A quadruple dissociation of reward-related behaviour in mice across excitatory inputs to the nucleus accumbens shell.

The nucleus accumbens shell (NAcSh) is critically important for reward valuations, yet it remains unclear how valuation information is integrated in this region to drive behaviour during reinforcement learning. Using an optogenetic spatial self-stimulation task in mice, here we show that contingent activation of different excitatory inputs to the NAcSh change expression of different reward-related behaviours. Our data indicate that medial prefrontal inputs support place preference via repeated actions, ventral hippocampal inputs consistently promote place preferences, basolateral amygdala inputs produce modest place preferences but as a byproduct of increased sensitivity to time investments, and paraventricular inputs reduce place preferences yet do not produce full avoidance behaviour. These findings suggest that each excitatory input provides distinct information to the NAcSh, and we propose that this reflects the reinforcement of different credit assignment functions. Our finding of a quadruple dissociation of NAcSh input-specific behaviours provides insights into how types of information carried by distinct inputs to the NAcSh could be integrated to help drive reinforcement learning and situationally appropriate behavioural responses.

Genotype-dependent and heat-induced grain chalkiness in rice correlates with the expression patterns of starch biosynthesis genes.

Starch biosynthesis is a complex process underlying grain chalkiness in rice in a genotype-dependent manner. Coordinated expression of starch biosynthesis genes is important for producing translucent rice grains, while disruption in this process leads to opaque or chalky grains. To better understand the dynamics of starch biosynthesis genes in grain chalkiness, six rice genotypes showing variable chalk levels were subjected to gene expression analysis during reproductive stages. In the chalky genotypes, peak expression of the large subunit genes of ADP-glucose pyrophosphorylase (AGPase), encoding the first key step in starch biosynthesis, occurred in the stages before grain filling commenced, creating a gap with the upregulation of starch synthase genes, granule bound starch synthase I (GBSSI) and starch synthase IIA (SSIIA). Whereas, in low-chalk genotypes, AGPase large subunit genes expressed at later stages, generally following the expression patterns of GBSSI and SSIIA. However, heat treatment altered the expression in a genotype-dependent manner that was accompanied by transformed grain morphology and increased chalkiness. The suppression of AGPase subunit genes during early grain filling stages was observed in the chalky genotypes or upon heat treatment, which could result in a limited pool of ADP-Glucose for synthesizing amylose and amylopectin, the major components of the starch. This suboptimal starch biosynthesis process could subsequently lead to inefficient grain filling and air pockets that contribute to chalkiness. In summary, this study suggests a mechanism of grain chalkiness based on the expression patterns of the starch biosynthesis genes in rice.

Sex Differences in Animal Models of Opioid Reward.

PURPOSE OF REVIEW: This review aims to discuss sex differences observed in preclinical rodent models of opioid reward. RECENT FINDINGS: Utilizing a variety of methodological approaches and drug regimens, no clear consensus has emerged regarding the effects of opiates between males and females. This is quite dissimilar to work examining psychostimulants, where female animals reliably exhibit stronger behavioral responses. SUMMARY: With opioid research quickly expanding to determine the neural underpinnings of opioid addiction, further research is essential to determine the conditions wherein sex differences may occur and how they may relate to the human condition.

Brivaracetam augments short-term depression and slows vesicle recycling.

OBJECTIVE: Brivaracetam (BRV) decreases seizure activity in a number of epilepsy models and binds to the synaptic vesicle glycoprotein 2A (SV2A) with a higher affinity than the antiepileptic drug levetiracetam (LEV). Experiments were performed to determine if BRV acted similarly to LEV to induce or augment short-term depression (STD) under high-frequency neuronal stimulation and slow synaptic vesicle recycling. METHODS: Electrophysiologic field excitatory postsynaptic potential (fEPSP) recordings were made from CA1 synapses in rat hippocampal slices loaded with BRV or LEV during intrinsic activity or with BRV actively loaded during hypertonic stimulation. STD was examined in response to 5 or 40 Hz stimulus trains. Presynaptic release of FM1-43 was visualized using two-photon microscopy to assess drug effects upon synaptic vesicle mobilization. RESULTS: When hippocampal slices were incubated in 0.1-30 µm BRV or 30 µm-1 mm LEV for 3 h, the relative CA1 field EPSPs decreased over the course of a high-frequency train of stimuli more than for control slices. This STD was frequency- and concentration-dependent, with BRV being 100-fold more potent than LEV. The extent of STD depended on the length of the incubation time for both drugs. Pretreatment with LEV occluded the effects of BRV. Repeated hypertonic sucrose treatments and train stimulation successfully unloaded BRV from recycling vesicles and reversed BRVs effects on STD, as previously reported for LEV. At their maximal concentrations, BRV slowed FM1-43 release to a greater extent than in slices loaded with LEV during prolonged stimulation. SIGNIFICANCE: BRV, similar to LEV, entered into recycling synaptic vesicles and produced a frequency-dependent decrement of synaptic transmission at 100-fold lower concentrations than LEV. In addition, BRV slowed synaptic vesicle mobilization more effectively than LEV, suggesting that these drugs may modify multiple functions of the synaptic vesicle protein SV2A to curb synaptic transmission and limit epileptic activity.

