Search

Subcellular targeting of opsins in optogenetics provides new possibilities for studying the function of nerve cells. One of the widely used motifs for central targeting of opsins is the motif of the potential-dependent potassium channel Kv2.1. In our study, ChR2-Venus-Kv2.1 construct was expressed in the layer 2/3 pyramidal neurons of murine cerebral cortex by means of in utero electroporation. We found that, although the majority of neurons expressing ChR2-Venus-Kv2.1 demonstrated mainly central localization of fluorescence, limited to the soma, proximal dendrites, and axon, a considerable quantity of neurons remarkably exhibited disrupted targeting, i.e., fluorescent protein distributed far beyond the soma and proximal parts of all processes. We hypothesized that the observed mislocalization might have been caused by overexpression of the construct. It was found that a decrease in the plasmid concentration during the in utero electroporation from 1 to 0.35 μg/μl resulted in almost complete disappearance of the neurons that had altered targeting. Moreover, we found a pronounced correlation between the absolute brightness of the somatic fluorescence and the visible length of the apical dendrite, which also provides evidence that indeed overexpression might be the reason for the mislocalization of ChR2-Venus-Kv2.1 construct. Thus, the possibility of disruption of central targeting of ChR2 by the potassium channel Kv2.1 motif should be taken into account when using this construct in optogenetic experiments when somatic localization of the opsin is necessary. Lowering plasmid concentration during transfection procedure could be a solution for the above problem.

Heterosynaptic plasticity is a poorly studied type of synaptic plasticity in which synapses not active during the plasticity induction process are modified. The main methodological problem in studying the mechanisms of heterosynaptic plasticity using conventional techniques are the need for large numbers of experiments because of the probabilistic nature of the development of plastic changes in the synaptic inputs to neurons after intracellular tetanization – the most widely used approach to inducing heterosynaptic plasticity. To overcome this problem, we report here use of a method of delivering optogenetic stimulation to a multitude of presynaptic neurons converging on a single postsynaptic neuron. Our studies using rat brain slices showed that intracellular tetanization of a pyramidal neuron in layer 5 of the neocortex leads to the development of depression or potentiation of a proportion of the synaptic inputs from above-lying pyramidal cells in layers 2/3 expressing light-activated protein channelrhodopsin-2. Use of this stimulation method was found to allow a single experiment to yield sufficient data for statistical evaluation of the correlation between the paired pulse ratio and the amplitude of changes in EPSP after intracellular tetanization. We also showed that so-called “silent” synapses were not activated on development of long-term potentiation induced by intracellular tetanization.

The article presents four branches of multi-year research of human breathing. They are (1) modeling and study the movement of the convective flows in the artificial models of nose; (2) numerical modeling of the convective flows movement on the computer models; (3) breathing research with the help of diagnostic equipment; (4) determination of the exhaled air composition. In all our studies the breathing was examined as a dynamical process. The results of pressure, temperature, velocity and concentration CO2 measuring in the nostrils and in the significant parts of the artificial model of nasal cavity are presented. For this measurements estimated the correlation entropy, correlation dimension. The 3D geometrical model of human nasal cavities obtained from computer-aided tomography data using Mercury Amira program is also given. The 3D unstructured mesh with 1,5×107 finite elements was constructed after the segmentation using Altair Hypermesh software had been finished. The mesh was used to set up an unsteady simulation of airflow inside the obtained geometrical model. Application of DES method on the mesh of a good quality made it possible to distinguish the small-scale turbulent swirls inside the flow. A gas sensor based on spontaneous Raman scattering is proposed for the compositional analysis of single breath events. The Raman sensor is able to detect all the major gas components, i.e. N2, O2, CO2, and H2O at ambient pressure with a high temporal resolution. Concentration fluctuations within a single breath event could be resolved.

Optogenetics is a powerful technique in neuroscience that provided a great success in studying the brain functions during the last decade. Progress of optogenetics crucially depends on development of new molecular tools. Light-activated cation-conducting channelrhodopsin2 was widely used for excitation of cells since the emergence of optogenetics. In 2015 a family of natural light activated chloride channels GtACR was identified which appeared to be a very promising tool for using in optogenetics experiments as a cell silencer. Here we examined properties of GtACR2 channel expressed in the rat layer 2/3 pyramidal neurons by means of in utero electroporation. We have found that despite strong inhibition the light stimulation of GtACR2-positive neurons can surprisingly lead to generation of action potentials, presumably initiated in the axonal terminals. Thus, when using the GtACR2 in optogenetics experiments, its ability to induce action potentials should be taken into account. Our results also open an interesting possibility of using the GtACR2 both as cell silencer and cell activator in the same experiment varying the pattern of light stimulation.

