evoked inward currents (EC(50) 6.2 µM) which decayed during the continued presence of agonist. UTP and ADP did not activate the receptor whereas αßmeATP was a weak agonist. BzATP was a partial agonist with an EC(50) of 2.4 µM and a maximal response 33% smaller than that of ATP. The general P2 receptor antagonists PPADS and suramin both inhibited LymP2X currents with IC(50) values of 8.1 and 27.4 µM respectively. LymP2X is inhibited by acidic pH whereas Zn(2+) and Cu(2+) ions exhibited a biphasic effect, potentiating currents up to 100 µM and inhibiting at higher concentrations. Quantitative RT-PCR and in situ hybridization detected expression of LymP2X mRNA in neurones of all CNS ganglia suggesting this ion channel may have widespread roles in Lymnaea CNS function.

A self-organizing state-space-model approach for parameter estimation in hodgkin-huxley-type models of single neurons.

Traditional approaches to the problem of parameter estimation in biophysical models of neurons and neural networks usually adopt a global search algorithm (for example, an evolutionary algorithm), often in combination with a local search method (such as gradient descent) in order to minimize the value of a cost function, which measures the discrepancy between various features of the available experimental data and model output. In this study, we approach the problem of parameter estimation in conductance-based models of single neurons from a different perspective. By adopting a hidden-dynamical-systems formalism, we expressed parameter estimation as an inference problem in these systems, which can then be tackled using a range of well-established statistical inference methods. The particular method we used was Kitagawa's self-organizing state-space model, which was applied on a number of Hodgkin-Huxley-type models using simulated or actual electrophysiological data. We showed that the algorithm can be used to estimate a large number of parameters, including maximal conductances, reversal potentials, kinetics of ionic currents, measurement and intrinsic noise, based on low-dimensional experimental data and sufficiently informative priors in the form of pre-defined constraints imposed on model parameters. The algorithm remained operational even when very noisy experimental data were used. Importantly, by combining the self-organizing state-space model with an adaptive sampling algorithm akin to the Covariance Matrix Adaptation Evolution Strategy, we achieved a significant reduction in the variance of parameter estimates. The algorithm did not require the explicit formulation of a cost function and it was straightforward to apply on compartmental models and multiple data sets. Overall, the proposed methodology is particularly suitable for resolving high-dimensional inference problems based on noisy electrophysiological data and, therefore, a potentially useful tool in the construction of biophysical neuron models.

The penultimate arginine of the carboxyl terminus determines slow desensitization in a P2X receptor from the cattle tick Boophilus microplus.

P2X ion channels have been functionally characterized from a range of eukaryotes. Although these receptors can be broadly classified into fast and slow desensitizing, the molecular mechanisms underlying current desensitization are not fully understood. Here, we describe the characterization of a P2X receptor from the cattle tick Boophilus microplus (BmP2X) displaying extremely slow current kinetics, little desensitization during ATP application, and marked rundown in current amplitude between sequential responses. ATP (EC(50), 67.1 µM) evoked concentration-dependent currents at BmP2X that were antagonized by suramin (IC(50), 4.8 µM) and potentiated by the antiparasitic drug amitraz. Ivermectin did not potentiate BmP2X currents, but the mutation M362L conferred ivermectin sensitivity. To investigate the mechanisms underlying slow desensitization we generated intracellular domain chimeras between BmP2X and the rapidly desensitizing P2X receptor from Hypsibius dujardini. Exchange of N or C termini between these fast- and slow-desensitizing receptors altered the rate of current desensitization toward that of the donor channel. Truncation of the BmP2X C terminus identified the penultimate residue (Arg413) as important for slow desensitization. Removal of positive charge at this position in the mutant R413A resulted in significantly faster desensitization, which was further accentuated by the negatively charged substitution R413D. R413A and R413D, however, still displayed current rundown to sequential ATP application. Mutation to a positive charge (R413K) reconstituted the wild-type phenotype. This study identifies a new determinant of P2X desensitization where positive charge at the end of the C terminal regulates current flow and further demonstrates that rundown and desensitization are governed by distinct mechanisms.

