Early-Life Gene Expression in Neurons Modulates Lasting Epigenetic States.

In mammals, the environment plays a critical role in promoting the final steps in neuronal development during the early postnatal period. While epigenetic factors are thought to contribute to this process, the underlying molecular mechanisms remain poorly understood. Here, we show that in the brain during early life, the DNA methyltransferase DNMT3A transiently binds across transcribed regions of lowly expressed genes, and its binding specifies the pattern of DNA methylation at CA sequences (mCA) within these genes. We find that DNMT3A occupancy and mCA deposition within the transcribed regions of genes is negatively regulated by gene transcription and may be modified by early-life experience. Once deposited, mCA is bound by the methyl-DNA-binding protein MECP2 and functions in a rheostat-like manner to fine-tune the cell-type-specific transcription of genes that are critical for brain function.

Longevity, demographic characteristics, and socio-economic status are linked to triiodothyronine levels in the general population.

The hypothalamic-pituitary-thyroid (HPT) axis is fundamental to human biology, exerting central control over energy expenditure and body temperature. However, the consequences of normal physiologic HPT-axis variation in populations without diagnosed thyroid disease are poorly understood. Using nationally representative data from the 2007 to 2012 National Health and Nutrition Examination Survey, we explore relationships with demographic characteristics, longevity, and socio-economic factors. We find much larger variation across age in free T3 than other HPT-axis hormones. T3 and T4 have opposite relationships to mortality: free T3 is inversely related and free T4 is positively related to the likelihood of death. Free T3 and household income are negatively related, particularly at lower incomes. Finally, free T3 among older adults is associated with labor both in terms of unemployment and hours worked. Physiologic TSH/T4 explain only 1.7% of T3 variation, and neither are appreciably correlated to socio-economic outcomes. Taken together, our data suggest an unappreciated complexity of the HPT-axis signaling cascade broadly such that TSH and T4 may not be accurate surrogates of free T3. Furthermore, we find that subclinical variation in the HPT-axis effector hormone T3 is an important and overlooked factor linking socio-economic forces, human biology, and aging.

Publisher Correction: A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.

In the version of this article originally published, the bottom of Figure 4f,g was partially truncated in the PDF. The error has been corrected in the PDF version of this article.

A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.

We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.

All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

Optical recording of action potentials in mammalian neurons using a microbial rhodopsin.

Reliable optical detection of single action potentials in mammalian neurons has been one of the longest-standing challenges in neuroscience. Here we achieved this goal by using the endogenous fluorescence of a microbial rhodopsin protein, Archaerhodopsin 3 (Arch) from Halorubrum sodomense, expressed in cultured rat hippocampal neurons. This genetically encoded voltage indicator exhibited an approximately tenfold improvement in sensitivity and speed over existing protein-based voltage indicators, with a roughly linear twofold increase in brightness between -150 mV and +150 mV and a sub-millisecond response time. Arch detected single electrically triggered action potentials with an optical signal-to-noise ratio >10. Arch(D95N) lacked endogenous proton pumping and had 50% greater sensitivity than wild type but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual action potentials. Microbial rhodopsin-based voltage indicators promise to enable optical interrogation of complex neural circuits and electrophysiology in systems for which electrode-based techniques are challenging.

Sub-clinical triiodothyronine levels predict health, demographic, and socioeconomic outcomes.

The Hypothalamic-Pituitary-Thyroid (HPT) axis is fundamental to human biology, exerting central control over energy expenditure, metabolic rate, and body temperature. However, the consequences of "normal" physiologic HPT-axis variation in non-clinical populations are poorly understood. Using nationally-representative data from the 2007-2012 NHANES, we explore relationships with demographics, mortality, and socio-economic factors. We find much larger variation across age in free T3 than other HPT-axis hormones. T3 and T4 have opposite effects on mortality: free T3 is inversely related and free T4 is positively related with likelihood of death. Free T3 and household income are negatively related, particularly at lower incomes. Finally, free T3 among older adults is associated with labor both on the extensive margin (unemployment) and intensive margin (hours worked). Physiologic TSH/T4 explain only 1% of T3 variation, and neither are appreciably correlated to socio-economic outcomes. Taken together, our data suggest an unappreciated complexity and non-linearity of the HPT-axis signaling cascade broadly such that TSH and T4 may not be accurate surrogates of free T3. Furthermore, we find that sub-clinical variation in the HPT-axis effector hormone T3 is an important and overlooked factor linking socio-economic forces, human biology, and aging.