To perform optogenetic prosthetics of the retinal ganglion cell receptive field, a bicistronic genetic construct carrying the genes encoding the excitatory (channelrhodopsin-2) and inhibitory (Guillardia theta anion channelrhodopsin GtACR2) rhodopsins was created. A characteristic feature of this construct was the combination of these two genes with a mutant IRES insertion between them, which ensures the exact ratio of expression levels of the first and second genes in each transfected cell. Illumination of the central part of the neuron with light with a wavelength of 470 nm induced the action potential generation in the cell. Stimulation of the peripheral neuronal region with light induced the inhibition of action potential generation. Thus, using optogenetics methods, we simulated the ON–OFF interaction in the retinal ganglion cell receptive field. Theoretically, this construct can be used for optogenetic prosthetics of degenerative retina in the case of its delivery to the ganglion cells with lentiviral vectors.

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.

Subcellular targeting of opsins in optogenetics provides new possibilities for investigating the function of nerve cells. One of the widely used motifs for central targeting of opsins is the motif of potential-dependent potassium channel Kv2.1. We have expressed construct CHR2-Venus-Kv2.1 in the layer 2/3 pyramidal neurons of the murine cerebral cortex by means of in utero electroporation. It was found that, although the majority of neurons expressing CHR2-Venus-Kv2.1 demonstrated mainly central localization of fluorescence in the soma, proximal dendrites and axon, there was also significant population of neurons with disruption of the “correct” targeting resulting in the fluorescent protein distributed uniformly throughout the entire cell surface. We have suggested that observed mislocalization was caused by overexpression of the construct. Indeed, a decrease in the plasmid concentration during the in utero electroporation procedure resulted in almost complete absence of neurons with altered targeting. Thus, the possibility of “incorrect” targeting of CHR2 by the potassium channel motif Kv2.1 should be taken into account when using this construct in optogenetic experiments.

The vestibular system receives a permanent influence from gravity and reflexively controls equilibrium. If we assume gravity has remained constant during the species' evolution, will its sensory system adapt to abrupt loss of that force? We address this question in the land snail Helix lucorum exposed to 30 days of near weightlessness aboard the Bion-M1 satellite, and studied geotactic behavior of postflight snails, differential gene expressions in statocyst transcriptome, and electrophysiological responses of mechanoreceptors to applied tilts. Each approach revealed plastic changes in the snail's vestibular system assumed in response to spaceflight. Absence of light during the mission also affected statocyst physiology, as revealed by comparison to dark-conditioned control groups. Readaptation to normal tilt responses occurred at ~20 h following return to Earth. Despite the permanence of gravity, the snail responded in a compensatory manner to its loss and readapted once gravity was restored.

Understanding the mechanism of synaptic transmission and its plasticity is one of the central goals of modern neuroscience. Neuronal culture is a very convenient model for studying mechanisms of synaptic transmission. However, investigation of two or more synaptically connected neurons in culture by conventional methods is a difficult task. In this study, we describe new protocol for studying the synaptic transmission between cultured neurons using in suspension electroporation for the expression of channelrhodopsin-2 (ChR2) which was applied to only certain subpopulation of cultured cells, leaving the majority of neurons completely untreated. We show that this technique allows reliable long-lasting (hours) recording of monosynaptic excitatory postsynaptic potentials (EPSPs) in cultured hippocampal neurons using a repeated light stimulation of neighboring ChR2-expressing neurons.

