Melanopsin and mechanisms of non-visual ocular photoreception.

In addition to rods and cones, the mammalian eye contains a third class of photoreceptor, the intrinsically photosensitive retinal ganglion cell (ipRGC). ipRGCs are heterogeneous irradiance-encoding neurons that primarily project to non-visual areas of the brain. Characteristics of ipRGC light responses differ significantly from those of rod and cone responses, including depolarization to light, slow on- and off-latencies, and relatively low light sensitivity. All ipRGCs use melanopsin (Opn4) as their photopigment. Melanopsin resembles invertebrate rhabdomeric photopigments more than vertebrate ciliary pigments and uses a G(q) signaling pathway, in contrast to the G(t) pathway used by rods and cones. ipRGCs can recycle chromophore in the absence of the retinal pigment epithelium and are highly resistant to vitamin A depletion. This suggests that melanopsin employs a bistable sequential photon absorption mechanism typical of rhabdomeric opsins.

Melanopsin is highly resistant to light and chemical bleaching in vivo.

Melanopsin is the photopigment of mammalian intrinsically photosensitive retinal ganglion cells, where it contributes to light entrainment of circadian rhythms, and to the pupillary light response. Previous work has shown that the melanopsin photocycle is independent of that used by rhodopsin (Tu, D. C., Owens, L. A., Anderson, L., Golczak, M., Doyle, S. E., McCall, M., Menaker, M., Palczewski, K., and Van Gelder, R. N. (2006) Inner retinal photoreception independent of the visual retinoid cycle. Proc. Natl. Acad. Sci. U.S.A. 103, 10426-10431). Here we determined the ability of apo-melanopsin, formed by ex vivo UV light bleaching, to use selected chromophores. We found that 9-cis-retinal, but not all-trans-retinal or 9-cis-retinol, is able to restore light-dependent ipRGC activity after bleaching. Melanopsin was highly resistant to both visible-spectrum photic bleaching and chemical bleaching with hydroxylamine under conditions that fully bleach rod and cone photoreceptor cells. These results suggest that the melanopsin photocycle can function independently of both rod and cone photocycles, and that apo-melanopsin has a strong preference for binding cis-retinal to generate functional pigment. The data support a model in which retinal is continuously covalently bound to melanopsin and may function through a reversible, bistable mechanism.

Pectin Methylesterase genes influence solid wood properties of Eucalyptus pilularis.

This association study of Eucalyptus pilularis populations provides empirical evidence for the role of Pectin Methylesterase (PME) in influencing solid wood characteristics of Eucalyptus. PME6 was primarily associated with the shrinkage and collapse of drying timber, which are phenotypic traits consistent with the role of pectin as a hydrophilic polysaccharide. PME7 was primarily associated with cellulose and pulp yield traits and had an inverse correlation with lignin content. Selection of specific alleles in these genes may be important for improving trees as sources of high-quality wood products. A heterozygote advantage was postulated for the PME7 loci and, in combination with haplotype blocks, may explain the absence of a homozygous class at all single-nucleotide polymorphisms investigated in this gene.

Increased expression of 5-HT6 receptors in the rat dorsomedial striatum impairs instrumental learning.

A number of studies have shown that systemic 5-HT(6) receptor antagonists can improve learning and memory, but the mechanism for these observations is not known. As striatum normally expresses 5-HT(6) receptors abundantly and is important in consolidating stimulus-response learning, we used targeted gene delivery to further increase the expression of 5-HT(6) receptors in rat striatum and then examined learning. Increased 5-HT(6) expression had no effect on performance in the Morris water maze, a hippocampal-dependent learning paradigm, and did not alter the latency to approach or consume sucrose tablets. However, rats with increased 5-HT(6) expression failed to acquire a reward-based instrumental learning task, a striatum-dependent learning model, during 3 days of successive sessions as compared to sham surgery or GFP-expressing control rats. This behavioral deficit was observed in rats overexpressing 5-HT(6) receptors in the dorsomedial striatum, but not in rats with increased dorsocentral striatal expression. The 5-HT(6) receptor-associated deficit was reversed by administration of a 5-HT(6) antagonist, SB-258585, before each training session. When animals learned the instrumental learning task before gene transfer, increased 5-HT(6) receptor expression had no effect on long-term recall or performance of the task or on extinction of operant responding. Thus, 5-HT(6) receptor activity in rat striatum disrupts acquisition of new instrumental learning but does not impair memory or performance of reward-motivated behavior once established.

