Adenosine A2A receptor modulation of hippocampal CA3-CA1 synapse plasticity during associative learning in behaving mice

© 2009 Nature Publishing Group All rights reservedPrevious in vitro studies have characterized the electrophysiological and molecular signaling pathways of adenosine tonic modulation on long-lasting synaptic plasticity events, particularly for hippocampal long-term potentiation(LTP). However, it remains to be elucidated whether the long-term changes produced by endogenous adenosine in the efficiency of synapses are related to those required for learning and memory formation. Our goal was to understand how endogenous activation of adenosine excitatory A2A receptors modulates the associative learning evolution in conscious behaving mice. We have studied here the effects of the application of a highly selective A2A receptor antagonist, SCH58261, upon a well-known associative learning paradigm - classical eyeblink conditioning. We used a trace paradigm, with a tone as the conditioned stimulus (CS) and an electric shock presented to the supraorbital nerve as the unconditioned stimulus(US). A single electrical pulse was presented to the Schaffer collateral–commissural pathway to evoke field EPSPs (fEPSPs) in the pyramidal CA1 area during the CS–US interval. In vehicle-injected animals, there was a progressive increase in the percentage of conditioning responses (CRs) and in the slope of fEPSPs through conditioning sessions, an effect that was completely prevented (and lost) in SCH58261 (0.5 mg/kg, i.p.)-injected animals. Moreover, experimentally evoked LTP was impaired in SCH58261- injected mice. In conclusion, the endogenous activation of adenosine A2A receptors plays a pivotal effect on the associative learning process and its relevant hippocampal circuits, including activity-dependent changes at the CA3-CA1 synapse.This study was supported by grants from the Spanish Ministry of Education and Research (BFU2005-01024 and BFU2005-02512), Spanish Junta de Andalucía (BIO-122 and CVI-02487), and the Fundación Conocimiento y Cultura of the Pablo de Olavide University (Seville, Spain).B. Fontinha was in receipt of a studentship from a project grant (POCI/SAU-NEU/56332/2004) supported by Fundação para a Ciência e Tecnologia (FCT, Portugal), and of an STSM from Cost B30 concerted action of the EU

TMT-Opsins differentially modulate medaka brain function in a context-dependent manner.

Vertebrate behavior is strongly influenced by light. Light receptors, encoded by functional opsin proteins, are present inside the vertebrate brain and peripheral tissues. This expression feature is present from fishes to human and appears to be particularly prominent in diurnal vertebrates. Despite their conserved widespread occurrence, the nonvisual functions of opsins are still largely enigmatic. This is even more apparent when considering the high number of opsins. Teleosts possess around 40 opsin genes, present from young developmental stages to adulthood. Many of these opsins have been shown to function as light receptors. This raises the question of whether this large number might mainly reflect functional redundancy or rather maximally enables teleosts to optimally use the complex light information present under water. We focus on tmt-opsin1b and tmt-opsin2, c-opsins with ancestral-type sequence features, conserved across several vertebrate phyla, expressed with partly similar expression in non-rod, non-cone, non-retinal-ganglion-cell brain tissues and with a similar spectral sensitivity. The characterization of the single mutants revealed age- and light-dependent behavioral changes, as well as an impact on the levels of the preprohormone sst1b and the voltage-gated sodium channel subunit scn12aa. The amount of daytime rest is affected independently of the eyes, pineal organ, and circadian clock in tmt-opsin1b mutants. We further focused on daytime behavior and the molecular changes in tmt-opsin1b/2 double mutants, and found that-despite their similar expression and spectral features-these opsins interact in part nonadditively. Specifically, double mutants complement molecular and behavioral phenotypes observed in single mutants in a partly age-dependent fashion. Our work provides a starting point to disentangle the highly complex interactions of vertebrate nonvisual opsins, suggesting that tmt-opsin-expressing cells together with other visual and nonvisual opsins provide detailed light information to the organism for behavioral fine-tuning. This work also provides a stepping stone to unravel how vertebrate species with conserved opsins, but living in different ecological niches, respond to similar light cues and how human-generated artificial light might impact on behavioral processes in natural environments