The ability of neocortical neurons to detect and encode rapid changes at their inputs is crucial for basic neuronal computations, such as coincidence detection, precise synchronization of activity and spike-timing dependent plasticity. Indeed, populations of cortical neurons can respond to subtle changes of the input very fast, on a millisecond time scale. Theoretical studies and model simulations linked the encoding abilities of neuronal populations to the fast onset dynamics of action potentials. Experimental results support this idea, however mechanisms of fast onset of action potentials in cortical neurons remain elusive. Studies in neuronal cultures, that are allowing for accurate control over conditions of growth and microenvironment during the development of neurons and provide better access to the spike initiation zone, may help to shed light on mechanisms of action potential generation and encoding. Here we characterize properties of action potential encoding in neocortical neurons grown for 11-25 days in culture. We show that encoding of high frequencies improves upon culture maturation, which is accompanied by the development of passive electrophysiological properties and action potential generation. The onset of action potentials becomes faster with culture maturation. Statistical analysis using correlations and linear model approaches identified the onset dynamics of action potentials as a major predictor of age-dependent changes of encoding. Encoding of high frequencies strongly correlated also with the input resistance of neurons. Finally, we show that maturation of encoding properties of neurons in cultures is similar to the maturation of encoding in neurons studied in slices. These results show that maturation of action potential generators and encoding is, to a large extent, determined genetically and takes place even without normal micro-environment and activity of the whole brain in vivo. This establishes neuronal cultures as a valid experimental model for studying mechanisms of action potential generation and encoding, and their maturation.

The speed of computations in neocortical networks critically depends on the ability of populations of spiking neurons to rapidly detect subtle changes in the input and translate them into firing rate changes. However, high sensitivity to perturbations may lead to explosion of noise and increased energy consumption. Can neuronal networks reconcile the requirements for high sensitivity, operation in a low-noise regime, and constrained energy consumption? Using intracellular recordings in slices from the rat visual cortex, we show that layer 2/3 pyramidal neurons are highly sensitive to minor input perturbations. They can change their population firing rate in response to small artificial excitatory postsynaptic currents (aEPSCs) immersed in fluctuating noise very quickly, within 2-2.5 ms. These quick responses were mediated by the generation of new, additional action potentials (APs), but also by shifting spikes into the response peak. In that latter case, the spike count increase during the peak and the decrease after the peak cancelled each other, thus producing quick responses without increases in total spike count and associated energy costs. The contribution of spikes from one or the other source depended on the aEPSCs timing relative to the waves of depolarization produced by ongoing activity. Neurons responded by shifting spikes to aEPSCs arriving at the beginning of a depolarization wave, but generated additional spikes in response to aEPSCs arriving towards the end of a wave. We conclude that neuronal networks can combine high sensitivity to perturbations and operation in a low-noise regime. Moreover, certain patterns of ongoing activity favor this combination and energy-efficient computations.

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.

We analyzed changes in the activity of individually identifiable neurons involved in the networks underlying feeding and withdrawal behaviors in snails before, during, and after aversive learning in vitro. Responses to food in the “reinforcing” serotonergic neurons involved in withdrawal changed significantly after training, implying that these serotonergic cells participate in the reactivation of memory and are involved in the reconsolidation process. In behavioral experiments it was shown that impairment of the functioning of the serotonergic system with the selective neurotoxin 5,7-DiHT did not change the memory, when tested once, but resulted in a complete extinction of the contextual memory after repeated reactivation of memory. Conversely, the cued memory to a specific type of food was significantly reduced but still present. Thus, we conclude that it is only for the context memory, that participation of the “reinforcing” serotonergic neurons in memory retrieval may be the gate condition for the choice between extinction/reconsolidation.

Visual stimulation often leads to elevated fluctuations of the membrane potential in the γ-frequency range (25-70 Hz) in visual cortex neurons. Recently, we have found that the strength of γ-band fluctuations is coupled to the oscillation of the membrane potential at the temporal frequency of the stimulus, so that the γ-band fluctuations are stronger at depolarization peaks, but weaker at troughs of the stimulus frequency oscillation of the membrane potential. We hypothesized that this coupling may improve stimulus encoding. Here, we tested this hypothesis by using a single-compartment conductance-based neuron model, with parameters of the input adjusted to reproduce typical features of membrane potential and spike responses, recorded in cat visual cortical neurons in vivo during the presentation of moving gratings. We show that modulation of the γ-range membrane potential fluctuations by the amplitude of the slow membrane depolarization greatly improves stimulus encoding. Moreover, changing the degree of modulation of the γ-activity by the low-frequency signal within the range typically observed in visual cortex cells had a stronger effect on both the firing rates and information rates than changing the amplitude of the low-frequency stimulus itself. Thus, modulation of the γ-activity represents an efficient mechanism for regulation of neuronal firing and encoding of the temporal characteristics of visual stimuli.

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.

SUMMARYBeating 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.

