Serotonergic cerebral cells control activity of cilia in the foregut of the pteropod mollusk Clione limacina.

Bilaterally symmetrical pair of serotonergic cells, named C1 in Clione, has been described in the cerebral ganglia of all gastropod species. Here we describe a new role of C1 cells in gastropod mollusks: control of activity of ciliated epithelium in the foregut. Detailed morphological investigation of C1 neurons in the pteropod mollusk Clione limacina revealed that these cells among other destinations send their neurites into foregut where they produce intense arborization with large varicosities along the processes. Intracellular stimulation of a single C1 induced pronounced activation (often followed by inhibition) of cilia lining the foregut. This activation was substantially reduced by serotonin antagonist mianserin. Bath application of serotonin also induced transient increase in ciliary transport rate, followed by inhibition of ciliary activity up to its full cessation in some areas of isolated foregut. These data suggest that C1 in Clione may use serotonin to influence cilia in the foregut. Taking into account high homology of serotonergic cerebral cells across studied species we can speculate that these cells may be involved in the neural control of cilia in the foregut in other gastropod mollusks.

Buccal neurons activate ciliary beating in the foregut of the pteropod mollusk Clione limacina.

Beating of cilia lining the foregut of gastropods facilitates the swallowing of food and, therefore, plays a role in feeding behavior. Despite the fact that neural control of feeding is well studied in mollusks, no neurons controlling ciliary beating in the foregut have been identified to date. Here we describe for the first time a pair of buccal neurons innervating the foregut of Clione. Intracellular stimulation of these neurons induced vigorous activation of cilia lining the foregut in a semi-intact preparation. Using immunochemistry labeling, buccal foregut cells were found to contain peptides similar to CNP neuropeptides of the terrestrial snail Helix lucorum. Application of DYPRL-amide, a member of the Helix CNP peptide family, mimicked the effect of buccal foregut cell stimulation on ciliary activity. Induction of fictive feeding in an isolated CNS preparation resulted in the activation of buccal foregut cells suggesting that these cells control ciliary beating in the foregut during feeding. Thus, cilia-activating buccal neurons may represent a new intrinsic element of the neural control of feeding in gastropods.

Neural control of heartbeat during two antagonistic behaviors: whole body withdrawal and escape swimming in the mollusk Clione limacina.

Two cardioexcitatory and one cardioinhibitory neural groups have been previously identified as the central cardioregulatory system in the pteropod mollusk Clione limacina. We describe in this study one additional element of the central cardioregulatory system, which consists of a large intestinal neuron named Z-cell with a novel effect on the heart activity. Intracellular stimulation of the Z-cell induced only auricle contractions with no effect on the ventricle activity. The Z-cell processes were traced down to the heart, and vigorous branching was found in the auricle tissue. Specific patterns of activity of the Z-cell as well as intestinal heart excitatory and inhibitory neurons were studied during initiation of two behaviors--whole body withdrawal and escape swimming. It was found that initiation of both behaviors was accompanied by activation of Z-cell and intestinal heart excitor neurons. The firing rate of neurons induced by sensory stimuli was sufficient to trigger auricle contractions in the semi-intact preparations. Video analysis of heart activity revealed that auricle indeed was activated during both active and passive avoidance reactions, though the intensity and delay of the activation were different. The possible physiological role of the auricle contractions during antagonistic forms of behavior is discussed.

NTnC-like genetically encoded calcium indicator with a positive and enhanced response and fast kinetics.

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells.

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi.

Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator in vitro, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP in vivo to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.

A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites.

Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca2+-binding sites. The engineered sensor, called NTnC, uses TnC as the Ca2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca2+. Using NTnC, we have visualized Ca2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca2+ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope.

Homolog of protein kinase Mζ maintains context aversive memory and underlying long-term facilitation in terrestrial snail Helix.