A P2X receptor from the tardigrade species Hypsibius dujardini with fast kinetics and sensitivity to zinc and copper.

BACKGROUND: Orthologs of the vertebrate ATP gated P2X channels have been identified in Dictyostelium and green algae, demonstrating that the emergence of ionotropic purinergic signalling was an early event in eukaryotic evolution. However, the genomes of a number of animals including Drosophila melanogaster and Caenorhabditis elegans, both members of the Ecdysozoa superphylum, lack P2X-like proteins, whilst other species such as the flatworm Schistosoma mansoni have P2X proteins making it unclear as to what stages in evolution P2X receptors were lost. Here we describe the functional characterisation of a P2X receptor (HdP2X) from the tardigrade Hypsibius dujardini demonstrating that purinergic signalling is preserved in some ecdysozoa. RESULTS: ATP (EC50 approximately 44.5 microM) evoked transient inward currents in HdP2X with millisecond rates of activation and desensitisation. HdP2X is antagonised by pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (IC50 15.0 microM) and suramin (IC50 22.6 microM) and zinc and copper inhibit ATP-evoked currents with IC50 values of 62.8 microM and 19.9 microM respectively. Site-directed mutagenesis showed that unlike vertebrate P2X receptors, extracellular histidines do not play a major role in coordinating metal binding in HdP2X. However, H306 was identified as playing a minor role in the actions of copper but not zinc. Ivermectin potentiated responses to ATP with no effect on the rates of current activation or decay. CONCLUSION: The presence of a P2X receptor in a tardigrade species suggests that both nematodes and arthropods lost their P2X genes independently, as both traditional and molecular phylogenies place the divergence between Nematoda and Arthropoda before their divergence from Tardigrada. The phylogenetic analysis performed in our study also clearly demonstrates that the emergence of the family of seven P2X channels in human and other mammalian species was a relatively recent evolutionary event that occurred subsequent to the split between vertebrates and invertebrates. Furthermore, several characteristics of HdP2X including fast kinetics with low ATP sensitivity, potentiation by ivermectin in a channel with fast kinetics and distinct copper and zinc binding sites not dependent on histidines make HdP2X a useful model for comparative structure-function studies allowing a better understanding of P2X receptors in higher organisms.

Characterization of NO-sensitive guanylyl cyclase: expression in an identified interneuron involved in NO-cGMP-dependent memory formation.

In a number of neuronal models of learning signalling by endogenous nitric oxide (NO), produced by the enzyme NO synthase (NOS), is essential for the formation of long-term memory (LTM). For example, in the molluscan model system Lymnaea, NO is required for LTM formation in the first few hours after one-trial reward conditioning. Furthermore, conditioning leads to transient up-regulation of the NOS gene in identified modulatory neurons, the cerebral giant cells (CGCs), which are known to be involved in LTM formation. In Lymnaea nothing is known however about the structure and localization of the major receptor for NO, the soluble guanylyl cyclase (sGC). Here we report on the cloning and characterization of both alpha and beta subunits of NO-sensitive sGC and show that they are coexpressed in the CGCs. Furthermore, our electrophysiological experiments on isolated CGCs show that these neurons respond to NO by generating a prolonged depolarization of the membrane potential. Moreover, we demonstrate that this depolarization is blocked by ODQ, supporting our hypothesis that it is mediated by sGC.

Dynamic control of a central pattern generator circuit: a computational model of the snail feeding network.

