Evgeny S. Nikitin

A Single Serotonergic Modulatory Cell Can Mediate Reinforcement in the Withdrawal Network of the Terrestrial Snail

A cluster of 40 serotonergic cells in the rostral part of pedal ganglia of the terrestrial snail Helix lucorum was shown previously to participate in the modulation of withdrawal behavior and to be necessary during the acquisition of aversive withdrawal conditioning in intact snails. Local extracellular stimulation of the serotonergic cells paired with a test stimulus elicited a pairing-specific increase (the difference between paired and explicitly unpaired sessions was significant, p <.01) of synaptic responses to test stimulation in the premotor interneurons involved in withdrawal. This result suggested participation of serotonergic cells in mediating the reinforcement in the withdrawal network. Intracellular stimulation of only one identified Pd4 cell from the pedal group of serotonergic neurons paired with a test stimulus also significantly increased (the difference between paired and explicitly unpaired sessions was significant, p <.05) synaptic responses to paired nerve stimulation in same premotor interneurons involved in withdrawal. Morphological investigation of a cluster of pedal serotonergic neurons showed that only the Pd4 cell had branches in the parietal ganglia neuropile where the synapses of premotor withdrawal interneurons and of presynaptic neurons are located. The data suggest that a single serotonergic cell can mediate the reinforcement in the withdrawal network of the terrestrial snail. Patterns of responses of the Pd4 cells to tactile and chemical stimuli conform to the suggestion.

Fluorescent ratiometric pH indicator SypHer2: Applications in neuroscience and regenerative biology.

BACKGROUND SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, the relatively low brightness of the indicator limits its use. METHODS Here we designed a new version of pH-sensor called SypHer-2, which has up to three times brighter fluorescence in cultured mammalian cells compared to the SypHer. RESULTS Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent transient neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop that occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate. CONCLUSIONS SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo. GENERAL SIGNIFICANCE The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.

Fine tuning of olfactory orientation behaviour by the interaction of oscillatory and single neuronal activity

We used a simple sensory and motor system to investigate the neuronal mechanisms of olfactory orientation behaviour. The main olfactory organs of terrestrial molluscs, the experimental animals used in this work, are located on the tips of their tentacles, which display complex movements when they explore a new environment. By reconstructing the trajectory of the tentacle tip (‘nose’) movements in three dimensions in freely moving snails, we showed that the protracted tentacles performed continuous scanning, both spontaneously and in response to odours. Odour applications elicited a brief startle‐like quiver of the tentacle in a concentration‐independent manner as well as a concentration‐dependent contraction. Previous work showed that activation of an identified cerebral motoneuron, MtC3, produces tentacle contraction. Here we showed that in semi‐intact preparations, MtC3 responded to odours in a concentration‐dependent manner, similar to the tentacle contraction response to the same odours in intact animals. This observation suggests that MtC3 is involved in the central control of the scanning area by regulating the tentacle length. Using voltage‐sensitive dyes and imaging, we demonstrated that during the hyperpolarizing phases of oscillations in the procerebral lobe, the main olfactory centre of the CNS of terrestrial molluscs, MtC3 spike frequency significantly decreased. We also showed that direct activation of the procerebral lobe resulted in the phasic inhibition of MtC3. This is therefore an example of an olfactory system in which the interaction of oscillatory and single neuronal activity plays an important role in the fine tuning of orientation behaviour to suit the particular odour environment.

Nonsynaptic Plasticity Underlies a Compartmentalized Increase in Synaptic Efficacy after Classical Conditioning

It is now well documented in both vertebrates and invertebrates that nonsynaptic as well as synaptic plasticity can be a substrate for long-term memory [1-4]. Little is known, however, about how learning-induced nonsynaptic plasticity can lead to compartmentalized presynaptic changes underlying specific memory traces while leaving other circuit functions of the neuron unaffected. Here, using behavioral, electrophysiological, and optical recording methods, we show that the previously described learning-induced depolarization of a modulatory neuron [5] of the Lymnaea feeding system affects its axonal terminals in a spatially segregated manner. In a side branch of the axon of the cerebral giant cells (CGCs), classical conditioning of intact animals reduced proximal-to-distal attenuation of spike-evoked calcium transients, providing a highly effective mechanism for a compartmentalized increase in synaptic efficacy. Somatic depolarization by current injection, which spreads onto the CGCs axonal side branch [5], and the blocking of A-type potassium channels with 4-aminopyridine had an effect similar to learning on the calcium transients. Both of these experimental manipulations also reduced axonal spike attenuation. These findings suggest that the voltage-dependent inactivation of an A-type potassium current links global nonsynaptic changes to compartmentalized synaptic changes.