Thyroid hormone rewires cortical circuits to coordinate body-wide metabolism and exploratory drive.

Animals adapt to varying environmental conditions by modifying the function of their internal organs, including the brain. To be adaptive, alterations in behavior must be coordinated with the functional state of organs throughout the body. Here we find that thyroid hormone- a prominent regulator of metabolism in many peripheral organs- activates cell-type specific transcriptional programs in anterior regions of cortex of adult mice via direct activation of thyroid hormone receptors. These programs are enriched for axon-guidance genes in glutamatergic projection neurons, synaptic regulators across both astrocytes and neurons, and pro-myelination factors in oligodendrocytes, suggesting widespread remodeling of cortical circuits. Indeed, whole-cell electrophysiology recordings revealed that thyroid hormone induces local transcriptional programs that rewire cortical neural circuits via pre-synaptic mechanisms, resulting in increased excitatory drive with a concomitant sensitization of recruited inhibition. We find that thyroid hormone bidirectionally regulates innate exploratory behaviors and that the transcriptionally mediated circuit changes in anterior cortex causally promote exploratory decision-making. Thus, thyroid hormone acts directly on adult cerebral cortex to coordinate exploratory behaviors with whole-body metabolic state.

Activity-dependent regulome of human GABAergic neurons reveals new patterns of gene regulation and neurological disease heritability.

Neuronal activity-dependent gene expression is essential for brain development. Although transcriptional and epigenetic effects of neuronal activity have been explored in mice, such an investigation is lacking in humans. Because alterations in GABAergic neuronal circuits are implicated in neurological disorders, we conducted a comprehensive activity-dependent transcriptional and epigenetic profiling of human induced pluripotent stem cell-derived GABAergic neurons similar to those of the early developing striatum. We identified genes whose expression is inducible after membrane depolarization, some of which have specifically evolved in primates and/or are associated with neurological diseases, including schizophrenia and autism spectrum disorder (ASD). We define the genome-wide profile of human neuronal activity-dependent enhancers, promoters and the transcription factors CREB and CRTC1. We found significant heritability enrichment for ASD in the inducible promoters. Our results suggest that sequence variation within activity-inducible promoters of developing human forebrain GABAergic neurons contributes to ASD risk.

Distinct Cortical-Thalamic-Striatal Circuits through the Parafascicular Nucleus.

The thalamic parafascicular nucleus (PF), an excitatory input to the basal ganglia, is targeted with deep-brain stimulation to alleviate a range of neuropsychiatric symptoms. Furthermore, PF lesions disrupt the execution of correct motor actions in uncertain environments. Nevertheless, the circuitry of the PF and its contribution to action selection are poorly understood. We find that, in mice, PF has the highest density of striatum-projecting neurons among all sub-cortical structures. This projection arises from transcriptionally and physiologically distinct classes of PF neurons that are also reciprocally connected with functionally distinct cortical regions, differentially innervate striatal neurons, and are not synaptically connected in PF. Thus, mouse PF contains heterogeneous neurons that are organized into parallel and independent associative, limbic, and somatosensory circuits. Furthermore, these subcircuits share motifs of cortical-PF-cortical and cortical-PF-striatum organization that allow each PF subregion, via its precise connectivity with cortex, to coordinate diverse inputs to striatum.

Single-cell analysis of experience-dependent transcriptomic states in the mouse visual cortex.

Activity-dependent transcriptional responses shape cortical function. However, a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease, is lacking. To investigate the breadth of transcriptional changes that occur across cell types in the mouse visual cortex after exposure to light, we applied high-throughput single-cell RNA sequencing. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, thus revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibited inter- and intralaminar heterogeneity in the induction of stimulus-responsive genes. Non-neuronal cells showed clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of the stimulus-dependent transcriptional changes occurring across cell types in the visual cortex; these changes are probably critical for cortical function and may be sites of deregulation in developmental brain disorders.