Variation in tolerance of rice to long-term stagnant flooding that submerges most of the shoot will aid in breeding tolerant cultivars.

Stagnant flooding (SF) is a major problem in rainfed lowlands where floodwater of 25-50 cm stagnates in the field for most of the season. We aimed to establish a system for phenotyping SF tolerance and identifying tolerant germplasm through screening of landraces. A total of 626 rice accessions were evaluated over 3 years under control conditions and two levels of SF. Floodwater was raised to 20 cm at 25 or 30 days after transplanting (DAT). In one trial, the depth was increased subsequently by 5 cm a week and in another (severe stress), it was increased to 40 cm at 37 DAT and to 50 cm at 42 DAT. In both trials, water depth was maintained at 50-60 cm until maturity. In all cases, no plant was completely submerged. Plant height, elongation rate and yield were measured at maturity. Genotypes best suited to SF showed moderate elongation of 1.3-2.3 cm day(-1) under SF. In contrast, semi-dwarf and fast-elongating types performed poorly. Subsequent trials using 18 genotypes, including six pairs of near isogenic lines (NILs) with or without SUB1 showed that all SUB1 NILs were sensitive to SF. Five of the other six genotypes contained SUB1 and were SF tolerant, suggesting the possibility of combining tolerances to complete submergence (SUB1) and SF. Stem starch and soluble sugar concentrations were similar under control conditions among the 18 genotypes, but starch was depleted by 37 % under SF, with less depletion in tolerant genotypes. SUB1 NILs contained similar concentrations of starch and sugars under SF. We conclude that survival and yield under SF are dependent on moderate elongation, high tillering, lesser carbohydrate depletion and higher fertility. The tolerant genotypes identified here performed strongly in both wet and dry seasons and will be used to identify tolerance mechanisms and alleles for use in marker-assisted breeding.

Determination of heterotic groups for tropical Indica hybrid rice germplasm.

KEY MESSAGE: Two heterotic groups and four heterotic patterns were identified for IRRI hybrid rice germplasm to develop hybrid rice in the tropics based on SSR molecular data and field trials. Information on heterotic groups and patterns is a fundamental prerequisite for hybrid crop breeding; however, no such clear information is available for tropical hybrid rice breeding after more than 30 years of hybrid rice commercialization. Based on a study of genetic diversity using molecular markers, 18 parents representing hybrid rice populations historically developed at the International Rice Research Institute (IRRI) were selected to form diallel crosses of hybrids and were evaluated in tropical environments. Yield, yield heterosis and combining ability were investigated with the main objectives of (1) evaluating the magnitude of yield heterosis among marker-based parental groups, (2) examining the consistency between marker-based group and heterotic performance of hybrids, and (3) identifying foundational hybrid parents in discrete germplasm pools to provide a reference for tropical indica hybrid rice breeding. Significant differences in yield, yield heterosis and combining ability were detected among parents and among hybrids. On average, the hybrids yielded 14.8 % higher than the parents. Results revealed that inter-group hybrids yielded higher, with higher yield heterosis than intra-group hybrids. Four heterotic patterns within two heterotic groups based on current IRRI B- and R-line germplasm were identified. Parents in two marker-based groups were identified with limited breeding value among current IRRI hybrid rice germplasm because of their lowest contribution to heterotic hybrids. Heterotic hybrids are significantly correlated with high-yielding parents. The efficiency of breeding heterotic hybrids could be enhanced using selected parents within identified marker-based heterotic groups. This information is useful for exploiting those widely distributed IRRI hybrid rice parents.

Retinal NMDA receptor function and expression are altered in a mouse lacking D-amino acid oxidase.