It has been shown that a variety of long-term memories in different regions of the brain and in different species are quickly erased by local inhibition of protein kinase Mζ (PKMζ), a persistently active protein kinase. Using antibodies to mammalian PKMζ, we describe in the present study the localization of immunoreactive molecules in the nervous system of the terrestrial snail Helix lucorum. Presence of a homolog of PKMζ was confirmed with transcriptomics. We have demonstrated in behavioral experiments that contextual fear memory disappeared under a blockade of PKMζ with a selective peptide blocker of PKMζ zeta inhibitory peptide (ZIP), but not with scrambled ZIP. If ZIP was combined with a "reminder" (20 min in noxious context), no impairment of the long-term contextual memory was observed. In electrophysiological experiments we investigated whether PKMζ takes part in the maintenance of long-term facilitation (LTF) in the neural circuit mediating tentacle withdrawal. LTF of excitatory synaptic inputs to premotor interneurons was induced by high-frequency nerve stimulation combined with serotonin bath applications and lasted at least 4 h. We found that bath application of 2 × 10(-6) M ZIP at the 90th min after the tetanization reduced the EPSP amplitude to the non-tetanized EPSP values. Applications of the scrambled ZIP peptide at a similar time and concentration didn't affect the EPSP amplitudes. In order to test whether effects of ZIP are specific to the synapses, we performed experiments with LTF of somatic membrane responses to local glutamate applications. It was shown earlier that serotonin application in such an "artificial synapse" condition elicits LTF of responses to glutamate. It was found that ZIP had no effect on LTF in these conditions, which may be explained by the very low concentration of PKMζ molecules in somata of these identified neurons, as evidenced by immunochemistry. Obtained results suggest that the Helix homolog of PKMζ might be involved in post-induction maintenance of long-term changes in the nervous system of the terrestrial snail.

Functional changes in the snail statocyst system elicited by microgravity.

BACKGROUND: The mollusk statocyst is a mechanosensing organ detecting the animal's orientation with respect to gravity. This system has clear similarities to its vertebrate counterparts: a weight-lending mass, an epithelial layer containing small supporting cells and the large sensory hair cells, and an output eliciting compensatory body reflexes to perturbations. METHODOLOGY/PRINCIPAL FINDINGS: In terrestrial gastropod snail we studied the impact of 16- (Foton M-2) and 12-day (Foton M-3) exposure to microgravity in unmanned orbital missions on: (i) the whole animal behavior (Helix lucorum L.), (ii) the statoreceptor responses to tilt in an isolated neural preparation (Helix lucorum L.), and (iii) the differential expression of the Helix pedal peptide (HPep) and the tetrapeptide FMRFamide genes in neural structures (Helix aspersa L.). Experiments were performed 13-42 hours after return to Earth. Latency of body re-orientation to sudden 90° head-down pitch was significantly reduced in postflight snails indicating an enhanced negative gravitaxis response. Statoreceptor responses to tilt in postflight snails were independent of motion direction, in contrast to a directional preference observed in control animals. Positive relation between tilt velocity and firing rate was observed in both control and postflight snails, but the response magnitude was significantly larger in postflight snails indicating an enhanced sensitivity to acceleration. A significant increase in mRNA expression of the gene encoding HPep, a peptide linked to ciliary beating, in statoreceptors was observed in postflight snails; no differential expression of the gene encoding FMRFamide, a possible neurotransmission modulator, was observed. CONCLUSIONS/SIGNIFICANCE: Upregulation of statocyst function in snails following microgravity exposure parallels that observed in vertebrates suggesting fundamental principles underlie gravi-sensing and the organism's ability to adapt to gravity changes. This simple animal model offers the possibility to describe general subcellular mechanisms of nervous system's response to conditions on Earth and in space.

Neural mechanisms underlying co-activation of functionally antagonistic motoneurons during a Clione feeding behavior.

The ability of some neural networks to produce multiple motor patterns required during different behaviors is a well-documented phenomenon. We describe here a dramatic transition from coordinated inhibition between two functionally antagonistic groups of motoneurons to their co-activation in the feeding neural network of the predatory mollusk Clione limacina. To seize its prey, Clione uses specialized oral appendages, called buccal cones, which are controlled by two groups of motoneurons: cerebral A (Cr-A) neurons controlling buccal cone protraction and cerebral B (Cr-B) neurons controlling buccal cone retraction. When Cr-A neurons are active, Cr-B neurons usually receive strong inhibitory inputs that terminate their firing, which leads to the full protraction and elongation of the buccal cones. We have found, however, that the Cr-A and Cr-B motoneurons sometimes burst simultaneously without any traces of inhibition in the Cr-B motoneurons. This transformation of the neural network activity from inhibitory interactions to co-activation presumably occurs during the late "extraction" period of the feeding behavior when buccal cones become partially retracted and rhythmically active. The transition from the inhibitory interaction to co-activation is controlled by the activity of a single pair of cerebral interneurons (Cr-Aint interneurons), which are electrically coupled to the Cr-A neurons and monosynaptically inhibit Cr-B neurons. Normally, the Cr-Aint interneurons are active along with Cr-A motoneurons and inhibit Cr-B motoneurons. During a period of co-activation, however, these interneurons do not produce spikes, thus allowing Cr-A motoneuron activation without inhibition of the Cr-B motoneurons.