In the terrestrial snail a direct monosynaptic glutamatergic connection between the primary sensory neuron and a premotor interneuron involved in withdrawal behaviour can be functionally identified using electrophysiological techniques. We investigated the involvement of cannabinoids in regulation of this synaptic contact. The results demonstrate that the specific binding sites for agonists to mammalian type 1 cannabinoid receptors (CB1Rs) exist in the snail's nervous system. Application of a synthetic cannabinoid agonist anandamide selectively changed the efficacy of synaptic contacts between the identified neurons. A decrease in the long-term synaptic facilitation of the synaptic contact elicited by high-frequency nerve tetanization in the presence of cannabinoid agonist anandamide was observed, suggesting a possible role of endocannabinoids in regulation of plasticity at this synaptic site. The selective antagonist of CB1Rs [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] AM251 bath application was changing the efficacy of the synaptic contact only when the postsynaptic neuron had been intracellularly activated before its application. This observation implies an involvement of endocannabinoids in plasticity phenomena induced by activity in the postsynaptic target. Additional support of endocannabinoid involvement in synaptic function at this site was given by experiments in which AM251 blocked the short-term suppression of synaptic excitation evoked by low-frequency nerve tetanization, a phenomenon qualitatively similar to cannabinoid-dependent synaptically evoked suppression of excitation demonstrated in the mammalian nervous system. The results of the present study suggest an involvement of cannabinoids in the regulation of synaptic efficacy. Further, anandamide could be a candidate for an endogenous neuromessenger involved in plasticity processes.

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.

Understanding single-neuron computations and encoding performed by spike-generation mechanisms of cortical neurons is one of the central challenges for cell electrophysiology and computational neuroscience. An established paradigm to study spike encoding in controlled conditions in vitro uses intracellular injection of a mixture of signals with fluctuating currents that mimic in vivo-like background activity. However this technique has two serious limitations: it uses current injection, while synaptic activation leads to changes of conductance, and current injection is technically most feasible in the soma, while the vast majority of synaptic inputs are located on the dendrites. Recent progress in optogenetics provides an opportunity to circumvent these limitations. Transgenic expression of light-activated ionic channels, such as Channelrhodopsin2 (ChR2), allows induction of controlled conductance changes even in thin distant dendrites. Here we show that photostimulation provides a useful extension of the tools to study neuronal encoding, but it has its own limitations. Optically induced fluctuating currents have a low cutoff (~70 Hz), thus limiting the dynamic range of frequency response of cortical neurons. This leads to severe underestimation of the ability of neurons to phase-lock their firing to high frequency components of the input. This limitation could be worked around by using short (2 ms) light stimuli which produce membrane potential responses resembling EPSPs by their fast onset and prolonged decay kinetics. We show that combining application of short light stimuli to different parts of dendritic tree for mimicking distant EPSCs with somatic injection of fluctuating current that mimics fluctuations of membrane potential in vivo, allowed us to study fast encoding of artificial EPSPs photoinduced at different distances from the soma. We conclude that dendritic photostimulation of ChR2 with short light pulses provides a powerful tool to investigate population encoding of simulated synaptic potentials generated in dendrites at different distances from the soma.

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.

1. The HCS2 (Helix command specific 2) gene expressed in giant command neurons for withdrawal behavior of the terrestrial snail Helix lucorum encodes a unique hybrid precursor protein that contains a Ca-binding (EF-hand motif) protein and four small peptides (CNP1-CNP4) with similar Tyr-Pro-Arg-X aminoacid sequence at the C terminus. Previous studies suggest that under conditions of increased intracellular Ca(2+) concentration the HCS2 peptide precursor may be cleaved, and small physiologically active peptides transported to the release sites. In the present paper, intracellular localization of putative peptide products of the HCS2-encoded precursor was studied immunocytochemically by means of light and electron microscopy. 2. Polyclonal antibodies against the CNP3 neuropeptide and a Ca-binding domain of the precursor protein were used for gold labeling of ultrathin sections of identified isolated neurons maintained in culture for several days, and in same identified neurons freshly isolated from the central nervous system. 3. In freshly isolated neurons, the gold particles were mainly localized over the cytoplasmic secretory granules, with the density of labeling for the CNP3 neuropeptide being two-fold higher than for the calcium-binding domain. In cultured neurons, both antibodies mostly labeled clusters of secretory granules in growth cones and neurites of the neuron. The density of labeling for cultured neurons was the same for both antibodies, and was two-fold higher than for the freshly isolated from the central nervous system neurons. 4. The immunogold particles were practically absent in the bodies of cultured neurons. 5. The data obtained conform to the suggestion that the HCS2 gene products are transported from the cell body to the regions of growth or release sites.