Overexpression of 5-HT1B receptor in dorsal raphe nucleus using Herpes Simplex Virus gene transfer increases anxiety behavior after inescapable stress.

5-HT(1B) autoreceptors have been implicated in animal models of stress and are regulated selectively by serotonin-selective reuptake inhibitors such as fluoxetine. These terminal autoreceptors regulate serotonin release from dorsal raphe nucleus (DRN) projections throughout rat forebrain. However, it has not been previously possible to manipulate 5-HT(1B) autoreceptor activity selectively without also changing 5-HT(1B) activity in other neurons mediating different behavioral responses. Therefore, we have developed a viral-mediated gene transfer strategy to express hemagglutinin-tagged 5-HT(1B) and manipulate these autoreceptors in DRN. Green fluorescent protein (GFP) was coexpressed from a separate transcriptional unit on the same amplicon to assist in monitoring infection and expression. We confirmed the expression and biological activity of both transgenic proteins in vitro. When injected directly into DRN using stereotaxic procedure, HA-5-HT(1B) receptors were expressed in serotonergic neurons and translocated to the forebrain. The effect of DRN expression of HA-5-HT(1B) on stress-induced behaviors was compared with control rats that received GFP-only amplicons. There was no change in immobility in the forced swim test. However, HA-5-HT(1B) expression significantly reduced entrances into the central region of an open-field arena after water-restraint stress without altering overall locomotor activity, but not in the absence of stress exposure. HA-5-HT(1B) expression also reduced entries into the open arms of the elevated plus maze after water restraint. Because these tests are sensitive to increases in anxiety-like behavior, our results suggest that overactivity of 5-HT(1B) autoreceptors in DRN neurons may be an important mediator of pathological responses to stressful events.

G-Protein Coupled Receptor Kinase 2 Minimally Regulates Melanopsin Activity in Intrinsically Photosensitive Retinal Ganglion Cells.

Phosphorylation is a primary modulator of mammalian G-protein coupled receptor (GPCR) activity. The GPCR melanopsin is the photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs) in the mammalian retina. Recent evidence from in vitro experiments suggests that the G-protein coupled receptor kinase 2 (GRK2) phosphorylates melanopsin and reduces its activity following light exposure. Using an ipRGC-specific GRK2 loss-of-function mouse, we show that GRK2 loss alters melanopsin response dynamics and termination time in postnatal day 8 (P8) ipRGCs but not in older animals. However, the alterations are small in comparison to the changes reported for other opsins with loss of their cognate GRK. These results suggest GRK2 contributes to melanopsin deactivation, but that other mechanisms account for most of modulation of melanopsin activity in ipRGCs.

Type I intrinsically photosensitive retinal ganglion cells of early post-natal development correspond to the M4 subtype.

BACKGROUND: Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate circadian light entrainment and the pupillary light response in adult mice. In early development these cells mediate different processes, including negative phototaxis and the timing of retinal vascular development. To determine if ipRGC physiologic properties also change with development, we measured ipRGC cell density and light responses in wild-type mouse retinas at post-natal days 8, 15 and 30. RESULTS: Melanopsin-positive cell density decreases by 17% between post-natal days 8 and 15 and by 25% between days 8 and 30. This decrease is due specifically to a decrease in cells co-labeled with a SMI-32, a marker for alpha-on ganglion cells (corresponding to adult morphologic type M4 ipRGCs). On multi-electrode array recordings, post-natal day 8 (P8) ipRGC light responses show more robust firing, reduced adaptation and more rapid recovery from short and extended light pulses than do the light responses of P15 and P30 ipRGCs. Three ipRGC subtypes - Types I-III - have been defined in early development based on sensitivity and latency on multielectrode array recordings. We find that Type I cells largely account for the unique physiologic properties of P8 ipRGCs. Type I cells have previously been shown to have relatively short latencies and high sensitivity. We now show that Type I cells show have rapid and robust recovery from long and short bright light exposures compared with Type II and III cells, suggesting differential light adaptation mechanisms between cell types. By P15, Type I ipRGCs are no longer detectable. Loose patch recordings of P8 M4 ipRGCs demonstrate Type I physiology. CONCLUSIONS: Type I ipRGCs are found only in early development. In addition to their previously described high sensitivity and rapid kinetics, these cells are uniquely resistant to adaptation and recover quickly and fully to short and prolonged light exposure. Type I ipRGCs correspond to the SMI-32 positive, M4 subtype and largely lose melanopsin expression in development. These cells constitute a unique morphologic and physiologic class of ipRGCs functioning early in postnatal development.

