Diurnal and circadian regulation of opsin-like transcripts in the eyeless cnidarian Hydra.

Opsins play a key role in the ability to sense light both in image-forming vision and in non-visual photoreception (NVP). These modalities, in most animal phyla, share the photoreceptor protein: an opsin-based protein binding a light-sensitive chromophore by a lysine (Lys) residue. So far, visual and non-visual opsins have been discovered throughout the Metazoa phyla, including the photoresponsive Hydra, an eyeless cnidarian considered the evolutionary sister species to bilaterians. To verify whether light influences and modulates opsin gene expression in Hydra, we utilized four expression sequence tags, similar to two classic opsins (SW rhodopsin and SW blue-sensitive opsin) and two non-visual opsins (melanopsin and peropsin), in investigating the expression patterns during both diurnal and circadian time, by means of a quantitative RT-PCR. The expression levels of all four genes fluctuated along the light hours of diurnal cycle with respect to the darkness one and, in constant dark condition of the circadian cycle, they increased. The monophasic behavior in the L12:D12 cycle turned into a triphasic expression profile during the continuous darkness condition. Consequently, while the diurnal opsin-like expression revealed a close dependence on light hours, the highest transcript levels were found in darkness, leading us to novel hypothesis that in Hydra, an "internal" biological rhythm autonomously supplies the opsins expression during the circadian time. In conclusion, in Hydra, both diurnal and circadian rhythms apparently regulate the expression of the so-called visual and non-visual opsins, as already demonstrated in higher invertebrate and vertebrate species. Our data confirm that Hydra is a suitable model for studying ancestral precursor of both visual and NVP, providing useful hints on the evolution of visual and photosensory systems.

In vivo neuromodulation of animal behavior with organic semiconducting oligomers.

Modulating neural activity with electrical or chemical stimulus can be used for fundamental and applied research. Typically, neuronal stimulation is performed with intracellular and extracellular electrodes that deliver brief electrical pulses to neurons. However, alternative wireless methodologies based on functional materials may allow clinical translation of technologies to modulate neuronal function. Here, we show that the organic semiconducting oligomer 4-[2-{2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)thiophen-3-yl}ethoxy]butane-1-sulfonate (ETE-S) induces precise behaviors in the small invertebrate Hydra, which were dissected through pharmacological and electrophysiological approaches. ETE-S-induced behavioral response relies on the presence of head neurons and calcium ions and is prevented by drugs targeting ionotropic channels and muscle contraction. Moreover, ETE-S affects Hydra's electrical activity enhancing the contraction burst frequency. The unexpected neuromodulatory function played by this conjugated oligomer on a simple nerve net opens intriguing research possibilities on fundamental chemical and physical phenomena behind organic bioelectronic interfaces for neuromodulation and on alternative methods that could catalyze a wide expansion of this rising technology for clinical applications.

Changes in Biophysical Properties of Undifferentiated SH-SY5Y Cells During Long-term Cultures.

The electrophysiological properties of undifferentiated SH-SY5Y cells were examined during cultures prolonged even to 20 days by measuring the passive and active membrane properties at 5 days interval, as well as the spontaneous spiking activity. The results showed that culturing this cell for long time affected not only membrane shape but also their electrophysiological properties. In particular, these cells considerably varied their sodium and potassium voltage-dependent currents, various channels kinetic features and their excitable properties. These modifications would synergically contribute to the bioelectrical conversion of these cells and could be part of a more complex machinery with which the tumoral cell would regulate its survival advantage and resilience. Understanding these processes could add a new clue to the exploitation of this preclinical human neuronal model.

Synaptic dendritic activity modulates the single synaptic event.

Synaptic transmission is the key system for the information transfer and elaboration among neurons. Nevertheless, a synapse is not a standing alone structure but it is a part of a population of synapses inputting the information from several neurons on a specific area of the dendritic tree of a single neuron. This population consists of excitatory and inhibitory synapses the inputs of which drive the postsynaptic membrane potential in the depolarizing (excitatory synapses) or depolarizing (inhibitory synapses) direction modulating in such a way the postsynaptic membrane potential. The postsynaptic response of a single synapse depends on several biophysical factors the most important of which is the value of the membrane potential at which the response occurs. The concurrence in a specific time window of inputs by several synapses located in a specific area of the dendritic tree can, consequently, modulate the membrane potential such to severely influence the single postsynaptic response. The degree of modulation operated by the synaptic population depends on the number of synapses active, on the relative proportion between excitatory and inbibitory synapses belonging to the population and on their specific mean firing frequencies. In the present paper we show results obtained by the simulation of the activity of a single Glutamatergic excitatory synapse under the influence of two different populations composed of the same proportion of excitatory and inhibitory synapses but having two different sizes (total number of synapses). The most relevant conclusion of the present simulations is that the information transferred by the single synapse is not and independent simple transition between a pre- and a postsynaptic neuron but is the result of the cooperation of all the synapses which concurrently try to transfer the information to the postsynaptic neuron in a given time window. This cooperativeness is mainly operated by a simple mechanism of modulation of the postsynaptic membrane potential which influences the amplitude of the different components forming the postsynaptic excitatory response.