Olfactory experience modifies the effect of odour on feeding behaviour in a goal-related manner

Natural food odours elicit different behavioural responses in snails. The tentacle carries an olfactory organ, and it either protracts toward a stimulating carrot odour or retraces in a startle-like fashion away from a cucumber odour. The tentacle retraction to cucumber was still present after the snails were fed cucumber during inter-trial periods. Also, snails without any food experience displayed a longer latency to the first bite of cucumber than of carrot and rejected cucumber more often. After tasting these foods, the latency to carrot was not affected while the latency to and number of rejections of cucumber decreased. These results suggest that initial repulsive features of food odour can be only partially compensated by olfactory learning and feeding experience. In the present study, we demonstrated that an invertebrate can be repulsed or attracted by the same natural odour at the same time and that these behavioural responses are likely aimed at achieving different physiologically relevant goals.

Optical recording of responses to odor in olfactory structures of the nervous system in the terrestrial mollusk Helix.

Electrophysiological methods and optical methods based on the use of potential-sensitive dyes were used to record stable rhythmic oscillations of local potentials in the olfactory structure (procerebrum) of the pulmonate mollusk Helix; these oscillations were generally similar to those previously observed in slugs. Odor had the effect of transiently altering rhythmic oscillations to generate an individual pattern. This is the first study describing the recording of procerebrum potentials evoked by presentation of odor, with mapping of the areas of propagation of these potentials relative to the areas of propagation of rhythmic oscillations. The boundary of the propagation of the evoked potential was essentially similar to the projection of the neuropil, and rhythmic oscillations were recorded in the projection layer of procerebrum cell bodies. Evoked potential waves appeared in areas corresponding to the site at which the olfactory nerve enters the cerebral ganglion (of which the procerebrum forms a part) and were propagated in the procerebrum neuropil towards the cell body layer. Evoked potentials did not provoke out-of-phase waves of rhythmic oscillations.

Thermogenetic neurostimulation with single-cell resolution

Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of neurons using thermosensitive transient receptor potential (TRP) cation channels. Broader application of this approach in neuroscience is, however, hindered by a limited variety of suitable ion channels, and by low spatial and temporal resolution of neuronal activation when TRP channels are activated by ambient temperature variations or chemical agonists. Here, we demonstrate rapid, robust and reproducible repeated activation of snake TRPA1 channels heterologously expressed in non-neuronal cells, mouse neurons and zebrafish neurons in vivo by infrared (IR) laser radiation. A fibre-optic probe that integrates a nitrogen−vacancy (NV) diamond quantum sensor with optical and microwave waveguide delivery enables thermometry with single-cell resolution, allowing neurons to be activated by exceptionally mild heating, thus preventing the damaging effects of excessive heat. The neuronal responses to the activation by IR laser radiation are fully characterized using Ca2+ imaging and electrophysiology, providing, for the first time, a complete framework for a thermogenetic manipulation of individual neurons using IR light.

Biolistic delivery of voltage-sensitive dyes for fast recording of membrane potential changes in individual neurons in rat brain slices

Optical recording of membrane potential changes with fast voltage-sensitive dyes (VSDs) in neurons is one of the very few available methods for studying the generation and propagation of electrical signals to the distant compartments of excitable cells. The more lipophilic is the VSD, the better signal-to-noise ratio of the optical signal can be achieved. At present there are no effective ways to deliver water-insoluble dyes into the membranes of live cells. Here, we report a possibility to stain individual live neurons with highly lipophilic VSDs in acute brain slices using biolistic delivery. We tested four ANEP-based VSDs with different lipophilic properties and showed their ability to stain single neurons in a slice area of up to 150 μm in diameter after being delivered by a biolistic apparatus. In the slices of neocortex and hippocampus, the two most lipophilic dyes, di-8-ANEPPS and di-12-ANEPPQ, showed cell-specific loading and Golgi-like staining patterns with minimal background fluorescence. Simultaneous patch-clamp and optical recording of biolistically stained neurons demonstrated a good match of optical and electrical signals both for spontaneous APs (action potentials) and stimulus-evoked events. Our results demonstrate the high efficiency of a fast and targeted method of biolistic delivery of lipophilic VSDs for optical signals recording from mammalian neurons in vitro.

Regulation of tentacle length in snails by odor concentration.

The upper tentacle of the snail, bearing the olfactory organ, produces complex movements when the snail explores a new environment. Tentacle trajectories were reconstructed in the presence and absence of odors using two simultaneous video recordings. Reconstructions showed that in the absence of odor, snails constantly scanned the surrounding space with the extended tentacles. Presentation of an odor elicited rapid flexion, independent of the odor concentration, accompanied by concentration-dependent tentacle contractions. Activation of identified motoneuron MtC3 is known to elicit tentacle contraction. Recordings made in semi-intact preparations showed that the dynamics and duration of the spike activity of MtC3 produced in response to odors correlated with the degree of tentacle contraction in response to odors. These data suggest that the central motoneuron MtC3, which triggers tentacle contraction, is involved in controlling the margins of the scanning field. Slow contraction or extension of the tentacle, associated with the level of MtC3 activity, may operate to tune the snails investigative behavior to the conditions of the sensory environment.

Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor

Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Förster resonance energy transfer (FRET).