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The last decade has seen the development of a wide set of tools, such as wavefront shaping, computational or fundamental methods, that allow us to understand and control light propagation in a complex medium, such as biological tissues or multimode fibers. A vibrant and diverse community is now working in this field, which has revolutionized the prospect of diffraction-limited imaging at depth in tissues. This roadmap highlights several key aspects of this fast developing field, and some of the challenges and opportunities ahead.

Genetically encoded calcium indicators and optogenetics have revolutionized neuroscience by enabling the detection and modulation of neural activity with single-cell precision using light. To fully leverage the immense potential of these techniques, advanced optical instruments that can place a light on custom ensembles of neurons with a high level of spatial and temporal precision are required. Modern light sculpting techniques that have the capacity to shape a beam of light are preferred because they can precisely target multiple neurons simultaneously and modulate the activity of large ensembles of individual neurons at rates that match natural neuronal dynamics. The most versatile approach, computer-generated holography (CGH), relies on a computer-controlled light modulator placed in the path of a coherent laser beam to synthesize custom three-dimensional (3D) illumination patterns and illuminate neural ensembles on demand. Here, we review recent progress in the development and implementation of fast and spatiotemporally precise CGH techniques that sculpt light in 3D to optically interrogate neural circuit functions.

Dysregulation in the neural circuitry that encodes physiological arousal responses is thought to contribute to the manifestation of the maladaptive behaviors observed in neuropsychiatric disorders. We previously found that prepronociceptin-expressing neurons in the bed nucleus of the stria terminalis (PnocBNST neurons) modulate rapid changes in physiological arousal upon presentation of motivationally salient stimuli (Rodriguez-Romaguera et al., 2020). However, whether PnocBNST neurons are necessary to regulate behavioral actions to motivationally salient stimuli is still unknown. Here, we investigated the role of PnocBNST neurons in encoding behavioral responses to motivationally salient stimuli using in vivo calcium imaging and optogenetic approaches in freely behaving mice. We find that the bulk activity of PnocBNST neurons increases when mice are near an aversive odor in comparison to a rewarding odor. However, optogenetic inhibition of PnocBNST neurons does not affect the amount of time mice spend near an aversive odor. Further analysis revealed that a subgroup of PnocBNST neurons that correlate with proximity to the aversive odor also correlate to darting away from the same aversive odor. Since these two behaviors are opposite to each other and since we previously found PnocBNST neurons correlate with arousal responses, we believe these results may be due in part to the encoding of arousal responses that occur when mice approach and dart away from aversive stimuli. Competing Interest Statement The authors have declared no competing interest.

Traumatic social experiences redefine socially motivated behaviors to enhance safety and survival. Although many brain regions have been implicated in signaling a social threat, the mechanisms by which global neural networks regulate such motivated behaviors remain unclear. To address this issue, we first combined traditional and modern behavioral tracking techniques in mice to assess both approach and avoidance, as well as sub-second behavioral changes, during a social threat learning task. We were able to identify previously undescribed body and tail movements during social threat learning and recognition that demonstrate unique alterations into the behavioral structure of social motivation. We then utilized inter-regional correlation analysis of brain activity after a mouse recognizes a social threat to explore functional communication amongst brain regions implicated in social motivation. Broad brain activity changes were observed within the nucleus accumbens, the paraventricular thalamus, the ventromedial hypothalamus, and the nucleus of reuniens. Inter-regional correlation analysis revealed a reshaping of the functional connectivity across the brain when mice recognize a social threat. Altogether, these findings suggest that reshaping of functional brain connectivity may be necessary to alter the behavioral structure of social motivation when a social threat is encountered.

The last decade has seen the development of a wide set of tools, such as wavefront shaping, computational or fundamental methods, that allow to understand and control light propagation in a complex medium, such as biological tissues or multimode fibers. A vibrant and diverse community is now working on this field, that has revolutionized the prospect of diffraction-limited imaging at depth in tissues. This roadmap highlights several key aspects of this fast developing field, and some of the challenges and opportunities ahead.

Although culture-independent techniques have shown that the lungs are not sterile, little is known about the lung microbiome in chronic obstructive pulmonary disease (COPD). We used pyrosequencing of 16S amplicons to analyze the lung microbiome in two ways: first, using bronchoalveolar lavage (BAL) to sample the distal bronchi and air-spaces; and second, by examining multiple discrete tissue sites in the lungs of six subjects removed at the time of transplantation. We performed BAL on three never-smokers (NS) with normal spirometry, seven smokers with normal spirometry (\"healthy smokers\", HS), and four subjects with COPD (CS). Bacterial 16 s sequences were found in all subjects, without significant quantitative differences between groups. Both taxonomy-based and taxonomy-independent approaches disclosed heterogeneity in the bacterial communities between HS subjects that was similar to that seen in healthy NS and two mild COPD patients. The moderate and severe COPD patients had very limited community diversity, which was also noted in 28% of the healthy subjects. Both approaches revealed extensive membership overlap between the bacterial communities of the three study groups. No genera were common within a group but unique across groups. Our data suggests the existence of a core pulmonary bacterial microbiome that includes Pseudomonas, Streptococcus, Prevotella, Fusobacterium, Haemophilus, Veillonella, and Porphyromonas. Most strikingly, there were significant micro-anatomic differences in bacterial communities within the same lung of subjects with advanced COPD. These studies are further demonstration of the pulmonary microbiome and highlight global and micro-anatomic changes in these bacterial communities in severe COPD patients.