Co-Expression of VAL- and TMT-Opsins Uncovers Ancient Photosensory Interneurons and Motorneurons in the Vertebrate Brain

<div><p>The functional principle of the vertebrate brain is often paralleled to a computer: information collected by dedicated devices is processed and integrated by interneuron circuits and leads to output. However, inter- and motorneurons present in today's vertebrate brains are thought to derive from neurons that combined sensory, integration, and motor function. Consistently, sensory inter­motorneurons have been found in the simple nerve nets of cnidarians, animals at the base of the evolutionary lineage. We show that light-sensory motorneurons and light-sensory interneurons are also present in the brains of vertebrates, challenging the paradigm that information processing and output circuitry in the central brain is shielded from direct environmental influences. We investigated two groups of nonvisual photopigments, VAL- and TMT-Opsins, in zebrafish and medaka fish; two teleost species from distinct habitats separated by over 300 million years of evolution. TMT-Opsin subclasses are specifically expressed not only in hypothalamic and thalamic deep brain photoreceptors, but also in interneurons and motorneurons with no known photoreceptive function, such as the typeXIV interneurons of the fish optic tectum. We further show that TMT-Opsins and Encephalopsin render neuronal cells light-sensitive. TMT-Opsins preferentially respond to blue light relative to rhodopsin, with subclass-specific response kinetics. We discovered that <i>tmt-opsins</i> co-express with <i>val-opsins</i>, known green light receptors, in distinct inter- and motorneurons. Finally, we show by electrophysiological recordings on isolated adult tectal slices that interneurons in the position of typeXIV neurons respond to light. Our work supports “sensory-inter-motorneurons” as ancient units for brain evolution. It also reveals that vertebrate inter- and motorneurons are endowed with an evolutionarily ancient, complex light-sensory ability that could be used to detect changes in ambient light spectra, possibly providing the endogenous equivalent to an optogenetic machinery.</p></div

Phylogenetic and sequence analyses of TMT-Opsins and Encephalopsins.

<p>(A) Maximum likelihood (ML) and neighbor joining (NJ) trees group TMT-Opsins into three distinct subclasses conserved across vertebrates with high branch support. Encephalopsins form a fourth, closely related group. The topology of the NJ tree is shown, and support values are given as NJ/ML next to critical branches. Grey box, ciliary-type opsins; yellow, TMT-Opsins; blue, Encephalopsins. (B, C, D, F) Conserved sequence stretches in the c-terminus of the indicated opsin subfamilies. Numbers on <i>x</i>-axis refer to the amino acid positions in bovine rhodopsin (B, C, D, F) or human Encephalopsin (G). (E) Comparative analysis of characteristic opsin sequence features critical for photopigment function. Bovine rhodopsin is used as reference for counterion position. (G) Conserved sequence stretch in the c-terminus of Encephalopsins.</p

A subset of tectal interneurons are intrinsically light sensitive.

<p>(A) Confocal ISH images of anti-ChAT staining (green) in the tectum of coronal whole-brain slices. Z-stack, 25.55 µm (B) Magnification of boxed area in (A). Z-stack, 25.55 µm; arrowheads, ChAT-positive interneurons; arrows, neurites projecting from ChAT-expressing interneurons. (C) Representative membrane potential traces from two tectal interneurons recorded under total darkness. (D) Membrane potential changes of interneurons recorded under darkness (<i>N</i> = 14). Values were calculated in relation to the reference point (0 min). No significant difference can be observed between t = −1 and t = +1. Example traces in (C) of one spiking (triangle) and one nonspiking (circle) neuron highlighted in red. Note that delta membrane changes of cells recorded under darkness never get close to 10 mV (dashed line). (E) Representative membrane potential traces from single non-light-responsive interneurons exposed to light (yellow box). (F) Membrane potential changes of interneurons exposed to 1 min light (<i>N</i> = 28). Example traces in (E) of one intrinsically spiking (triangle) and a nonspiking (circle) neuron that do not respond to light are highlighted in blue. Light-responsive (circle) and intrinsically spiking light-responsive (triangle) interneurons in (G) are marked in green. A significant difference can be observed between t = −1 and t = +1. (G) Representative membrane potential traces from single light-responsive interneurons exposed to light. The <i>p</i> values were assessed by paired Student's <i>t</i> test.</p

<i>TMT-Opsin 1B</i> is expressed in inter- and motorneuron nuclei on mRNA and protein level.

