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Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse activity in dopamine axons responsive to both reward and aversive stimuli. Some axons exhibited a preference for reward, while others favored aversive stimuli, and there was a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings indicate that a group of mesocortical dopamine axons encodes aversive-related signals, which are modulated by both classical conditioning across days and trial-by-trial discrimination within a day.

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Significance: The initiation of goal-directed actions is a complex process involving the medial prefrontal cortex and dopaminergic inputs through the mesocortical pathway. However, it is unclear what information the mesocortical pathway conveys and how it impacts action initiation. In this study, we unveiled the indispensable role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Aim: To investigate the role of mesocortical axon terminals in encoding the execution of movements in self-initiated actions. Approach: We designed a lever-press task in which mice internally determine the timing of the press, receiving a larger reward for longer waiting periods. Results: Our study revealed that self-initiated actions depend on dopaminergic signaling mediated by D2 receptors, whereas sensory-triggered lever-press actions do not involve D2 signaling. Microprism-mediated two-photon calcium imaging further demonstrated ramping activity in mesocortical axon terminals approximately 0.5 s before the self-initiated lever press. Remarkably, the ramping patterns remained consistent whether the mice responded to cues immediately for a smaller reward or held their response for a larger reward. Conclusions: We conclude that mesocortical dopamine axon terminals encode the timing of self-initiated actions, shedding light on a crucial aspect of the intricate neural mechanisms governing goal-directed behavior.

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Some species of the genus Aegilops, a wild relative of wheat, carry chromosomes that after introducing to wheat exhibit preferential transmission to progeny. Their selective retention is a result of the abortion of gametes lacking them due to induced chromosomal aberrations. These chromosomes are termed Gametocidal (Gc) and, based on their effects, they are categorized into three types: mild, intense or severe, and very strong. Gc elements within the same homoeologous chromosome groups of Aegilops (II, III, or IV) demonstrate similar Gc action. This review explores the intriguing dynamics of Gc chromosomes and encompasses comprehensive insights into their source species, behavioral aspects, mode of action, interactions, suppressions, and practical applications of the Gc system in wheat breeding. By delving into these areas, this work aims to contribute to the development of novel plant genetic resources for wheat breeding. The insights provided herein shed light on the utilization of Gc chromosomes to produce chromosomal rearrangements in wheat and its wild relatives, thereby facilitating the generation of chromosome deletions, translocations, and telosomic lines. The Gc approach has significantly advanced various aspects of wheat genetics, including the introgression of novel genes and alleles, molecular markers and gene mapping, and the exploration of homoeologous relationships within Triticeae species. The mystery lies in why gametes possessing Gc genes maintain their normality while those lacking Gc genes suffer abnormalities, highlighting an unresolved research gap necessitating deeper investigation.

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Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse activity in dopamine axons responsive to both reward and aversive stimuli. Some axons exhibited a preference for reward, while others favored aversive stimuli, and there was a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings indicate that a group of mesocortical dopamine axons encodes aversive-related signals, which are modulated by both classical conditioning across days and trial-by-trial discrimination within a day.

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Goal-directed behavior often involves temporal separation and flexible context-dependent association between sensory input and motor output. The control of goal-directed behavior is proposed to lie in the frontoparietal network, but the computational architecture of this network remains elusive. Based on recent rodent studies that measured and manipulated projection neurons in the frontoparietal network together with findings from earlier primate studies, we propose a canonical scheme of information flows in this network. The parietofrontal pathway transmits the spatial information of a sensory stimulus or internal motor bias to drive motor programs in the frontal areas. This pathway might consist of multiple parallel connections, each controlling distinct motor effectors. The frontoparietal pathway sends the spatial information of cognitively processed motor plans through multiple parallel connections. Each of these connections could support distinct spatial functions that use the motor target information, including attention allocation, multi-body part coordination, and forward estimation of movement state (i.e., forward models). The parallel pathways in the frontoparietal network enable dynamic interactions between regions that are tuned for specific goal-directed behaviors. This scheme offers a promising framework within which the computational architecture of the frontoparietal network and the underlying circuit mechanisms can be delineated in a systematic way, providing a holistic understanding of information processing in this network. Clarifying this network may also improve the diagnosis and treatment of behavioral deficits associated with dysfunctional frontoparietal connectivity in various neurological disorders including Alzheimer's disease.