Obligate symbioses involving intracellular bacteria have transformed eukaryotic life, from providing aerobic respiration and photosynthesis to enabling colonization of previously inaccessible niches, such as feeding on xylem and phloem, and surviving in deep-sea hydrothermal vents. A major challenge in the study of obligate symbioses is to understand how they arise. Because the best studied obligate symbioses are ancient, it is especially challenging to identify early or intermediate stages. Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium, a well-studied nematode parasite of Drosophila flies. We have found that H. aoronymphium and its sister species harbor a maternally inherited intracellular bacterial symbiont. We never find the symbiont in nematode-free flies, and virtually all nematodes in the field and the laboratory are infected. Treating nematodes with antibiotics causes a severe reduction in fly infection success. The association is recent, as more distantly related insect-parasitic tylenchid nematodes do not host these endosymbionts. We also report that the Howardula nematode symbiont is a member of a widespread monophyletic group of invertebrate host-associated microbes that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, and that is sister to Pectobacterium, a lineage of plant pathogenic bacteria. Comparative genomic analysis of this group, which we name Candidatus Symbiopectobacterium, shows signatures of genome erosion characteristic of early stages of symbiosis, with the Howardula symbiont’s genome containing over a thousand predicted pseudogenes, comprising a third of its genome.

No studies have examined the relationships between bacterial communities along sites of the upper aerodigestive tract of an individual subject. Our objective was to perform an intrasubject and intersite analysis to determine the contributions of two upper mucosal sites (mouth and nose) as source communities for the bacterial microbiome of lower sites (lungs and stomach). Oral wash, bronchoalveolar lavage (BAL) fluid, nasal swab, and gastric aspirate samples were collected from 28 healthy subjects. Extensive analysis of controls and serial intrasubject BAL fluid samples demonstrated that sampling of the lungs by bronchoscopy was not confounded by oral microbiome contamination. By quantitative PCR, the oral cavity and stomach contained the highest bacterial signal levels and the nasal cavity and lungs contained much lower levels. Pyrosequencing of 16S rRNA gene amplicon libraries generated from these samples showed that the oral and gastric compartments had the greatest species richness, which was significantly greater in both than the richness measured in the lungs and nasal cavity. The bacterial communities of the lungs were significantly different from those of the mouth, nose, and stomach, while the greatest similarity was between the oral and gastric communities. However, the bacterial communities of healthy lungs shared significant membership with the mouth, but not the nose, and marked subject-subject variation was noted. In summary, microbial immigration from the oral cavity appears to be the significant source of the lung microbiome during health, but unlike the stomach, the lungs exhibit evidence of selective elimination of Prevotella bacteria derived from the upper airways. IMPORTANCE We have demonstrated that the bacterial communities of the healthy lung overlapped those found in the mouth but were found at lower concentrations, with lower membership and a different community composition. The nasal microbiome, which was distinct from the oral microbiome, appeared to contribute little to the composition of the lung microbiome in healthy subjects. Our studies of the nasal, oral, lung, and stomach microbiomes within an individual illustrate the microbiological continuity of the aerodigestive tract in healthy adults and provide culture-independent microbiological support for the concept that microaspiration is common in healthy individuals. We have demonstrated that the bacterial communities of the healthy lung overlapped those found in the mouth but were found at lower concentrations, with lower membership and a different community composition. The nasal microbiome, which was distinct from the oral microbiome, appeared to contribute little to the composition of the lung microbiome in healthy subjects. Our studies of the nasal, oral, lung, and stomach microbiomes within an individual illustrate the microbiological continuity of the aerodigestive tract in healthy adults and provide culture-independent microbiological support for the concept that microaspiration is common in healthy individuals.

The transcription factor BMAL1 is a clock protein that generates daily or circadian rhythms in physiological functions including the inflammatory response of macrophages. Intracellular metabolic pathways direct the macrophage inflammatory response, however whether the clock is impacting intracellular metabolism to direct this response is unclear. Specific metabolic reprogramming of macrophages controls the production of the potent pro-inflammatory cytokine IL-1β. We now describe that the macrophage molecular clock, through Bmal1 , regulates the uptake of glucose, its flux through glycolysis and the Krebs cycle, including the production of the metabolite succinate to drive Il-1β production. We further demonstrate that BMAL1 modulates the level and localisation of the glycolytic enzyme PKM2, which in turn activates STAT3 to further drive Il-1β mRNA expression. Overall, this work demonstrates that BMAL1 is a key metabolic sensor in macrophages, and its deficiency leads to a metabolic shift of enhanced glycolysis and mitochondrial respiration, leading to a heightened pro-inflammatory state. These data provide insight into the control of macrophage driven inflammation by the molecular clock, and the potential for time-based therapeutics against a range of chronic inflammatory diseases.