<p>ISH (A, C) and immunohistochemistry (B, D, E, F) of TMT-Opsin 1b on coronal adult medaka brain sections. Magnification of black boxes in insets. Scale bars, 50 µm. (A) mRNA expression of <i>tmtops1b</i> in the dorsal tegmental nucleus. (B) Protein expression of TMTopsin1b in cells of the dorsal tegmental nucleus, the same area as in (A). Arrowheads indicate TMTopsin1b+ cells. (C) Multiple domains of mRNA expression of <i>tmtops1b</i> in the hindbrain. (D) TMTopsin1b+ cells localize to sites of mRNA expression indicated by a yellow box in (C). (E) Overview of TMTopsin1b protein expression in the hindbrain. Arrows and asterisks indicate protein expression domains that correspond to mRNA expression in (C). (F) Magnification of box in (E), <i>z</i>-stack: 13.87 µm. Note the projections (arrowheads) extending from a TMTopsin1b+ cell verifying its neuronal nature.</p

<i>TMT-Opsin</i> expression in inter- and motorneuron nuclei is maintained from larvae to adult stages.

<p>(Left topmost panel) Dorsal view of a schematized medaka larva; red boxes indicate positions of sections displayed below. (Right topmost panel) lateral view of an adult medaka brain, transversal planes corresponding to sections below. ISH on 7 dpf larvae (A, C, E, G) and coronal sections of the adult brain (B, D, F, H). Magnifications of boxed areas on the right; corresponding expression domains in larvae are indicated with arrowheads. Scale bars, 50 µm. Expression domains: <i>tmtops1b</i>, granular layer of the olfactory bulb (A, B); <i>tmtops3a</i>, semicircular torus (C, D); <i>tmtops2</i>, dorsal tegmental nucleus (E, F), facial nerve nucleus of the hindbrain (G, H).</p

TMT-Opsins and Encephalopsin are functional light receptors.

<p>(A) Example traces of Neuro2A (N2A) cell stimulation by two consecutive light pulses of 12 min and 10 min. Data are presented as means (<i>N</i> = 4). (B–D) Traces of N2A cells transfected with medaka <i>tmt-opsins</i> (red) versus mutated (L294A) <i>tmt-opsins</i> (black), 10 min light stimulation. Data are presented as mean ± SEM (grey lines) (<i>N</i> = 4). (E) Different kinetics of TMT-2 (blue) compared to TMT-1B and TMT-3A in N2A cells (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001585#pbio.1001585.s003" target="_blank">Figure S3</a>). Baseline normalized CI values were normalized to the maximum and data are presented as means (<i>N</i> = 4). (F) Quantification of opsin-dependent N2A cell responses to light. Relative light responses are displayed as mean ± SEM (<i>N</i> = 72–136). (G) Quantification of opsin-dependent N2A cell responses to different spectra compared to human rhodopsin. Data represent mean ± SEM (<i>N</i> = 12–36). (H) HEK cells transfected with medaka <i>encephalopsin</i> (red) versus Schiff base mutant version (black). Data represent mean ± SEM (grey lines) (<i>N</i> = 32). (I) Quantification of Encephalopsin-dependent cell responses in HEK and N2A cells. Data represent mean ± SEM (<i>N</i> = 44–120); **** <i>p</i><0.0001; *** <i>p</i><0.0005; ns, not significant; yellow background box, light stimulation. See Figures S4 and S13 for analyses details.</p