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.

Increase of intracellular [Ca 2 + ] evoked by action potentials in a cell can induce long-term synaptic plasticity even without concomitant presynaptic stimulation. We used optical recording of the fluorescence of a Ca 2 + -indicator Oregon Green to investigate whether differences in results obtained with modifications of that purely postsynaptic induction protocol could be due to differential Ca 2 + influx. We compared changes of the somatic [Ca 2 + ] in layer II-III pyramidal cells in slices of rat visual cortex evoked by bursts of depolarization pulses and long depolarizing steps. During weak depolarizations, the Ca 2 + influx was proportional to the amplitude and duration of the depolarization. With suprathreshold depolarizations, the Ca 2 + influx was proportional to the number of action potentials. Because the burst depolarizations evoked more spikes than did the long duration steps, this burst protocol led to a larger Ca 2 + influx. With all stimulation protocols, the spike-induced Ca 2 + influx was reduced during blockade of N-methyl-D-aspartate (NMDA) receptors. Differences in intracellular [Ca 2 + ] increases thus may be one reason for differential effects of purely postsynaptic challenges on synaptic transmission.

In the present study, the applicability of antisense morpholino oligos for loss-of-function experiments in neurobiology was investigated. The identified withdrawal interneurons of the parietal ganglia expressing helix command neuron-specific 2 (HCS2) gene were pressure injected with HCS2 antisense or control morpholino oligo solution at a final concentration 1–4 μM. No toxic or side effects for the neural functioning were noted immediately or several hours after injection. The changes in the concentration of HCS2-encoded protein in neurons after injection were monitored by two methods, Western blotting and immunostaining of the brain. The amount of the peptide immunoreactive with the HCS2 antibody started to decline in the injected cells at day 2 post-injection, decreased four- to five-fold at day 4, and stayed at this low level thereafter. Similar results obtained by both methods suggest significant selective blockade of production of the HCS2-encoded peptide. In contrast, no substantial decrease of the HCS2-encoded polypeptide was observed after injection with control oligos. Due to the high stability of the morpholino oligos in the cell, they represent a highly efficient tool for a specific long-term blockade of gene expression in molluscan neurons.

The properties of the monosynaptic input from mechanosensory neurons to withdrawal interneurons were examined in Helix lucorum . The instantaneous I–V relation of the excitatory postsynaptic current in withdrawal interneurons was nonlinear, having a plateau region between −40 and −60 mV. On application of the blocker of vertebrate N -methyl- d -aspartate (NMDA) receptors AP5, or reduction of the Mg 2+ concentration, the current–voltage relation became more linear, suggesting that Mg 2+ may partially block the ion channel underlying the EPSC at voltages ranging from −40 to around −60 mV and the involvement of NMDA-like receptors. DNQX and 6-cyano-7-nitroquinoxaline-2,3-dione, which are known to block the glutamate non-NMDA receptors in mammals, significantly depress in a dose-dependent manner the actions of the natural transmitter. Exogenous l -glutamate applications mimicked the action of the mechanosensory neuron transmitter.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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 novel gene named Helix command-specific 2 (HCS2) was shown to be expressed predominantly in four giant parietal interneurons involved in withdrawal behavior of the terrestrial snail Helix lucorum L. and several single neurons in other ganglia. Decrease in spontaneous electrophysiological activity of neurons in the isolated CNS by 24 h incubation in saline with elevated Mg 2+ concentration significantly decreased the number of HCS2-expressing neurons. Five short-term serotonin applications (each of 10 μM), during a 24 h incubation of the nervous system in saline induced expression of the HCS2 gene in many cells in cerebral, parietal, pleural and pedal ganglia. Dopamine applications under similar conditions were not effective. Application of anisomycin or cycloheximide, known to block protein synthesis, did not prevent the induction of HCS2 expression under serotonin influence. Skin injury elicited a significant increase in the number of HCS2-expressing cells 24 h later in pleural and cerebral ganglia. Incubation of the isolated nervous system preparations for three days in culture medium elicited close to a maximum increase in number of HCS2-expressing cells. Elevation of the normal Mg 2+ concentration in the culture medium significantly decreased the number of cells demonstrating HCS2 expression. Application of the cAMP activator forskolin (10 μM) increased the expression under Mg 2+ , indicating that cAMP was involved in the up-regulation of HCS2. Application of thapsigargin (10 μM), known to release Ca 2+ from intracellular stores, was also effective in increasing expression, suggesting participation of Ca 2+ in regulation of HCS2 expression. Cellular groups expressing the HCS2 gene under different conditions seem to be functionally related since it was demonstrated earlier that some neurons constituting these clusters are involved in the withdrawal behavior and the response of the organism to stress stimuli. From these results we suggest that the HCS2 pattern of expression can be down-regulated by a decrease in synaptic activity in the nervous system, and up-regulated by external noxious inputs, as well as the application of neurotransmitters and second messengers known to be involved in the withdrawal behavior and maintenance of isolated ganglia in culture medium. When up-regulated, the HCS2 expression appears, at least in part in neurons, to be involved in the withdrawal behavior.