Treating TB human neuroectodermal cell line with retinoic acid induces the appearance of neuron-like voltage-gated ionic currents.

TB is a cell line derived from the cerebrospinal fluid sample of a patient with primary leptomeningeal melanomatosis. Our previous immunological and ultrastructural analysis revealed that TB cells differentiate towards a neuronal phenotype when grown in vitro up to 7 days in presence of 10 µM all-trans retinoic acid (RA). Recently, we reported that TB cells are sensitive to the cytotoxic effects of ß-amyloid peptides, activating the cytosolic phospholipase A2. To date, it is not known if RA, in addition to inducing morphological changes, also causes functional modification in TB cells, by regulating voltage-gated ionic currents. To this purpose, we performed electrophysiological characterization of undifferentiated (TB) and differentiated (RA-TB) cells by means of whole-cell patch clamp recordings. Upon depolarizing stimuli, both groups displayed voltage-gated K+ outward currents of similar amplitude. By contrast, the low amplitude voltage-gated Na+ currents recorded in undifferentiated TB cells were largely up-regulated by RA exposure. This current was strongly reduced by TTX and lidocaine and completely abolished by removal of extracellular sodium. Furthermore, treatment with RA caused the appearance of a late-onset inward current carried by Ca2+ ions in a subpopulation of TB cells. This current was not affected by removal of extracellular Na+ and was completely blocked by Cd2+, a broad-spectrum blocker of Ca2+ currents. Altogether, our results indicate that RA-differentiation of TB cells induces functional changes by augmenting the amplitude of voltage-gated sodium current and by inducing, in a subpopulation of treated cells, the appearance of a voltage-gated calcium current.

Influence of active synaptic pools on the single synaptic event.

The activity of the single synapse is the base of information processing and transmission in the brain as well as of important phenomena as the Long Term Potentiation which is the main mechanism for learning and memory. Although usually considered as independent events, the single quantum release gives variable postsynaptic responses which not only depend on the properties of the synapses but can be strongly influenced by the activity of other synapses. In the present paper we show the results of a series of computational experiments where pools of active synapses, in a compatible time window, influence the response of a single synapse of the considered pool. Moreover, our results show that the activity of the pool, by influencing the membrane potential, can be a significant factor in the NMDA unblocking from Mg2+ increasing the contribution of this receptor type to the Excitatory Post Synaptic Current. We consequently suggest that phenomena like the LTP, which depend on NMDA activation, can occur also in subthreshold conditions due to the integration of the dendritic synaptic activity.

Multisynaptic cooperation shapes single glutamatergic synapse response.

The activity of thousands of excitatory synapse in the dendritic tree produces variations of membrane potential which, while can produce the spike generation at soma (hillock), can also influence the output of a single glutamatergic synapse. We used a model of synaptic diffusion and EPSP generation to simulate the effect of different number of active synapses on the output of a single one. Our results show that, also in subthreshold conditions, the excitatory dendritic activity can influence several parameters of the single synaptic output such as its amplitude, its time course, the NMDA-component activation and consequently phenomena like STP and LTP.

Stochastic, structural and functional factors influencing AMPA and NMDA synaptic response variability: a review.

Synaptic transmission is the basic mechanism of information transfer between neurons not only in the brain, but along all the nervous system. In this review we will briefly summarize some of the main parameters that produce stochastic variability in the synaptic response. This variability produces different effects on important brain phenomena, like learning and memory, and, alterations of its basic factors can cause brain malfunctioning.

A model of cooperative effect of AMPA and NMDA receptors in glutamatergic synapses.

Glutamatergic synapses play a pivotal role in brain excitation. The synaptic response is mediated by the activity of two receptor types (AMPA and NMDA). In the present paper we propose a model of glutamatergic synaptic activity where the fast current generated by the AMPA conductance produces a local depolarization which activates the voltage- and [Mg(2+)]-dependent NMDA conductance. This cooperative effect is dependent on the biophysical properties of the synaptic spine which can be considered a high input resistance specialized compartment. Herein we present results of simulations where different values of the spine resistance and of the Mg(2+) concentrations determine different levels of cooperativeness between AMPA and NMDA receptors in shaping the post-synaptic response.

AMPA/NMDA cooperativity and integration during a single synaptic event.

Coexistence of AMPA and NMDA receptors in glutamatergic synapses leads to a cooperative effect that can be very complex. This effect is dependent on many parameters including the relative and absolute number of the two types of receptors and biophysical parameters that can vary among synapses of the same cell. Herein we simulate the AMPA/NMDA cooperativity by using different number of the two types of receptors and considering the effect of the spine resistance on the EPSC production. Our results show that the relative number of NMDA with respect to AMPA produces a different degree of cooperation which depends also on the spine resistance.