A Hybrid of Amodiaquine and Primaquine Linked by Gold(I) Is a Multistage Antimalarial Agent Targeting Heme Detoxification and Thiol Redox Homeostasis

Hybrid-based drugs linked through a transition metal constitute an emerging concept for Plasmodium intervention. To advance the drug design concept and enhance the therapeutic potential of this class of drugs, we developed a novel hybrid composed of quinolinic ligands amodiaquine (AQ) and primaquine (PQ) linked by gold(I), named [AuAQPQ]PF6. This compound demonstrated potent and efficacious antiplasmodial activity against multiple stages of the Plasmodium life cycle. The source of this activity was thoroughly investigated by comparing parasite susceptibility to the hybrid’s components, the annotation of structure–activity relationships and studies of the mechanism of action. The activity of [AuAQPQ]PF6 for the parasite’s asexual blood stages was influenced by the presence of AQ, while its activity against gametocytes and pre-erythrocytic parasites was influenced by both quinolinic components. Moreover, the coordination of ligands to gold(I) was found to be essential for the enhancement of potency, as suggested by the observation that a combination of quinolinic ligands does not reproduce the antimalarial potency and efficacy as observed for the metallic hybrid. Our results indicate that this gold(I) hybrid compound presents a dual mechanism of action by inhibiting the beta-hematin formation and enzymatic activity of thioredoxin reductases. Overall, our findings support the potential of transition metals as a dual chemical linker and an antiplasmodial payload for the development of hybrid-based drugs

Spiro-ß-lactam BSS-730A Displays Potent Activity against HIV and Plasmodium

The high burden of malaria and HIV/AIDS prevents economic and social progress in developing countries. A continuing need exists for development of novel drugs and treatment regimens for both diseases in order to address the tolerability and long-term safety concerns associated with current treatment options and the emergence of drug resistance. We describe new spiro-β-lactam derivatives with potent (nM) activity against HIV and Plasmodium and no activity against bacteria and yeast. The best performing molecule of the series, BSS-730A, inhibited both HIV-1 and HIV-2 replication with an IC50 of 13 ± 9.59 nM and P. berghei hepatic infection with an IC50 of 0.55 ± 0.14 μM with a clear impact on parasite development. BSS-730A was also active against the erythrocytic stages of P. falciparum, with an estimated IC50 of 0.43 ± 0.04 μM. Time-of-addition studies showed that BSS-730A potentially affects all stages of the HIV replicative cycle, suggesting a complex mechanism of action. BSS-730A was active against multidrug-resistant HIV isolates, with a median 2.4-fold higher IC50 relative to control isolates. BSS-730A was equally active against R5 and X4 HIV isolates and displayed strong synergism with the entry inhibitor AMD3100. BSS-730A is a promising candidate for development as a potential therapeutic and/or prophylactic agent against HIV and Plasmodium.Coimbra Chemistry Centre (CQC), University of Coimbra, Portugal, is supported by the Portuguese Agency for Scientific Research, “Fundação para a Ciência e a Tecnologia” (FCT) through Projects UIDB/00313/2020 and UIDP/00313/2020, cofunded by COMPETE2020-UE. iMed. ULisboa, Faculdade de Farmacia de Lisboa, Portugal, is supported by the Portuguese Agency for Scientific Research, “Fundação para a Ciência e a Tecnologia” (FCT) through Projects UIDB/04138/2020 and UIDP/04138/2020. FCT is also acknowledged for Project PTDC-SAU-INF-29550-2017 to M.P., for a postdoc fellowship to I.B. (SFRH/BPD/76225/2011) and for Ph.D. fellowships to N.A. (PD/BD/135287/2017) and A.A. (SFRH/BD/128910/2017). The funders had no role in study design, data collection, and interpretation, nor the decision to submit the work for publication. We acknowledge the UC1010 NMR facility for producing the NMR data (www.nmrccc.uc.pt) and Filipa Teixeira for producing Plasmodium-infected Anopheles mosquitoes for sporozoite isolation.info:eu-repo/semantics/acceptedVersio