Publisher Correction: Single-cell analysis of experience-dependent transcriptomic states in the mouse visual cortex.

In the version of this article initially published, the x-axis labels in Fig. 3c read Vglut, Gad1/2, Aldh1l1 and Pecam1; they should have read Vglut+, Gad1/2+, Aldh1l1+ and Pecam1+. In Fig. 4, the range values were missing from the color scales; they are, from left to right, 4-15, 0-15, 4-15 and 0-15 in Fig. 4a and 4-15, 4-15 and 4-8 in Fig. 4h. In the third paragraph of the main text, the phrase reading "Previous approaches have analyzed a limited number of inhibitory cell types, thus masking the full diversity of excitatory populations" should have read "Previous approaches have analyzed a limited number of inhibitory cell types and masked the full diversity of excitatory populations." In the second paragraph of Results section "Diversity of experience-regulated ERGs," the phrase reading "thus suggesting considerable divergence within the gene expression program responding to early stimuli" should have read "thus suggesting considerable divergence within the early stimulus-responsive gene expression program." In the fourth paragraph of Results section "Excitatory neuronal LRGs," the sentence reading "The anatomical organization of these cell types into sublayers, coupled with divergent transcriptional responses to a sensory stimulus, suggested previously unappreciated functional subdivisions located within the laminae of the mouse visual cortex and resembling the cytoarchitecture in higher mammals" should have read "The anatomical organization of these cell types into sublayers, coupled with divergent transcriptional responses to a sensory stimulus, suggests previously unappreciated functional subdivisions located within the laminae of the mouse visual cortex, resembling the cytoarchitecture in higher mammals." In the last sentence of the Results, "sensory-responsive genes" should have read "sensory-stimulus-responsive genes." The errors have been corrected in the HTML and PDF versions of the article.

Measuring membrane voltage with microbial rhodopsins.

Membrane voltage (Vm) is a fundamental biological parameter that is essential for neuronal communication, cardiac activity, transmembrane transport, regulation of signaling, and bacterial motility. Optical measurements of Vm promise new insights into how voltage propagates within and between cells, but effective optical contrast agents have been lacking. Microbial rhodopsin-based fluorescent voltage indicators are exquisitely sensitive and fast, but very dim, necessitating careful attention to experimental procedures. This chapter describes how to make optical voltage measurements with microbial rhodopsins.

Bright and fast multicoloured voltage reporters via electrochromic FRET.

Genetically encoded fluorescent reporters of membrane potential promise to reveal aspects of neural function not detectable by other means. We present a palette of multicoloured brightly fluorescent genetically encoded voltage indicators with sensitivities from 8-13% ΔF/F per 100 mV, and half-maximal response times from 4-7 ms. A fluorescent protein is fused to an archaerhodopsin-derived voltage sensor. Voltage-induced shifts in the absorption spectrum of the rhodopsin lead to voltage-dependent nonradiative quenching of the appended fluorescent protein. Through a library screen, we identify linkers and fluorescent protein combinations that report neuronal action potentials in cultured rat hippocampal neurons with a single-trial signal-to-noise ratio from 7 to 9 in a 1 kHz imaging bandwidth at modest illumination intensity. The freedom to choose a voltage indicator from an array of colours facilitates multicolour voltage imaging, as well as combination with other optical reporters and optogenetic actuators.

Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein.

Bacteria have many voltage- and ligand-gated ion channels, and population-level measurements indicate that membrane potential is important for bacterial survival. However, it has not been possible to probe voltage dynamics in an intact bacterium. Here we developed a method to reveal electrical spiking in Escherichia coli. To probe bacterial membrane potential, we engineered a voltage-sensitive fluorescent protein based on green-absorbing proteorhodopsin. Expression of the proteorhodopsin optical proton sensor (PROPS) in E. coli revealed electrical spiking at up to 1 hertz. Spiking was sensitive to chemical and physical perturbations and coincided with rapid efflux of a small-molecule fluorophore, suggesting that bacterial efflux machinery may be electrically regulated.