Central pattern generators (CPGs) are networks underlying rhythmic motor behaviours and they are dynamically regulated by neuronal elements that are extrinsic or intrinsic to the rhythmogenic circuit. In the feeding system of the pond snail, Lymnaea stagnalis, the extrinsic slow oscillator (SO) interneuron controls the frequency of the feeding rhythm and the N3t (tonic) has a dual role; it is an intrinsic CPG interneuron, but it also suppresses CPG activity in the absence of food, acting as a decision-making element in the feeding circuit. The firing patterns of the SO and N3t neurons and their synaptic connections with the rest of the CPG are known, but how these regulate network function is not well understood. This was investigated by building a computer model of the feeding network based on a minimum number of cells (N1M, N2v and N3t) required to generate the three-phase motor rhythm together with the SO that was used to activate the system. The intrinsic properties of individual neurons were represented using two-compartment models containing currents of the Hodgkin-Huxley type. Manipulations of neuronal activity in the N3t and SO neurons in the model produced similar quantitative effects to food and electrical stimulation in the biological network indicating that the model is a useful tool for studying the dynamic properties of the feeding circuit. The model also predicted novel effects of electrical stimulation of two CPG interneurons (N1M and N2v). When tested experimentally, similar effects were found in the biological system providing further validation of our model.

Modulation of serotonergic neurotransmission by nitric oxide.

Nitric oxide (NO) and serotonin (5-HT) are two neurotransmitters with important roles in neuromodulation and synaptic plasticity. There is substantial evidence for a morphological and functional overlap between these two neurotransmitter systems, in particular the modulation of 5-HT function by NO. Here we demonstrate for the first time the modulation of an identified serotonergic synapse by NO using the synapse between the cerebral giant cell (CGC) and the B4 neuron within the feeding network of the pond snail Lymnaea stagnalis as a model system. Simultaneous electrophysiological recordings from the pre- and postsynaptic neurons show that blocking endogenous NO production in the intact nervous system significantly reduces the B4 response to CGC activity. The blocking effect is frequency dependent and is strongest at low CGC frequencies. Conversely, bath application of the NO donor DEA/NONOate significantly enhances the CGC-B4 synapse. The modulation of the CGC-B4 synapse is mediated by the soluble guanylate cyclase (sGC)/cGMP pathway as demonstrated by the effects of the sGC antagonist 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). NO modulation of the CGC-B4 synapse can be mimicked in cell culture, where application of 5-HT puffs to isolated B4 neurons simulates synaptic 5-HT release. Bath application of diethylamine NONOate (DEA/NONOate) enhances the 5-HT induced response in the isolated B4 neuron. However, the cell culture experiment provided no evidence for endogenous NO production in either the CGC or B4 neuron suggesting that NO is produced by an alternative source. Thus we conclude that NO modulates the serotonergic CGC-B4 synapse by enhancing the postsynaptic 5-HT response.

Role of delayed nonsynaptic neuronal plasticity in long-term associative memory.

BACKGROUND: It is now well established that persistent nonsynaptic neuronal plasticity occurs after learning and, like synaptic plasticity, it can be the substrate for long-term memory. What still remains unclear, though, is how nonsynaptic plasticity contributes to the altered neural network properties on which memory depends. Understanding how nonsynaptic plasticity is translated into modified network and behavioral output therefore represents an important objective of current learning and memory research. RESULTS: By using behavioral single-trial classical conditioning together with electrophysiological analysis and calcium imaging, we have explored the cellular mechanisms by which experience-induced nonsynaptic electrical changes in a neuronal soma remote from the synaptic region are translated into synaptic and circuit level effects. We show that after single-trial food-reward conditioning in the snail Lymnaea stagnalis, identified modulatory neurons that are extrinsic to the feeding network become persistently depolarized between 16 and 24 hr after training. This is delayed with respect to early memory formation but concomitant with the establishment and duration of long-term memory. The persistent nonsynaptic change is extrinsic to and maintained independently of synaptic effects occurring within the network directly responsible for the generation of feeding. Artificial membrane potential manipulation and calcium-imaging experiments suggest a novel mechanism whereby the somal depolarization of an extrinsic neuron recruits command-like intrinsic neurons of the circuit underlying the learned behavior. CONCLUSIONS: We show that nonsynaptic plasticity in an extrinsic modulatory neuron encodes information that enables the expression of long-term associative memory, and we describe how this information can be translated into modified network and behavioral output.

