Matvey Roshchin

Nitric oxide is necessary for labilization of a consolidated context memory during reconsolidation in terrestrial snails

Nitric oxide (NO) is known to be involved in associative memory formation. We investigated the influence of blocking NO function on the reconsolidation of context memory in terrestrial snails (Helix lucorum L.). After a 10 day session of electric shocks in one context only, context memory in snails was observed in test sessions as the significant difference of amplitudes of withdrawal responses to tactile stimuli in two different contexts. After a 1 day rest, a session of ‘reminding’ was performed, preceded by injection in different groups of the snails with either vehicle or combination of the protein synthesis blocker anisomycin (ANI) with one of the following drugs: the NO scavenger carboxy‐PTIO, the NO‐synthase inhibitors N‐omega‐nitro‐L‐arginin, nitroindazole and NG‐nitro‐L‐arginine methyl ester hydrochloride, or the NO donor S‐nitroso‐N‐acetyl‐DL‐penicillamine. Testing the context memory at different time intervals after the reminder under ANI injection showed that the context memory was impaired at 24 h and later, whereas the reminder under combined injection of ANI and each of the NO‐synthase inhibitors used or the NO scavenger showed no impairment of long‐term context memory. Injection of the NO donor S‐nitroso‐N‐acetyl‐DL‐penicillamine with or without reminder had no effect on context memory. The results obtained demonstrated that NO is necessary for labilization of a consolidated context memory.

Functional Changes in the Snail Statocyst System Elicited by Microgravity

Background The mollusk statocyst is a mechanosensing organ detecting the animals 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 organisms ability to adapt to gravity changes. This simple animal model offers the possibility to describe general subcellular mechanisms of nervous systems response to conditions on Earth and in space.

Chloride conducting light activated channel GtACR2 can produce both cessation of firing and generation of action potentials in cortical neurons in response to light

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.

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.

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.

Neural control of olfaction and tentacle movements by serotonin and dopamine in terrestrial snail

We investigated the role of serotonin (5HT) and dopamine (DA) in the regulation of olfactory system function and odor-evoked tentacle movements in the snail Helix. Preparations of the posterior tentacle (including sensory pad, tentacular ganglion and olfactory nerve) or central ganglia with attached posterior tentacles were exposed to cineole odorant and the evoked responses were affected by prior application of 5HT or DA or their precursors 5-hydroxytryptophan (5HTP) and l-DOPA, respectively. 5HT applications decreased cineole-evoked responses recorded in the olfactory nerve and hyperpolarized the identified tentacle retractor muscle motoneuron MtC3, while DA applications led to the opposite changes. 5HTP and l-DOPA modified MtC3 activity comparable to 5HT and DA action. DA was also found to decrease the amplitude of spontaneous local field potential oscillations in the procerebrum, a central olfactory structure. In vivo studies demonstrated that injection of 5HTP in freely moving snails reduced the tentacle withdrawal response to aversive ethyl acetate odorant, whereas the injection of l-DOPA increased responses to “neutral” cineole and aversive ethyl acetate odorants. Our data suggest that 5HT and DA affect the peripheral (sensory epithelium and tentacular ganglion), the central (procerebrum), and the single motor neuron (withdrawal motoneuron MtC3) level of the snail’s nervous system.

Nitric Oxide Upregulates Proteasomal Protein Degradation in Neurons.

Nitric oxide (NO) is involved in many neuronal functions such as neuromodulation and intracellular signaling. Recent studies have demonstrated that nitric oxide is involved in regulation of proteasomal protein degradation. However, its role in neuronal protein degradation still remains unclear. In our study, we investigated the influence of endogenous nitric oxide production in this process. We have shown that nitric oxide synthase blockade prevents decline of the UbG76V-GFP fluorescence (GFP-based proteasomal protein degradation reporter) in neuronal processes of the cultured hippocampal neurons. It suggests that nitric oxide may regulate ubiquitin-dependent proteasomal protein degradation in neurons. Also, we have confirmed that the NO synthesis blockade alone significantly impairs long-term potentiation, and demonstrated for the first time that simultaneous blockade of the NO and proteins synthesis leads to the long-term potentiation amplitude rescue to the control values. Obtained results suggest that nitric oxide is involved in the protein degradation in proteasomes in physiological conditions.

Anion-selective channelrhodopsin expressed in neuronal cell culture and in vivo in murine brain: Light-induced inhibition of generation of action potentials.

Anionic channelrhodopsin slow ChloC was expressed in the culture of nerve cells and in vivo in mouse brain. We demonstrated ability of slow ChloC to suppress effectively the activity of the neuron in response to the illumination with the visible light. It has been shown for a first time that slow ChloC works equally efficiently in both neuronal culture and in the whole brain being expressed in vivo. Thus, slow ChloC could be considered as an effective optogenetic tool capable in response to light stimulation to inhibit the generation of action potentials in the neuron.

A BK channel–mediated feedback pathway links single-synapse activity with action potential sharpening in repetitive firing

Activated single synapses sharpen future action potential shape in repetitive firing. Action potential shape is a major determinant of synaptic transmission, and mechanisms of spike tuning are therefore of key functional significance. We demonstrate that synaptic activity itself modulates future spikes in the same neuron via a rapid feedback pathway. Using Ca2+ imaging and targeted uncaging approaches in layer 5 neocortical pyramidal neurons, we show that the single spike–evoked Ca2+ rise occurring in one proximal bouton or first node of Ranvier drives a significant sharpening of subsequent action potentials recorded at the soma. This form of intrinsic modulation, mediated by the activation of large-conductance Ca2+/voltage-dependent K+ channels (BK channels), acts to maintain high-frequency firing and limit runaway spike broadening during repetitive firing, preventing an otherwise significant escalation of synaptic transmission. Our findings identify a novel short-term presynaptic plasticity mechanism that uses the activity history of a bouton or adjacent axonal site to dynamically tune ongoing signaling properties.