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Summary The discovery of the default mode network (DMN), a large-scale brain network that is suppressed during attention-demanding tasks, had major impact in neuroscience. This network exhibits an antagonistic relationship with attention-related networks. A better understanding of the processes underlying modulation of DMN is imperative, as this network is compromised in several neurological diseases. Cholinergic neuromodulation is one of the major regulatory networks for attention, and studies suggest a role in regulation of the DMN. In this study, we unilaterally activated the right basal forebrain cholinergic neurons and observed decreased right intra-hemispheric and interhemispheric FC in the default mode like network (DMLN). Our findings provide critical insights into the interplay between cholinergic neuromodulation and DMLN, demonstrate that differential effects can be exerted between the two hemispheres by unilateral stimulation, and open windows for further studies involving directed modulations of DMN in treatments for diseases demonstrating compromised DMN activity.

TnSeq, or transposon (Tn) insertion sequencing, is a powerful method for identifying the essential-as well as conditionally essential-regions in a genome, both coding and noncoding. The advent of accessible massively parallel DNA sequencing technologies in particular has resulted in the increased use of TnSeq-based approaches to elucidate various aspects of bacterial physiology and metabolism. Moreover, the availability of detailed protocols has enabled even nonspecialist laboratories to adapt and develop TnSeq approaches to address specific research questions. In this chapter, we describe a recently modified experimental protocol used in our laboratory for TnSeq in the major human pathogen, Mycobacterium tuberculosis, as well as the related non-pathogenic mycobacterium, M. smegmatis. The method, which was developed in close consultation with pioneers in the field of mycobacterial genetics, includes the steps involved in preparing a phage stock, generating a mutant library, selection of the library under a specific experimental condition, isolation of genomic DNA from the pooled population of mutants, amplification of the sites of Tn insertion and, finally, determining the essential genomic regions by next-generation sequencing.

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Song learning in zebra finches (Taeniopygia guttata) is a prototypical example of a complex learned behavior, yet knowledge of the underlying molecular processes is limited. Therefore, we characterized transcriptomic (RNA-sequencing) and epigenomic (RRBS, reduced representation bisulfite sequencing; immunofluorescence) dynamics in matched zebra finch telencephalon samples of both sexes from 1 day post hatching (1 dph) to adulthood, spanning the critical period for song learning (20 and 65 dph). We identified extensive transcriptional neurodevelopmental changes during postnatal telencephalon development. DNA methylation was very low, yet increased over time, particularly in song control nuclei. Only a small fraction of the massive differential expression in the developing zebra finch telencephalon could be explained by differential CpG and CpH DNA methylation. However, a strong association between DNA methylation and age-dependent gene expression was found for various transcription factors (i.e., OTX2, AR, and FOS) involved in neurodevelopment. Incomplete dosage compensation, independent of DNA methylation, was found to be largely responsible for sexually dimorphic gene expression, with dosage compensation increasing throughout life. In conclusion, our results indicate that DNA methylation regulates neurodevelopmental gene expression dynamics through steering transcription factor activity, but does not explain sexually dimorphic gene expression patterns in zebra finch telencephalon.

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In latitudinal avian migrants, increasing photoperiods induce fat deposition and body mass increase, and subsequent night‐time migratory restlessness in captive birds, but the underlying mechanisms remain poorly understood. We hypothesized that an enhanced hypothalamic neuronal plasticity was associated with the photostimulated spring migration phenotype. We tested this idea in adult migratory red‐headed buntings (Emberiza bruniceps), as compared with resident Indian weaverbirds (Ploceus philippinus). Birds were exposed to a stimulatory long photoperiod (14L:10D, LP), while controls were kept on a short photoperiod (10L:14D, SP). Under both photoperiods, one half of birds also received a high calorie, protein‐ and fat‐rich diet (SP‐R, LP‐R) while the other half stayed on the normal diet (SP‐N, LP‐N). Thirty days later, as expected, the LP had induced multiple changes in the behaviour and physiology in migratory buntings. Photostimulated buntings also developed a preference for the rich food diet. Most interestingly, the LP and the rich diet, both separately and in association, increased neurogenesis in the mediobasal hypothalamus (MBH), as measured by an increased number of cells immunoreactive for doublecortin (DCX), a marker of recently born neurons, in buntings, but not weaverbirds. This neurogenesis was associated with an increased density of fibres immunoreactive for the orexigenic neuropeptide Y (NPY). This hypothalamic plasticity observed in a migratory, but not in a non‐migratory, species in response to photoperiod and food quality might represent an adaptation to the pre‐migratory fattening, as required to support the extensive energy expenses that incur during the migratory flight.