Melanopsin elevates locomotor activity during the wake state of the diurnal zebrafish.

Mammalian and fish pineals play a key role in adapting behaviour to the ambient light conditions through the release of melatonin. In mice, light inhibits nocturnal locomotor activity via the non-visual photoreceptor Melanopsin. In contrast to the extensively studied function of Melanopsin in the indirect regulation of the rodent pineal, its role in the intrinsically photosensitive zebrafish pineal has not been elucidated. Therefore, it is not evident if the light signalling mechanism is conserved between distant vertebrates, and how Melanopsin could affect diurnal behaviour. A double knockout of melanopsins (opn4.1-opn4xb) was generated in the diurnal zebrafish, which manifests attenuated locomotor activity during the wake state. Transcriptome sequencing gave insight into pathways downstream of Melanopsin, implying that sustained repression of the melatonin pathway is required to elevate locomotor activity during the diurnal wake state. Moreover, we show that light induces locomotor activity during the diurnal wake state in an intensity-dependent manner. These observations suggest a common Melanopsin-driven mechanism between zebrafish and mammals, while the diurnal and nocturnal chronotypes are inversely regulated downstream of melatonin.

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.

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 inter-motorneurons 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 tmt-opsins co-express with val-opsins, 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.

Transgenic mouse models enabling photolabeling of individual neurons in vivo.

One of the biggest tasks in neuroscience is to explain activity patterns of individual neurons during behavior by their cellular characteristics and their connectivity within the neuronal network. To greatly facilitate linking in vivo experiments with a more detailed molecular or physiological analysis in vitro, we have generated and characterized genetically modified mice expressing photoactivatable GFP (PA-GFP) that allow conditional photolabeling of individual neurons. Repeated photolabeling at the soma reveals basic morphological features due to diffusion of activated PA-GFP into the dendrites. Neurons photolabeled in vivo can be re-identified in acute brain slices and targeted for electrophysiological recordings. We demonstrate the advantages of PA-GFP expressing mice by the correlation of in vivo firing rates of individual neurons with their expression levels of the immediate early gene c-fos. Generally, the mouse models described in this study enable the combination of various analytical approaches to characterize living cells, also beyond the neurosciences.

Enhancement of LTP in aged rats is dependent on endogenous BDNF.

Long-term potentiation (LTP), considered the neurophysiological basis for learning and memory, is facilitated by brain-derived neurotrophic factor (BDNF), an action more evident when LTP is evoked by weak θ-burst stimuli and dependent on co-activation of adenosine A(2A) receptors (A(2A)R), which are more expressed in aged rats. As θ-burst stimuli also favor LTP in aged animals, we hypothesized that enhanced LTP in aging could be related to changes in neuromodulation by BDNF. The magnitude of CA1 LTP induced by a weak θ-burst stimuli delivered to the Schaffer collaterals was significantly higher in hippocampal slices taken from 36 to 38 and from 70 to 80-week-old rats, when compared with LTP magnitude in slices from 4 or 10 to 15-week-old rats; this enhancement does not impact in cognitive improvement as aged rats revealed an impairment on hippocampal-dependent learning and memory performance, as assessed by the Morris water maze tests. The scavenger for BDNF, TrkB-Fc, and the inhibitor of Trk phosphorylation, K252a, attenuated LTP in slices from 70 to 80-week-old rats, but not from 10 to 15-week-old rats. When exogenously added, BDNF significantly increased LTP in slices from 4 and 10 to 15-week-old rats, but did not further increased LTP in 36 to 38 or 70 to 80-week-old rats. The effects of exogenous BDNF on LTP were prevented by the A(2A)R antagonist, SCH58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine). These results indicate that the higher LTP magnitude observed upon aging, which does not translate into improved spatial memory performance, is a consequence of an increase in the tonic action of endogenous BDNF.

Adenosine A(2A) receptor modulation of hippocampal CA3-CA1 synapse plasticity during associative learning in behaving mice.

Previous 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 A(2A) receptors modulates the associative learning evolution in conscious behaving mice. We have studied here the effects of the application of a highly selective A(2A) 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 A(2A) receptors plays a pivotal effect on the associative learning process and its relevant hippocampal circuits, including activity-dependent changes at the CA3-CA1 synapse.