Associative memory stored by functional novel pathway rather than modifications of preexisting neuronal pathways.

Associative conditioning involves changes in the processing pathways activated by sensory information to link the conditioned stimulus (CS) to the conditioned behavior. Thus, conditioning can recruit neuronal elements to form new pathways for the processing of the CS and/or can change the strength of existing pathways. Using a behavioral and systems level electrophysiological approach on a tractable invertebrate circuit generating feeding in the mollusk Lymnaea stagnalis, we identified three independent pathways for the processing of the CS amyl acetate used in appetitive conditioning. Two of these pathways, one suppressing and the other stimulating feeding, mediate responses to the CS in naive animals. The effects of these two pathways on feeding behavior are unaltered by conditioning. In contrast, the CS response of a third stimulatory pathway is significantly enhanced after conditioning, becoming an important contributor to the overall CS response. This is unusual because, in most of the previous examples in which naive animals already respond to the CS, memory formation results from changes in the strength of pathways that mediate the existing response. Here, we show that, in the molluscan feeding system, both modified and unmodified pathways are activated in parallel by the CS after conditioning, and it is their integration that results in the conditioned response.

Central localization of plasticity involved in appetitive conditioning in Lymnaea.

Learning to associate a conditioned (CS) and unconditioned stimulus (US) results in changes in the processing of CS information. Here, we address directly the question whether chemical appetitive conditioning of Lymnaea feeding behavior involves changes in the peripheral and/or central processing of the CS by using extracellular recording techniques to monitor neuronal activity at two stages of the sensory processing pathway. Our data show that appetitive conditioning does not affect significantly the overall CS response of afferent nerves connecting chemosensory structures in the lips and tentacles to the central nervous system (CNS). In contrast, neuronal output from the cerebral ganglia, which represent the first central processing stage for chemosensory information, is enhanced significantly in response to the CS after appetitive conditioning. This demonstrates that chemical appetitive conditioning in Lymnaea affects the central, but not the peripheral processing of chemosensory information. It also identifies the cerebral ganglia of Lymnaea as an important site for neuronal plasticity and forms the basis for detailed cellular studies of neuronal plasticity.

Endogenous and network properties of Lymnaea feeding central pattern generator interneurons.

Understanding central pattern generator (CPG) circuits requires a detailed knowledge of the intrinsic cellular properties of the constituent neurons. These properties are poorly understood in most CPGs because of the complexity resulting from interactions with other neurons of the circuit. This is also the case in the feeding network of the snail, Lymnaea, one of the best-characterized CPG networks. We addressed this problem by isolating the interneurons comprising the feeding CPG in cell culture, which enabled us to study their basic intrinsic electrical and pharmacological cellular properties without interference from other network components. These results were then related to the activity patterns of the neurons in the intact feeding network. The most striking finding was the intrinsic generation of plateau potentials by medial N1 (N1M) interneurons. This property is probably critical for rhythm generation in the whole feeding circuit because the N1M interneurons are known to play a pivotal role in the initiation of feeding cycles in response to food. Plateau potential generation in another cell type, the ventral N2 (N2v), appeared to be conditional on the presence of acetylcholine. Examination of the other isolated feeding CPG interneurons [lateral N1 (N1L), dorsal N2 (N2d), phasic N3 (N3p)] and the modulatory slow oscillator (SO) revealed no significant intrinsic properties in relation to pattern generation. Instead, their firing patterns in the circuit appear to be determined largely by cholinergic and glutamatergic synaptic inputs from other CPG interneurons, which were mimicked in culture by application of these transmitters. This is an example of a CPG system where the initiation of each cycle appears to be determined by the intrinsic properties of a key interneuron, N1M, but most other features of the rhythm are probably determined by network interactions.