The problem of stability of long-lasting synaptic facilitation postsynaptically induced by intracellular current pulses without concomitant presynaptic activation was addressed. A short (15-20 min) phase of synaptic facilitation induced by intracellular tetanization in identified snail neurones was stable and present in all experiments, while a long-term phase (lasting > or = 50 min) was observed only some experiments. Data analysis revealed dependence of the long-term phase on season. Dependence of the long-term phase of facilitation on serotonin concentration in hemolymph, which is known to change with season, was shown using the selective neurotoxin 5,7-dihydroxytriptamine. Dependence of habituation rate in the same synaptic connection on season and serotonin level was shown.

The abilities of neuronal populations to encode rapidly varying stimuli and respond quickly to abrupt input changes are crucial for basic neuronal computations, such as coincidence detection, grouping by synchrony, and spike-timing-dependent plasticity, as well as for the processing speed of neuronal networks. Theoretical analyses have linked these abilities to the fast-onset dynamics of action potentials (APs). Using a combination of whole-cell recordings from rat neocortical neurons and computer simulations, we provide the first experimental evidence for this conjecture and prove its validity for the case of distal AP initiation in the axon initial segment (AIS), typical for cortical neurons. Neocortical neurons with fast-onset APs in the soma can phase-lock their population firing to signal frequencies up to ∼300–400 Hz and respond within 1–2 ms to subtle changes of input current. The ability to encode high frequencies and response speed were dramatically reduced when AP onset was slowed by experimental manipulations or was intrinsically slow due to immature AP generation mechanisms. Multicompartment conductance-based models reproducing the initiation of spikes in the AIS could encode high frequencies only if AP onset was fast at the initiation site (e.g., attributable to cooperative gating of a fraction of sodium channels) but not when fast onset of somatic AP was produced solely by backpropagation. We conclude that fast-onset dynamics is a genuine property of cortical AP generators. It enables fast computations in cortical circuits that are rich in recurrent connections both within each region and across the hierarchy of areas.

The processing speed of the brain depends on the ability of neurons to rapidly relay input changes. Previous theoretical and experimental studies of the timescale of population firing rate responses arrived at controversial conclusions, some advocating an ultrafast response scale but others arguing for an inherent disadvantage of mean encoded signals for rapid detection of the stimulus onset. Here we assessed the timescale of population firing rate responses of neocortical neurons in experiments performed in the time domain and the frequency domainin vitroandin vivo. We show that populations of neocortical neurons can alter their firing rate within 1 ms in response to somatically delivered weak current signals presented on a fluctuating background. Signals with amplitudes of miniature postsynaptic currents can be robustly and rapidly detected in the population firing. We further show that population firing rate of neurons of rat visual cortexin vitroand cat visual cortexin vivocan reliably encode weak signals varying at frequencies up to ∼200–300 Hz, or ∼50 times faster than the firing rate of individual neurons. These results provide coherent evidence for the ultrafast, millisecond timescale of cortical population responses. Notably, fast responses to weak stimuli are limited to the mean encoding. Rapid detection of current variance changes requires extraordinarily large signal amplitudes. Our study presents conclusive evidence showing that cortical neurons are capable of rapidly relaying subtle mean current signals. This provides a vital mechanism for the propagation of rate-coded information within and across brain areas.