A model of dopamine regulation of glutamatergic synapse on medium size spiny neurons.

Spiny neurons of striatum receive glutamatergic synapses on dendritic spines on the neck of which project dopaminergic synapses. Dopamine modulates, by D1 type receptors, the glutamatergic synapses by inducing the phosphorylation of AMPA and NMDA receptors which produces an increased amplitude response. Herein we present a model where, in addition to phosphorylation, the direct modulation by dopamine of the spine resistance can cooperate in producing the observed effect on some of these synapses.

A model of dopamine modulated glutamatergic synapse.

The dopamine neurotransmitter regulates important neural pathways and its action in the brain is very complex. When dopaminergic neurons make synapses on spiny neurons of the striatum nucleus, they tune the responsiveness of glutamatergic synapses by means of the dopamine D1 and D2 receptors. We studied the effect of dopamine D1 receptors on glutamatergic synapse of GABAergic spiny neurons in striatum nucleus where they are located on the neck of the same spine. The action of dopamine consists essentially in promoting the phosphorylation of AMPA and NMDA receptors thus increasing the Excitatory Post Synaptic Current peak amplitude. The consequence is a cooperative effect of glutamatergic and dopaminergic synapses for the regulation of the GABAergic neuronal code. The mechanisms by which the phosphorylation induces the increase of the EPSC amplitude still remain unclear although the lack of this regulation can be involved in several pathologies as, for example, the Parkinson's disease. We tested, by computational experiments based on our model of glutamatergic synapse, three parameters of the synaptic function that could be involved in dopamine action: (a) time binding of glutamate to receptors; (b) open probability of the receptors; and (c) single receptor conductance. For different reasons, any of the three parameters could be responsible of the increased EPSC-dopamine-dependent. Our computational results were compared and discussed with experimental results found in literature. Although for our model both the open probability and the single receptor conductance can reproduce the phosphorylation effect of dopamine, we argue that the dopamine effect consists essentially in an increase of the single receptor conductance due to a 3D rearrangement of the phosphorylated receptors.

Electrophysiological variability in the SH-SY5Y cellular line.

The basic electrophysiological properties of SH-SY5Y tumour cells have been studied by whole cell patch-clamp in voltage clamp configuration. The results shown the existence of a large variability of the response among individual cells in the same experimental conditions. Experiments conducted by using different ionic concentrations of the recording pipette filling solution, yielded a significant variability of peak current amplitude for all the filling solutions used. In addition, variability among groups was detected. The inter-group variability was not dependent on the different ionic components among the groups. Our results confirm earlier findings that this cell line is not constituted of an uniform population of cells. Moreover, from the present results we can conclude that these cells have diverse regulatory patterns of membrane conductance, probably due to both the expression as well as a non precise regulation of the membrane density of the different channels.

Nitric oxide signaling pathways at neural level in invertebrates: functional implications in cnidarians.

Nitric oxide (NO) is a small molecule with unconventional properties. It is found in organisms throughout the phylogenetic scale, from fungi to mammals, in which it acts as an intercellular messenger of main physiological events, or even as an intracellular messenger in invertebrates. In both vertebrates and invertebrates, NO is involved in many processes, regulated in part by cyclic guanosine monophosphate (cGMP), and reacts with different oxygen molecular species. The presence of NO in the early-diverging metazoan phylum of Cnidaria, of which Hydra represents the first known species having a nervous system, supports a role of this molecule as an ancestral neural messenger with physiological roles that remain to be largely elucidated. Therefore, our novel findings on the presence of NO in Hydra are here integrated in such a comparative frame.

Evolving visual pigments: hints from the opsin-based proteins in a phylogenetically old "eyeless" invertebrate.

Visual pigments are photosensitive receptor proteins that trigger the transduction process producing the visual excitation once they have absorbed photons. In spite of the molecular and morpho-functional complexity that has characterized the development of animal eyes and eyeless photoreceptive systems, opsin-based protein family appears ubiquous along metazoan visual systems. Moreover, in addition to classic rhodopsin photoreceptors, all Metazoa have supplementary non-visual photosensitive structures, mainly located in the central nervous system, that sense light without forming an image and that rather regulate the organism's temporal physiology. The investigation of novel non-visual photopigments exerting extraretinal photoreception is a challenging field in vision research. Here we propose the cnidarian Hydra as a useful tool of investigation for molecular and functional differences between these pigment families. Hydra is the first metazoan owning a nervous system and it is an eyeless invertebrate showing only an extraocular photoreception, as it has no recognized visual or photosensitive structures. In this paper we provide an overview of the molecular and functional features of the opsin-based protein subfamilies and preliminary evidences in a phylogenetically old species of both image-forming and non-visual opsins. Then we give new insights on the molecular biology of Hydra photoreception and on the evolutionary pathways of visual pigments.