D-serine is present in the vertebrate retina and serves as a coagonist for the N-methyl-D-aspartate (NMDA) receptors of ganglion cells. Although the enzyme D-amino acid oxidase (DAO) has been implicated as a pathway for d-serine degradation, its role in the retina has not been established. In this study, we investigated the role of DAO in regulating D-serine levels using a mutant mouse line deficient in DAO (ddY/DAO(-)) and compared these results with their wild-type counterparts (ddY/DAO(+)). Our results show that DAO is functionally present in the mouse retina and normally serves to reduce the background levels of D-serine. The enzymatic activity of DAO was restricted to the inner plexiform layer as determined by histochemical analysis. Using capillary electrophoresis, we showed that mutant mice had much higher levels of D-serine. Whole cell recordings from identified retinal ganglion cells demonstrated that DAO-deficient animals had light-evoked synaptic activity strongly biased toward a high NMDA-to-AMPA receptor ratio. In contrast, recordings from wild-type ganglion cells showed a more balanced ratio between the two receptor subclasses. Immunostaining for AMPA and NMDA receptors was carried out to compare the two receptor ratios by quantitative immunofluorescence. These studies revealed that the mutant mouse had a significantly higher representation of NMDA receptors compared with the wild-type controls. We conclude that 1) DAO is an important regulatory enzyme and normally functions to reduce D-serine levels in the retina, and 2) D-serine levels play a role in the expression of NMDA receptors and the NMDA-to-AMPA receptor ratio.

Serine racemase deletion abolishes light-evoked NMDA receptor currents in retinal ganglion cells.

Glycine and/or D-serine are obligatory coagonists of the N-methyl-D-aspartate receptor (NMDAR). Serine racemase, the D-serine-synthesizing enzyme, is expressed by astrocytes and Müller cells of the retina, but little is known about its role in retinal signalling. In this study, we utilize a serine racemase knockout (SRKO) mouse to explore the contribution of D-serine to inner-retinal function. Retinal tissue levels of D-serine in SRKO mice are reduced by 85%. Whole-cell recordings from SRKO retinal ganglion cells showed markedly reduced coagonist occupancy of NMDARs and consequently a dramatic reduction in the NMDAR component of light-evoked responses. NMDAR currents in SRKOs could be rescued by applying exogenous coagonist, but SRKO ganglion cells still displayed lower NMDA/AMPA receptor ratios than wild-type (WT) controls when the coagonist site was saturated. Despite having abnormalities in synaptic glutamatergic transmission, SRKO mice displayed no obvious signs of visual impairment in behavioural testing. These findings raise interesting questions about the role of D-serine in inner-retinal function and development.

Light-evoked NMDA receptor-mediated currents are reduced by blocking D-serine synthesis in the salamander retina.

Experiments were carried out in the retina of the tiger salamander (Ambystoma tigrinum) to evaluate the importance of D-serine synthesis on light-evoked N-methyl D-aspartate (NMDA) receptor-mediated components of ganglion cells and contributions to the proximal negative field potential. We blocked the synthesis of D-serine through brief exposures of the retina to phenazine ethosulfate and validated the changes in the tissue levels of D-serine using capillary electrophoresis methods to separate and measure the amino acid enantiomers. Ten minute exposures to phenazine ethosulfate decreased D-serine levels in the retina by about 50% and significantly reduced the NMDA receptor contribution to light responses of the inner retina. This is the first report of a linkage between D-serine synthesis and NMDA receptor activity in the vertebrate retina.

The glycine transporter GlyT1 controls N-methyl-D-aspartic acid receptor coagonist occupancy in the mouse retina.

We examined the role of GlyT1, the high-affinity glycine transporter, in the mouse retina with an emphasis on the role of glycine as a coagonist of N-methyl-D-aspartic acid (NMDA) receptors. We pursued this objective by studying heterozygote mice deficient in the GlyT1 transporter (GlyT1(-/+)) and compared those results with wild-type (WT) littermate controls (GlyT1(+/+)). Capillary electrophoresis was used to separate and quantitatively measure glycine release from isolated retina preparations; pharmacologically blocking GlyT1 with N-[3-([1,1-biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine in the WT retina generated a significantly larger accumulation of glycine into the bathing environment when compared with the GlyT1(-/+) retinas. The relative occupancy state of the NMDA receptor coagonist sites was tested using whole-cell recordings from ganglion cells while bath applying D-serine or D-serine + NMDA. The interpretation of these studies was simplified by blocking post-synaptic inhibition with picrotoxinin and strychnine. NMDA receptor coagonist sites were more saturated and less enhanced by D-serine in the GlyT1(-/+) mice compared with the WT controls. Immunoblots of NMDA receptor subunits (NR1, NR2A and NR2B) in WT and GlyT1(-/+) animals showed that the NR1 subunits were identical. These observations are discussed in view of contemporary issues about NMDA receptor coagonist function in the vertebrate retina and the role of glycine vs. D-serine as the endogenous coagonist.

Monitoring neurotransmitter release from isolated retinas using online microdialysis-capillary electrophoresis.