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Most deletions for the short arm of chromosome 2A (2AS), and the telocentric chromosome for the long arm of chromosome 2A (2AL), are available only in the heterozygous condition in 'Chinese Spring' hexaploid wheat. This is due to the female sterility, and therefore self-sterility, of their homozygotes, caused by the partial or entire loss of the 2AS chromosome arm on which genes for normal synapsis and female fertility are located. On the other hand, a D-genome disomic substitution line 2D(2A) of 'Langdon' tetraploid wheat, in which chromosome 2D is disomically substituted for chromosome 2A, is available (i.e., self-fertile) despite chromosome 2A being missing in this line. This fact indicates that another gene for female fertility must be present in Langdon 2D(2A). We attempted to develop self-fertile 2AS homozygous deletion and ditelosomic 2AL lines by transferring this female fertility gene, through a series of crosses and cytological screening, from Langdon 2D(2A) to the two aneuploid lines. We finally obtained self-fertile 2AS homozygous deletion and ditelosomic 2AL lines. These lines displayed normal meiotic chromosome pairing and lacked all 12 of the 2AS markers used for PCR analysis.

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Cortical plasticity is fundamental to motor recovery following cortical perturbation. However, it is still unclear how this plasticity is induced at a functional circuit level. Here, we investigated motor recovery and underlying neural plasticity upon optogenetic suppression of a cortical area for eye movement. Using a visually-guided eye movement task in mice, we suppressed a portion of the secondary motor cortex (MOs) that encodes contraversive eye movement. Optogenetic unilateral suppression severely impaired contraversive movement on the first day. However, on subsequent days the suppression became inefficient and capability for the movement was restored. Longitudinal two-photon calcium imaging revealed that the regained capability was accompanied by an increased number of neurons encoding for ipsiversive movement in the unsuppressed contralateral MOs. Additional suppression of the contralateral MOs impaired the recovered movement again, indicating a compensatory mechanism. Our findings demonstrate that repeated optogenetic suppression leads to functional recovery mediated by the contralateral hemisphere.

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Three transgenic HOSUT lines of winter wheat, HOSUT12, HOSUT20, and HOSUT24, each harbor a single copy of the cDNA for the barley sucrose transporter gene HvSUT1 (SUT), which was fused to the barley endosperm-specific Hordein B1 promoter (HO; the HOSUT transgene). Previously, flow cytometry combined with PCR analysis demonstrated that the HOSUT transgene had been integrated into different wheat chromosomes: 7A, 5D, and 4A in HOSUT12, HOSUT20, and HOSUT24, respectively. In order to confirm the chromosomal location of the HOSUT transgene by a cytological approach using wheat aneuploid stocks, we crossed corresponding nullisomic-tetrasomic lines with the three HOSUT lines, namely nullisomic 7A-tetrasomic 7B with HOSUT12, nullisomic 5D-tetrasomic 5B with HOSUT20, and nullisomic 4A-tetrasomic 4B with HOSUT24. We examined the resulting chromosomal constitutions and the presence of the HOSUT transgene in the F2 progeny by means of chromosome banding and PCR. The chromosome banding patterns of the critical chromosomes in the original HOSUT lines showed no difference from those of the corresponding wild type chromosomes. The presence or absence of the critical chromosomes completely corresponded to the presence or absence of the HOSUT transgene in the F2 plants. Investigating telocentric chromosomes occurred in the F2 progeny, which were derived from the respective critical HOSUT chromosomes, we found that the HOSUT transgene was individually integrated on the long arms of chromosomes 4A, 7A, and 5D in the three HOSUT lines. Thus, in this study we verified the chromosomal locations of the transgene, which had previously been determined by flow cytometry, and moreover revealed the chromosome-arm locations of the HOSUT transgene in the HOSUT lines.

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The identification of genes of agronomic interest in bread wheat (Triticum aestivum L.) is hampered by its allopolyploid nature (2n = 6x = 42; AABBDD) and its very large genome, which is largely covered by transposable elements. However, owing to this complex structure, aneuploid stocks can be developed in which fragments or entire chromosomes are missing, sometimes resulting in visible phenotypes that help in the cloning of affected genes. In this study, the 2C gametocidal chromosome from Aegilops cylindrica was used to develop a set of 113 deletion lines for chromosome 3D in the reference cultivar Chinese Spring. Eighty-four markers were used to show that the deletions evenly covered chromosome 3D and ranged from 6.5 to 357 Mb. Cytogenetic analyses confirmed that the physical size of the deletions correlated well with the known molecular size deduced from the reference sequence. This new genetic stock will be useful for positional cloning of genes on chromosome 3D, especially for Ph2 affecting homoeologous pairing in bread wheat.