Coordinated excitatory effect of GABAergic interneurons on three feeding motor programs in the mollusk Clione limacina.

Coordination between different motor centers is essential for the orderly production of all complex behaviors. Understanding the mechanisms of such coordination during feeding behavior in the carnivorous mollusk Clione limacina is the main goal of the current study. A bilaterally symmetrical interneuron identified in the cerebral ganglia and designated Cr-BM neuron produced coordinated activation of neural networks controlling three main feeding structures: prey capture appendages called buccal cones, chitinous hooks used for prey extraction from the shell, and the toothed radula. The Cr-BM neuron produced strong excitatory inputs to motoneurons controlling buccal cone protraction. It also induced a prominent activation of the neural networks controlling radula and hook rhythmic movements. In addition to the overall activation, Cr-BM neuron synaptic inputs to individual motoneurons coordinated their activity in a phase-dependent manner. The Cr-BM neuron produced depolarizing inputs to the radula protractor and hook retractor motoneurons, which are active in one phase, and hyperpolarizing inputs to the radula retractor and hook protractor motoneurons, which are active in the opposite phase. The Cr-BM neuron used GABA as its neurotransmitter. It was found to be GABA-immunoreactive in the double-labeling experiments. Exogenous GABA mimicked the effects produced by Cr-BM neuron on the postsynaptic neurons. The GABA antagonists bicuculline and picrotoxin blocked Cr-BM neuron-induced PSPs. The prominent coordinating effect produced by the Cr-BM neuron on the neural networks controlling three major elements of the feeding behavior in Clione suggests that this interneuron is an important part of the higher-order system for the feeding behavior.

Identification of mechanoafferent neurons in terrestrial snail: response properties and synaptic connections.

In this study, we describe the putative mechanosensory neurons, which are involved in the control of avoidance behavior of the terrestrial snail Helix lucorum. These neurons, which were termed pleural ventrolateral (PlVL) neurons, mediated part of the withdrawal response of the animal via activation of the withdrawal interneurons. Between 15 and 30 pleural mechanosensory neurons were located on the ventrolateral side of each pleural ganglion. Intracellular injection of neurobiotin revealed that all PlVL neurons sent their axons into the skin nerves. The PlVL neurons had no spontaneous spike activity or fast synaptic potentials. In the reduced "CNS-foot" preparations, mechanical stimulation of the skin covering the dorsal surface of the foot elicited spikes in the PlVL neurons without any noticeable prepotential activity. Mechanical stimulus-induced action potentials in these cells persisted in the presence of high-Mg(2+)/zero-Ca(2+) saline. Each neuron had oval-shaped receptive field 5-20 mm in length located on the dorsal surface of the foot. Partial overlapping of the receptive fields of different neurons was observed. Intracellular stimulation of the PlVL neurons produced excitatory inputs to the parietal and pleural withdrawal interneurons, which are known to control avoidance behavior. The excitatory postsynaptic potentials (EPSPs) in the withdrawal interneurons were induced in 1:1 ratio to the PlVL neuron spikes, and spike-EPSP latency was short and highly stable. These EPSPs also persisted in the high-Mg(2+)/high-Ca(2+) saline, suggesting monosynaptic connections. All these data suggest that PlVL cells were the primary mechanosensory neurons.

Phase-locked coordination between two rhythmically active feeding structures in the mollusk Clione limacina. I. Motor neurons.

Coordination between different motor centers is essential for the orderly production of all complex behaviors, in both vertebrates and invertebrates. The current study revealed that rhythmic activities of two feeding structures of the pteropod mollusk Clione limacina, radula and hooks, which are used to extract the prey from its shell, are highly coordinated in a phase-dependent manner. Hook protraction always coincided with radula retraction, while hook retraction coincided with radula protraction. Thus hooks and radula were always moving in the opposite phases, taking turns grabbing and pulling the prey tissue out of the shell. Identified buccal ganglia motor neurons controlling radula and hooks protraction and retraction were rhythmically active in the same phase-dependent manner. Hook protractor motor neurons were active in the same phase with radula retractor motor neurons, while hook retractor motor neurons burst in phase with radula protractor motor neurons. One of the main mechanisms underlying the phase-locked coordination was electrical coupling between hook protractor and radula retractor motor neurons. In addition, reciprocal inhibitory synaptic connections were found between hook protractor and radula protractor motor neurons. These electrical and inhibitory synaptic connections ensure that rhythmically active hooks and radula controlling motor neurons are coordinated in the specific phase-dependent manner described above. The possible existence of a single multifunctional central pattern generator for both radula and hook motor centers is discussed.