We used genome-wide sequencing methods to study stimulus-dependent enhancer function in neurons. We identified ∼12,000 neuronal activity-regulated enhancers that are bound by the general transcriptional co-activator CBP in an activity-dependent manner. A function of CBP at enhancers may be to recruit RNA polymerase II (RNAPII), as we also observed activity-regulated RNAPII binding to thousands of enhancers. Remarkably, RNAPII at enhancers transcribes bi-directionally a novel class of enhancer RNAs (eRNAs) within enhancer domains defined by the presence of histone H3 that is mono-methylated at lysine 4 (H3K4me1). The level of eRNA expression at neuronal enhancers positively correlates with the level of mRNA synthesis at nearby genes, suggesting that eRNA synthesis occurs specifically at enhancers that are actively engaged in promoting mRNA synthesis. These findings reveal that a widespread mechanism of enhancer activation involves RNAPII binding and eRNA synthesis.

Caenorhabditis elegans explores its environment by interrupting its forward movement with occasional turns and reversals. Turns and reversals occur at stable frequencies but irregular intervals, producing probabilistic exploratory behaviors. Here we dissect the roles of individual sensory neurons, interneurons, and motor neurons in exploratory behaviors under different conditions. After animals are removed from bacterial food, they initiate a local search behavior consisting of reversals and deep omega-shaped turns triggered by AWC olfactory neurons, ASK gustatory neurons, and AIB interneurons. Over the following 30 min, the animals disperse as reversals and omega turns are suppressed by ASI gustatory neurons and AIY interneurons. Interneurons and motor neurons downstream of AIB and AIY encode specific aspects of reversal and turn frequency, amplitude, and directionality. SMD motor neurons help encode the steep amplitude of omega turns, RIV motor neurons specify the ventral bias of turns that follow a reversal, and SMB motor neurons set the amplitude of sinusoidal movement. Many of these sensory neurons, interneurons, and motor neurons are also implicated in chemotaxis and thermotaxis. Thus, this circuit may represent a common substrate for multiple navigation behaviors.chemosensation ͉ exploratory behavior ͉ neural circuit A s an animal travels through its environment, its nervous system detects sensory cues, evaluates them based on context and the experience of the animal, and converts this information into adaptive movement. For simple behaviors, sensory neurons sometimes communicate directly with motor neurons, but, in more complex behavioral circuits, several layers of interneurons integrate sensory information and relay it to motor neurons. The path from sensory input to motor output has been defined in only a few cases, including circuits for crustacean feeding (1) and circuits for rapid escape in fish, flies, and nematodes (2-4). In the nematode Caenorhabditis elegans, the escape circuit was defined by using a complete synaptic wiring diagram of the 302 neurons in its nervous system (4, 5). Six mechanosensory neurons that detect noxious stimuli synapse onto four pairs of interneurons called forward and backward command neurons. The command neurons synapse in turn onto motor neurons responsible for forward and backward locomotion, leading to rapid withdrawal from the stimulus. The definition of the escape circuit has enabled analysis of its development, regulation, and modification by experience (6-11). The C. elegans wiring diagram provides an opportunity to define many complete neuronal paths from sensory stimulus to behavior.In contrast with the escape circuit, the neuronal control of locomotion during exploratory behavior is poorly characterized. C. elegans navigates to favorable conditions by chemotaxis, thermotaxis, and aerotaxis. In these sensory behaviors and in exploratory behaviors in the absence of informative sensory cues, the animal moves forward and occasionally changes its direction of move...

Although many properties of the nervous system are shared among animals and systems, it is not known whether different neuronal circuits use common strategies to guide behaviour. Here we characterize information processing by Caenorhabditis elegans olfactory neurons (AWC) and interneurons (AIB and AIY) that control food- and odour-evoked behaviours. Using calcium imaging and mutations that affect specific neuronal connections, we show that AWC neurons are activated by odour removal and activate the AIB interneurons through AMPA-type glutamate receptors. The level of calcium in AIB interneurons is elevated for several minutes after odour removal, a neuronal correlate to the prolonged behavioural response to odour withdrawal. The AWC neuron inhibits AIY interneurons through glutamate-gated chloride channels; odour presentation relieves this inhibition and results in activation of AIY interneurons. The opposite regulation of AIY and AIB interneurons generates a coordinated behavioural response. Information processing by this circuit resembles information flow from vertebrate photoreceptors to 'OFF' bipolar and 'ON' bipolar neurons, indicating a conserved or convergent strategy for sensory information processing.