Visual stimulation often leads to elevated fluctuations of the membrane potential in the γ-frequency range (25-70 Hz) in visual cortex neurons. Recently, we have found that the strength of γ-band fluctuations is coupled to the oscillation of the membrane potential at the temporal frequency of the stimulus, so that the γ-band fluctuations are stronger at depolarization peaks, but weaker at troughs of the stimulus frequency oscillation of the membrane potential. We hypothesized that this coupling may improve stimulus encoding. Here, we tested this hypothesis by using a single-compartment conductance-based neuron model, with parameters of the input adjusted to reproduce typical features of membrane potential and spike responses, recorded in cat visual cortical neurons in vivo during the presentation of moving gratings. We show that modulation of the γ-range membrane potential fluctuations by the amplitude of the slow membrane depolarization greatly improves stimulus encoding. Moreover, changing the degree of modulation of the γ-activity by the low-frequency signal within the range typically observed in visual cortex cells had a stronger effect on both the firing rates and information rates than changing the amplitude of the low-frequency stimulus itself. Thus, modulation of the γ-activity represents an efficient mechanism for regulation of neuronal firing and encoding of the temporal characteristics of visual stimuli.

We study how threshold model neurons transfer temporal and interneuronal input correlations to correlations of spikes. We find that the low common input regime is governed by firing rate dependent spike correlations which are sensitive to the detailed structure of input correlation functions. In the high common input regime the spike correlations are insensitive to the firing rate and exhibit a universal peak shape independent of input correlations. Rate heterogeneous pairs driven by common inputs in general exhibit asymmetric spike correlations. All predictions are confirmed in in vitro experiments with cortical neurons driven by synthesized fluctuating input currents., 5 pages, 10 figures

Address: 1Department of Nonlinear Dynamics, Max-Planck-Institute for Dynamics and Self-Organization, D37073 Gottingen, Germany, 2Bernstein Center for Computational Neuroscience, D37073 Gottingen, Germany, 3Gottingen Graduate School for Neurosciences and Molecular Biosciences and Faculty of Physics, University of Gottingen, D37073 Gottingen, Germany, 4Institute of Higher Nervous Activity and Neurophysiology, RAS, 117485 Moscow, Russia, 5Department of Psychology, University of Connecticut, Storrs, CT 06269, USA and 6Department of Neurophysiology, Ruhr-University, D44780 Bochum, Germany

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.

The generation of action potentials (APs) is a key process in the operation of nerve cells and the communication between neurons. Action potentials in mammalian central neurons are characterized by an exceptionally fast onset dynamics, which differs from the typically slow and gradual onset dynamics seen in identified snail neurons. Here we describe a novel method of analysis which provides a quantitative measure of the onset dynamics of action potentials. This method captures the difference between the fast, step-like onset of APs in rat neocortical neurons and the gradual, exponential-like AP onset in identified snail neurons. The quantitative measure of the AP onset dynamics, provided by the method, allows us to perform quantitative analyses of factors influencing the dynamics. peerReviewed

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.

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-Mg2+/zero-Ca2+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-Mg2+/high-Ca2+saline, suggesting monosynaptic connections. All these data suggest that PlVL cells were the primary mechanosensory neurons.

A group of four cardioexcitatory neurons has been identified in the intestinal ganglia of the mollusc Clione limacina. Relatively weak stimulation of the intestinal neurons induced auricle contractions only, while strong stimulation produced initial auricle contractions followed by full-cycle auricle-ventricle contractions. Intestinal cardioexcitatory neurons probably utilized as their transmitter a peptide similar to Tritonia pedal peptide – they showed pedal peptide-like immunoreactivity, and their effects were mimicked by application of the exogenous pedal peptide. The pedal cardioexcitatory neuron was found to produce strong excitatory effects only on the ventricle contractions. Its stimulation induced ventricle contractions in the quiescent heart or significantly accelerated the rate of ventricle contractions in the rhythmically active heart. The pedal cardioexcitatory neuron apparently utilized serotonin as a neurotransmitter, based upon serotonin immunoreactivity, blocking effect of serotonin antagonists mianserin and methysergide, and the observation that exogenous serotonin mimicked its effect. A dense network of pedal peptide-like immunoreactivity was found both in the auricle and ventricle tissue. Serotonin immunoreactivity was densely present in the ventricle, while the auricle contained only a separate serotonin-immunoreactive unbranched axon. Thus, there are two separate groups of central cardioexcitatory neurons with different effects on heart activity, which together might provide a complex cardio-regulatory function in Clione.

BackgroundThe 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 findingsIn 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/significanceUpregulation 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.