Bicistronic Construct for Optogenetic Prosthesis of Ganglion Cell Receptive Field of Degenerative Retina.

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.

Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics.

BACKGROUND: The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC's high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+/F+Ca2+) and slow Ca2+ dissociation kinetics. RESULTS: Herein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca2+ (i.e. becoming dimmer with an increase of Ca2+ concentration). In the presence of Mg2+ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca2+ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca2+ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca2+ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope. CONCLUSIONS: We demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca2+ ions buffering capacity as compared with commonly used GECIs.

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.

Cannabinoid regulation in identified synapse of terrestrial snail.

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.

Intracellular localization of the HCS2 gene products in identified snail neurons in vivo and in vitro.

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.

Synaptic contact between mechanosensory neuron and withdrawal interneuron in terrestrial snail is mediated by L-glutamate-like transmitter.

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.

Participation of GABA in establishing behavioral hierarchies in the terrestrial snail.

GABA-immunoreactive fibers were observed in the neuropile of each ganglion of Helix lucorum, while GABA-immunoreactive neural somata were found only in the buccal, cerebral, and pedal ganglia. Bath application of 10(-5) M GABA to the preparation "buccal mass-buccal ganglia" elicited a sequence of radula movements characteristic of feeding behavior. Corresponding bursts of activity were recorded in the buccal nerves under GABA application and in the buccal neurons recorded optically. In preparations of isolated central nervous system, the bath applications of GABA (10(-5) to 10(-4) M) elicited no changes in synaptic input of the premotor interneurons involved in the withdrawal behavior. However, a significant decrease in amplitude of the synaptic input and in the number of spikes in responses elicited by the test nerve stimulation was observed in metacerebral serotonergic neurons involved in modulating the feeding behavior. GABA application inhibited the spontaneous spike activity in some pedal serotonergic neurons involved in the network underlying withdrawal responses and evoked bursting activity in the other neurons of this functional group. The effects of GABA application on mechanically isolated serotonergic neurons suggest that the primary effect of GABA is inhibition. Thus, our results give evidence of the putative role of GABA in activating the feeding behavior and in the synergistic suppression of serotonergic modulation of the withdrawal behavior and serotonergic modulation of feeding, which has corresponded to the observed behavioral suppression of withdrawal reactions during feeding.

Synaptic facilitation in Helix neurons depends upon postsynaptic calcium and nitric oxide.

Intracellular tetanization of premotor interneurons for withdrawal in the snail. Helix lucorum produces long-term facilitation of synaptic inputs of these neurons. Using this model of plasticity we investigated the role of calcium in postsynaptic induction of synaptic facilitation. It was shown that, from the one hand, postsynaptic injection of calcium chelator EGTA completely abolishes potentiation and, from the other hand, injection of calcium chloride into the interneuron produces facilitation-like changes in the EPSP amplitude in this neuron. Therefore, not only necessity but also sufficiency of increasing of postsynaptic calcium for induction of synaptic potentiation was demonstrated. We also showed that inhibitor of nitric oxide synthase N-omega-nitro-L-arginine prevents development of postsynaptically induced facilitation what suggests that nitric oxide may participate in investigated synaptic facilitation as a retrograde messenger. These results support the idea that both invertebrates and vertebrates have common mechanisms underlying synaptic plasticity.

Differential effects of serotonergic and peptidergic cardioexcitatory neurons on the heart activity in the pteropod mollusc, Clione limacina.

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.