Increased expression of 5-HT1B receptor in dorsal raphe nucleus decreases fear-potentiated startle in a stress dependent manner.

5-HT(1B) autoreceptors regulate serotonin release from terminals of dorsal raphe nucleus (DRN) projections. Due to postsynaptic 5-HT(1B) receptors in DRN terminal fields, it has not previously been possible to manipulate 5-HT(1B) autoreceptor activity without also changing 5-HT(1B) heteroreceptor activity. We have developed a viral gene transfer strategy to express epitope-tagged 5-HT(1B) and green fluorescent protein in vivo, allowing us to increase 5-HT(1B) expression in DRN neurons. We have shown that increased 5-HT(1B) autoreceptor expression reduced anxiety in unstressed animals but increased anxiety following inescapable stress. These findings suggest that effects of increased 5-HT(1B) autoreceptor expression are dependent on stress context. To better understand the mechanisms underlying these observations, we have used fear-potentiated startle (FPS). FPS is especially sensitive to the activity of the amygdala, which shares reciprocal connections with DRN. In the absence of an inescapable stressor, increased 5-HT(1B) autoreceptor expression attenuated FPS response compared with animals injected with a virus expressing only green fluorescent protein. Administration of the 5-HT(1B) antagonist SB224289 (5 mg/kg i.p.) before startle testing blocked the effects of increased 5-HT(1B) autoreceptor expression. Since SB224289 had no effect on FPS in the absence of viral gene transfer, these results suggest that the antagonist reversed the behavioral effects of increased 5-HT(1B) autoreceptor expression through blockade of transgenic receptors. When tested 24 h following water-restraint stress, animals with increased 5-HT(1B) autoreceptors demonstrated restoration of robust FPS response. These results extend our previous studies and suggest explanations for the complex relationship between 5-HT(1B) autoreceptor expression, stress, and anxiety behavior.

5-HT1B receptor mRNA levels in dorsal raphe nucleus: inverse association with anxiety behavior in the elevated plus maze.

Serotonergic neurons in the dorsal raphe nucleus, the major source of forebrain serotonin projections, synthesize a terminal autoreceptor that inhibits serotonin release-the 5-HT(1B) autoreceptor. Overexpression of this autoreceptor is hypothesized to contribute to anxiety. Antidepressants decrease (while learned helplessness increases) 5-HT(1B) mRNA in dorsal raphe neurons, and viral-mediated overexpression of 5-HT(1B) here increases anxiety behavior after stress. However, 5-HT(1B) mRNA levels in dorsal raphe are substantially elevated in unstressed rats in two models of stress resistance. Thus, the role of dorsal raphe 5-HT(1B) autoreceptors in anxiety is complex. Therefore, we tested whether different stressors differentially affect dorsal raphe 5-HT(1B) mRNA [via in situ hybridization histochemistry] and anxiety behavior (using the elevated plus maze). Rats were assigned to a stressor (either forced swim, water restraint, dry restraint, or electric tail shock) or a control condition, then were tested and sacrificed 24 h later. Overall, controls exhibited less anxiety than stressed rats as indicated by a higher ratio of open arm to total arm entries (OTR). The stressors did not differentially affect the OTR, nor did any alter dorsal raphe 5-HT(1B) mRNA levels. There was, however, a significant positive correlation between the OTR and 5HT(1B) mRNA intensity in controls (r=.64; P=.006), but not in stressed rats (r=.16, P=.36), providing further evidence that elevated dorsal raphe 5-HT(1B) levels are associated with reduced anxiety in animals that have not been exposed to stress.

Application of large-scale sequencing to marker discovery in plants.