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Despite a long history, the production of useful alien introgression lines in wheat remains difficult mainly due to linkage drag and incomplete genetic compensation. In addition, little is known about the molecular mechanisms underlying the impact of foreign chromatin on plant phenotype. Here, a comparison of the transcriptomes of barley, wheat and a wheat-barley 7HL addition line allowed the transcriptional impact both on 7HL genes of a non-native genetic background and on the wheat gene complement as a result of the presence of 7HL to be assessed. Some 42% (389/923) of the 7HL genes assayed were differentially transcribed, which was the case for only 3% (960/35 301) of the wheat gene complement. The absence of any transcript in the addition line of a suite of chromosome 7A genes implied the presence of a 36 Mbp deletion at the distal end of the 7AL arm; this deletion was found to be in common across the full set of Chinese Spring/Betzes barley addition lines. The remaining differentially transcribed wheat genes were distributed across the whole genome. The up-regulated barley genes were mostly located in the proximal part of the 7HL arm, while the down-regulated ones were concentrated in the distal part; as a result, genes encoding basal cellular functions tended to be transcribed, while those encoding specific functions were suppressed. An insight has been gained into gene transcription in an alien introgression line, thereby providing a basis for understanding the interactions between wheat and exotic genes in introgression materials.

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Cortical computation is distributed across multiple areas of the cortex by networks of reciprocal connectivity. However, how such connectivity contributes to the communication between the connected areas is not clear. In this study, we examine the communication between sensory and motor cortices. We develop an eye movement task in mice and combine it with optogenetic suppression and two-photon calcium imaging techniques. We identify a small region in the secondary motor cortex (MOs) that controls eye movements and reciprocally connects with a rostrolateral part of the higher visual areas (VRL/A/AL). These two regions encode both motor signals and visual information; however, the information flow between the regions depends on the direction of the connectivity: motor information is conveyed preferentially from the MOs to the VRL/A/AL, and sensory information is transferred primarily in the opposite direction. We propose that reciprocal connectivity streamlines information flow, enhancing the computational capacity of a distributed network.

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Prepared movements are more efficient than those that are not prepared for. Although changes in cortical activity have been observed prior to a forthcoming action, the circuits involved in motor preparation remain unclear. Here, we use in vivo two-photon calcium imaging to uncover changes in the motor cortex during variable waiting periods prior to a forepaw reaching task in mice. Consistent with previous reports, we observed a subset of neurons with increased activity during the waiting period; however, these neurons did not account for the degree of preparation as defined by reaction time (RT). Instead, the suppression of activity of distinct neurons in the same cortical area better accounts for RT. This suppression of neural activity resulted in a distinct and reproducible pattern when mice were well prepared. Thus, the selective suppression of network activity in the motor cortex may be a key feature of prepared movements.

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Sleep is characterized by unique patterns of cortical activity alternating between the stages of slow-wave sleep (SWS) and rapid-eye movement (REM) sleep. How these patterns relate to the balanced activity of excitatory pyramidal cells and inhibitory interneurons in cortical circuits is unknown. We investigated cortical network activity during wakefulness, SWS, and REM sleep globally and locally using in vivo calcium imaging in mice. Wide-field imaging revealed a reduction in pyramidal cell activity during SWS compared with wakefulness and, unexpectedly, a further profound reduction in activity during REM sleep. Two-photon imaging on local circuits showed that this suppression of activity during REM sleep was accompanied by activation of parvalbumin (PV)+ interneurons, but not of somatostatin (SOM)+ interneurons. PV+ interneurons most active during wakefulness were also most active during REM sleep. Our results reveal a sleep-stage-specific regulation of the cortical excitation/inhibition balance, with PV+ interneurons conveying maximum inhibition during REM sleep, which might help shape memories in these networks.

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Flow cytometry enables chromosomes to be sorted into different groups based on their characteristics, such as relative DNA content and the presence of repetitive DNA sequences. Despite the recent progress in the analysis of plant genome organization and chromosome structure, there is a need for easy methods to assign DNA sequences to individual chromosomes. Here, we describe an easy way to allocate genes or DNA sequences to chromosomes in wheat using flow-sorted chromosomes combined with fluorescence in situ hybridization and PCR analyses.

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BACKGROUND: There is no curative treatment for wheat-dependent exercise-induced anaphylaxis (WDEIA). ω-5 Gliadin is one of the dominant allergens affecting WDEIA patients. The use of ω-5 gliadin-free wheat flour in the regular diet is considered one of the prophylactic approaches against the elicitation of allergic symptoms and sensitization to ω-5 gliadin. We sought to find hypoallergenic bread wheat (or common wheat) that lacked the genes encoding ω-5 gliadin and to evaluate its in vitro allergenicity. We also aimed to evaluate the sensitization ability of one of the selected hypoallergenic wheat lines by using a possible animal model of wheat allergy. METHODS: We screened the deletion lines of bread wheat by western blotting to ascertain common wheat lines lacking the ω-5 gliadin locus. The deletion lines we used have partial deficiency of chromosome 1B (Endo and Gill, 1996). To assess sensitization ability of gluten from the selected deletion line, guinea pigs were fed with either the gluten from the selected deletion line or commercially available gluten, and allergic score was evaluated after challenging the same gluten preparations. RESULTS: We found that a deletion line 1BS-18 had the least deficiency of chromosome 1B among the deletion stocks lacking the ω-5 gliadin locus. The challenge test using the guinea pigs revealed that the symptoms induced by application of the 1BS-18 gluten were much less than that of commercially available gluten. CONCLUSIONS: The deletion line 1BS-18, which lacked the ω-5 gliadin locus, is likely to have a low sensitization capacity in the guinea pig. The use of the wheat products of the 1BS-18 line in daily life may provide a feasible solution for the onset of wheat allergy.