Release of neurotransmitters and other primary amine-containing analytes from intact, isolated larval salamander (Ambystoma tigrinum) retinas maintained in a 6.5-microL perfusion chamber was monitored using online microdialysis-capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Primary amines were derivatized online with o-phthaldialdehyde (OPA) and beta-mercaptoethanol. With the use of overlapping injections, the perfusate was sampled every approximately 10 s. Although separation conditions were optimized using 20 mM hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) for a number of important neuromessengers including D- and L-serine, D- and L-asparate, glutamate, GABA, serotonin, dopamine, norepinephrine, and taurine, only glutamate (0.48 +/- 0.27 microM), GABA (0.25 +/- 0.12 microM), taurine (5.5 +/- 2.1 microM), and l-serine (2.8 +/- 1.0 microM) were identified in the perfusate. Elevated levels of glutamate, GABA, and taurine were detected during stimulation with 60 mM K+. This method is the first to directly sample multiple neurotransmitters from perfused, isolated retinas and to observe changes in efflux of these neurotransmitters as a result of pharmacological stimulation.

D-serine and serine racemase are present in the vertebrate retina and contribute to the physiological activation of NMDA receptors.

d-serine has been proposed as an endogenous modulator of N-methyl-d-aspartate (NMDA) receptors in many brain regions, but its presence and function in the vertebrate retina have not been characterized. We have detected d-serine and its synthesizing enzyme, serine racemase, in the retinas of several vertebrate species, including salamanders, rats, and mice and have localized both constituents to Müller cells and astrocytes, the two major glial cell types in the retina. Physiological studies in rats and salamanders demonstrated that, in retinal ganglion cells, d-serine can enhance excitatory currents elicited by the application of NMDA, as well as the NMDA receptor component of light-evoked synaptic responses. Application of d-amino acid oxidase, which degrades d-serine, reduced the magnitude of NMDA receptor-mediated currents, raising the possibility that endogenous d-serine serves as a ligand for setting the sensitivity of NMDA receptors under physiological conditions. These observations raise exciting new questions about the role of glial cells in regulating the excitability of neurons through release of d-serine.

A high-throughput on-line microdialysis-capillary assay for D-serine.

A high-throughput method is described for the analysis of D-serine and other neurotransmitters in tissue homogenates. Analysis is performed by microdialysis-capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection in a sheath flow detection cell. Sample pretreatment is not required as microdialysis sampling excludes proteins and cell fragments. Primary amines are derivatized on-line with o-phthaldialdehyde (OPA) in the presence of beta-mercaptoethanol followed by on-line CE-LIF analysis. Under the separation conditions described here, D-serine is resolved from L-serine and other primary amines commonly found in biological samples. Each separation requires less than 22 s. Eliminating the need for sample pretreatment and performing the high-speed CE analysis on-line significantly reduces the time required for D-serine analysis when compared with traditional methods. This method has been used to quantify D-serine levels in larval tiger salamander retinal homogenates, as well as dopamine, gamma-amino-n-butyric acid (GABA), glutamate and L-aspartate. D-serine release from an intact retina was also detected.

CD38 expression and NAD+-induced intracellular Ca+ mobilization in isolated retinal Müller cells.

Müller cells of the vertebrate retina are prominent radial glia that provide essential support to sustain homeostasis of the tissue, including redistribution of external potassium, uptake and metabolism of neurotransmitters, and secretion of factors that stabilize the retina. Meeting this diversity of functional supports requires that Müller cells express numerous receptors, transporters, enzymes, and tissue factors. In this study, we provide evidence that adds to the dimensions of Müller cell function by demonstrating a unique relationship between external NAD(+) and the mobilization of internal calcium, expressed in the form of calcium waves. The cellular mechanism that supports internal mobilization of calcium appears to depend on a complex multifunctional ectoenzyme, CD38, which converts NAD(+) into the intracellular Ca(2+)-mobilizing second-messenger cyclic ADP-ribose (cADPR) and could function as a detector for extracellular NAD(+), thus providing a novel signal detection system for evaluating the extracellular environment. Our results are consistent with a model of intracellular Ca(2+) mobilization in which membrane-bound CD38 binds extracellular NAD(+) and triggers intracellular Ca(2+) waves either by direct conversion of NAD(+) to cADPR or by activating intracellular cADPR synthesis. Our preliminary results indicate that the Ca(2+) waves induced by external NAD(+) propagate through an internal pathway that depends on the activation of ryanodine receptors, which appear to be distributed throughout the Müller cell cytosol. Because NAD(+) is likely to be enhanced when cells are stress or damaged, CD38 could enable Müller cells to detect NAD(+) under these circumstances and respond appropriately. Alternatively, NAD(+) could also represent a novel extracellular, paracrine function that mediates signaling between glial cells and/or other cellular elements of the retina.