Advances in DNA sequencing provide tools for efficient large-scale discovery of markers for use in plants. Discovery options include large-scale amplicon sequencing, transcriptome sequencing, gene-enriched genome sequencing and whole genome sequencing. Examples of each of these approaches and their potential to generate molecular markers for specific applications have been described. Sequencing the whole genome of parents identifies all the polymorphisms available for analysis in their progeny. Sequencing PCR amplicons of sets of candidate genes from DNA bulks can be used to define the available variation in these genes that might be exploited in a population or germplasm collection. Sequencing of the transcriptomes of genotypes varying for the trait of interest may identify genes with patterns of expression that could explain the phenotypic variation. Sequencing genomic DNA enriched for genes by hybridization with probes for all or some of the known genes simplifies sequencing and analysis of differences in gene sequences between large numbers of genotypes and genes especially when working with complex genomes. Examples of application of the above-mentioned techniques have been described.

Increased expression of 5-HT(1B) receptors by Herpes simplex virus gene transfer in septal neurons: New in vitro and in vivo models to study 5-HT(1B) receptor function.

Serotonergic modulation of acetylcholine (ACh) release after neuron-specific increase of the expression of 5-HT(1B) receptors by gene transfer was studied in vitro and in vivo. The increased expression of the 5-HT(1B) receptor in vitro was induced by treating rat primary fetal septal cell cultures for 3 days with a viral vector inducing the expression of green fluorescent protein (GFP) vector alone, or, in addition, of 5-HT(1B) receptors (HA1B/GFP vector). The transfection resulted in a high number of GFP-positive cells, part of which being immunopositive for choline acetyltransferase. In HA1B/GFP-cultures (vs. GFP-cultures), electrically evoked ACh release was significantly more sensitive to the inhibitory action of the 5-HT(1B) agonist CP-93,129. Increased expression of the 5-HT(1B) receptor in vivo was induced by stereotaxic injections of the vectors into the rat septal region. Three days later, electrically evoked release of ACh in hippocampal slices of HA1B/GFP-treated rats was lower than in their GFP-treated counterparts, showing a higher inhibitory efficacy of endogenous 5-HT on cholinergic terminals after transfection. Moreover, CP-93,129 had a higher inhibitory potency. In conclusion, the HA1B/GFP vector reveals a useful tool to induce a targeted increase of 5-HT(1B) heteroreceptors on cholinergic neurons in selected CNS regions, which provides interesting perspectives for functional approaches at more integrated levels.

Crassulacean acid metabolism in the ZZ plant, Zamioculcas zamiifolia (Araceae).

Zamioculcas zamiifolia (Araceae), a terrestrial East African aroid, with two defining attributes of crassulacean acid metabolism (CAM) (net CO(2) uptake in the dark and diel fluctuations of titratable acidity) is the only CAM plant described within the Araceae, a mainly tropical taxon that contains the second largest number of epiphytes of any vascular plant family. Within the Alismatales, the order to which the Araceae belong, Z. zamiifolia is the only documented nonaquatic CAM species. Zamioculcas zamiifolia has weak CAM that is upregulated in response to water stress. In well-watered plants, day-night fluctuations in titratable acidity were 2.5 µmol H(+)·(g fresh mass)(-1), and net CO(2) uptake in the dark contributed less than 1% to daily carbon gain. Following 10 d of water stress, net CO(2) uptake in the light fell 94% and net CO(2) uptake in the dark increased 7.5-fold, such that its contribution increased to 19% of daily carbon gain. Following rewatering, dark CO(2) uptake returned to within 5% of prestressed levels. We postulate that CAM assists survival of Z. zamiifolia by reducing water loss and maintaining carbon gain during seasonal droughts characteristic of its natural habitat.

Catabolic action of insulin in rat arcuate nucleus is not enhanced by exogenous "tub" expression.

The central nervous system (CNS) protein "tub" has been identified from the genetically obese "tubby" mouse. Although the native function of tub in situ is not understood, cell-based studies suggest that one of its roles may be as an intracellular signaling target for insulin. In normal animals, insulin acts at the hypothalamic arcuate nucleus (ARC) to regulate energy balance. Here we used a Herpes Simplex viral expression system to evaluate whether tub overexpression in the ARC of normal rats enhances this action of insulin. In chow-fed rats, tub overexpression had no effect on insulin action. In rats fed a high-fat diet snack in addition to chow, simulating the diet of Westernized societies, the body weight regulatory action of insulin was impaired, and tub overexpression further impaired insulin action. Thus an excess of tub at the ARC does not enhance the in vivo effectiveness of insulin and is not able to compensate for the "downstream" consequences of a high-fat diet to impair CNS body weight regulatory mechanisms.