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BACKGROUND: Identification of transgene insertion sites in plant genomes has practical implications for crop breeding and is a stepping stone to analyze transgene function. However, single copy sequences are not always easy to localize in large plant genomes by standard approaches. RESULTS: We employed flow cytometric chromosome sorting to determine chromosomal location of barley sucrose transporter construct in three transgenic lines of common wheat. Flow-sorted chromosomes were used as template for PCR and fluorescence in situ hybridization to identify chromosomes with transgenes. The chromosomes carrying the transgenes were then confirmed by PCR using DNA amplified from single flow-sorted chromosomes as template. CONCLUSIONS: Insertion sites of the transgene were unambiguously localized to chromosomes 4A, 7A and 5D in three wheat transgenic lines. The procedure presented in this study is applicable for localization of any single-copy sequence not only in wheat, but in any plant species where suspension of intact mitotic chromosomes suitable for flow cytometric sorting can be prepared.

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BACKGROUND: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. RESULTS: We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91% of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87% of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. CONCLUSIONS: We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

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In order to assess the contribution of a central clock in the hypothalamic suprachiasmatic nucleus (SCN) to circadian behavior and the organization of peripheral clocks, we generated forebrain/SCN-specific Bmal1 knockout mice by using floxed Bmal1 and pan-neuronal Cre lines. The forebrain knockout mice showed >90% deletion of BMAL1 in the SCN and exhibited an immediate and complete loss of circadian behavior in constant conditions. Circadian rhythms in peripheral tissues persisted but became desynchronized and damped in constant darkness. The loss of synchrony was rescued by light/dark cycles and partially by restricted feeding (only in the liver and kidney but not in the other tissues) in a distinct manner. These results suggest that the forebrain/SCN is essential for internal temporal order of robust circadian programs in peripheral clocks, and that individual peripheral clocks are affected differently by light and feeding in the absence of a functional oscillator in the forebrain.

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Common wheat (Triticum aestivum L.) is one of the most important cereals in the world. To improve wheat quality and productivity, the genomic sequence of wheat must be determined. The large genome size (∼17 Gb/1 C) and the hexaploid status of wheat have hampered the genome sequencing of wheat. However, flow sorting of individual chromosomes has allowed us to purify and separately shotgun-sequence a pair of telocentric chromosomes. Here, we describe a result from the survey sequencing of wheat chromosome 6B (914 Mb/1 C) using massively parallel 454 pyrosequencing. From the 4.94 and 5.51 Gb shotgun sequence data from the two chromosome arms of 6BS and 6BL, 235 and 273 Mb sequences were assembled to cover ∼55.6 and 54.9% of the total genomic regions, respectively. Repetitive sequences composed 77 and 86% of the assembled sequences on 6BS and 6BL, respectively. Within the assembled sequences, we predicted a total of 4798 non-repetitive gene loci with the evidence of expression from the wheat transcriptome data. The numbers and chromosomal distribution patterns of the genes for tRNAs and microRNAs in wheat 6B were investigated, and the results suggested a significant involvement of DNA transposon diffusion in the evolution of these non-protein-coding RNA genes. A comparative analysis of the genomic sequences of wheat 6B and monocot plants clearly indicated the evolutionary conservation of gene contents.

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Despite remarkable recent progress in the analysis of plant genome organization and chromosome structure, there is a need for methods enabling DNA sequences to be mapped by fluorescence in situ hybridization (FISH) at high spatial resolution. We sorted mitotic metaphase chromosomes of wheat by flow cytometry and observed the occurrence of hyperexpanded chromosomes among them. However, this phenomenon was not reproducible in subsequent experiments. An investigation into the procedures of flow cytometry revealed that the hyperexpansion of chromosomes became reproducible when the concentration of formaldehyde used in sample fixation was reduced. We conducted FISH analysis with 45S rDNA, 5S rDNA and wheat centromeric repeat sequences as probes on flow-sorted chromosomes and also on chromosomes from squash preparations. We measured the length of chromosomes 1B and 6B, identified by FISH. On average, the hyperexpanded 1B and 6B chromosomes were 7.26 and 7.53 times longer, respectively, than the same chromosomes from the squash preparations. The most stretched 1B and 6B chromosomes both exceeded 100 micrometers.

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