Agrobacterium-mediated insertional mutagenesis in the mycorrhizal fungus Laccaria bicolor.

Agrobacterium-mediated gene transfer (AMT) is extensively employed as a tool in fungal functional genomics and accordingly, in previous studies we used AMT on a dikaryotic strain of the ectomycorrhizal basidiomycete Laccaria bicolor. The interest in this fungus derives from its capacity to establish a symbiosis with tree roots, thereby playing a major role in nutrient cycling of forest ecosystems. The ectomycorrhizal symbiosis is a highly complex interaction involving many genes from both partners. To advance in the functional characterization of fungal genes, AMT was used on a monokaryotic L. bicolor. A collection of over 1200 transgenic strains was produced, of which 200 randomly selected strains were analyzed for their genomic T-DNA insertion patterns. By means of insertional mutagenesis, a number of transgenic strains were obtained displaying differential growth features. Moreover, mating with a compatible strain resulted in dikaryons that retained altered phenotypic features of the transgenic monokaryon. The analysis of the T-DNA integration pattern revealed mostly similar results to those reported in earlier studies, confirming the usefulness of AMT on different genetic backgrounds of L. bicolor. Taken together, our studies display the great versatility and potentiality of AMT as a tool for the genetic characterization of L. bicolor.

Ghrelin directly targets the ventral tegmental area to increase food motivation.

Ghrelin, a circulating orexigenic stomach-derived hormone, has recently been implicated in extra-homeostatic feeding, increasing food reward and food-motivated behavior. The precise target site(s) for ghrelin's effects on food reward have yet to be elucidated. The neurocircuitry underpinning food-motivated behavior involves, in particular, the dopamine cells of the ventral tegmental area (VTA) that project to the nucleus accumbens (NAcc). Ghrelin stimulation in both of these mesolimbic reward areas increases chow intake. Here we sought to determine if ghrelin acts directly within these mesolimbic reward areas to increase food reward/motivation in studies that combine feeding behavior, pharmacology, and neuroanatomy. We found that motivated behavior for a sucrose reward, assessed in an operant conditioning paradigm in rats, was increased when ghrelin was microinjected directly into the VTA but not into the NAcc. By contrast, ghrelin administration to both areas increased the free feeding of chow. Importantly, in a state of overnight food restriction, where endogenous levels of ghrelin are increased, ghrelin receptor (GHS-R1A) blockade in the VTA was sufficient to decrease the motivation to work for a sugar reward. Blockade of the GHS-R1A in VTA or NAcc was not sufficient to reduce fasting-induced chow hyperphagia. Taken together our data identify the VTA but not the NAcc as a direct, necessary, and sufficient target site for ghrelin's action on food motivation.

Blockade of central nicotine acetylcholine receptor signaling attenuate ghrelin-induced food intake in rodents.

Here we sought to determine whether ghrelin's central effects on food intake can be interrupted by nicotine acetylcholine receptor (nAChR) blockade. Ghrelin regulates mesolimbic dopamine neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens, partly via cholinergic VTA afferents originating in the laterodorsal tegmental area (LDTg). Given that these cholinergic projections to the VTA have been implicated in natural as well as drug-induced reinforcement, we sought to investigate the role of cholinergic signaling in ghrelin-induced food intake as well as fasting-induced food intake, for which endogenous ghrelin has been implicated. We found that i.p. treatment with the non-selective centrally active nAChR antagonist, mecamylamine decreased fasting-induced food intake in both mice and rats. Moreover, central administration of mecamylamine decreased fasting-induced food intake in rats. I.c.v. ghrelin-induced food intake was suppressed by mecamylamine i.p. but not by hexamethonium i.p., a peripheral nAChR antagonist. Furthermore, mecamylamine i.p. blocked food intake following ghrelin injection into the VTA. Expression of the ghrelin receptor, the growth hormone secretagogue receptor 1A, was found to co-localize with choline acetyltransferase, a marker of cholinergic neurons, in the LDTg. Finally, mecamylamine treatment i.p. decreased the ability of palatable food to condition a place preference. These data suggest that ghrelin-induced food intake is partly mediated via nAChRs and that nicotinic blockade decreases the rewarding properties of food.