Specialized oxygen-sensing cells in the nervous system generate rapid behavioural responses to oxygen. We show here that the nematode Caenorhabditis elegans exhibits a strong behavioural preference for 5-12% oxygen, avoiding higher and lower oxygen levels. 3',5'-cyclic guanosine monophosphate (cGMP) is a common second messenger in sensory transduction and is implicated in oxygen sensation. Avoidance of high oxygen levels by C. elegans requires the sensory cGMP-gated channel tax-2/tax-4 and a specific soluble guanylate cyclase homologue, gcy-35. The GCY-35 haem domain binds molecular oxygen, unlike the haem domains of classical nitric-oxide-regulated guanylate cyclases. GCY-35 and TAX-4 mediate oxygen sensation in four sensory neurons that control a naturally polymorphic social feeding behaviour in C. elegans. Social feeding and related behaviours occur only when oxygen exceeds C. elegans' preferred level, and require gcy-35 activity. Our results suggest that GCY-35 is regulated by molecular oxygen, and that social feeding can be a behavioural strategy for responding to hyperoxic environments.

The ability to differentiate stimuli predicting positive or negative outcomes is critical for survival, and perturbations of emotional processing underlie many psychiatric disease states. Synaptic plasticity in the basolateral amygdala complex (BLA) mediates the acquisition of associative memories, both positive1,2 and negative3–7. Different populations of BLA neurons may encode fearful or rewarding associations8–10, but the identifying features of these populations and the synaptic mechanisms of differentiating positive and negative emotional valence have remained an enigma. Here, we show that BLA neurons projecting to the nucleus accumbens (NAc projectors) or the centromedial amygdala (CeM projectors) underwent opposing synaptic changes following fear or reward conditioning. We found that photostimulation of NAc projectors supports positive reinforcement while photostimulation of CeM projectors mediates negative reinforcement. Photoinhibition of CeM projectors impaired fear conditioning and enhanced reward conditioning. We then characterized these functionally-distinct neuronal populations by comparing their electrophysiological, morphological and genetic features. We provide a mechanistic explanation for the representation of positive and negative associations within the amygdala.

Summary Homeostatic sensory systems detect small deviations in temperature, water balance, pH and energy needs to regulate adaptive behavior and physiology. In C. elegans, a homeostatic preference for intermediate oxygen (O2) levels requires cGMP signaling through soluble guanylate cyclases (sGCs), proteins that bind gases through an associated heme group. Here we use behavioral analysis, functional imaging, and genetics to show that reciprocal changes in O2 levels are encoded by sensory neurons that express alternative sets of sGCs. URX sensory neurons are activated by increases in O2 levels, and require the sGCs gcy-35 and gcy-36. BAG sensory neurons are activated by decreases in O2 levels, and require the sGCs gcy-31 and gcy-33. The sGCs are instructive O2 sensors, as forced expression of URX sGC genes causes BAG neurons to detect O2 increases. Both sGC expression and cell-intrinsic dynamics contribute to the differential roles of URX and BAG in O2 -dependent behaviors.

Activation of complex molecular programs in specific cell lineages governs mammalian heart development, from a primordial linear tube to a four-chamber organ. To characterize lineage-specific, temporal-spatial developmental programs, we performed single-cell RNA sequencing of >1200 murine cells isolated at seven time points spanning E9.5 (primordial heart tube) to P21 (mature heart). Using unbiased transcriptional data we classified cardiomyocytes (CM), endothelial cells (EC), and fibroblast-enriched cells, thus identifying markers for temporal and chamber-specific developmental programs. Harnessing these datasets, we defined developmental ages of human and mouse pluripotent stem cell-derived CMs and characterized lineage-specific maturation defects in hearts of mice with heterozygous mutations in Nkx2.5 that cause human heart malformations. This spatial-temporal transcriptome analysis of heart development reveals lineage-specific gene programs underlying normal cardiac development and congenital heart disease.

SUMMARY Although many transcription factors are known to control important aspects of neural development, the genome-wide programs that are directly regulated by these factors are not known. We have characterized the genetic program that is activated by MEF2, a key regulator of activity-dependent synapse development. These MEF2 target genes have diverse functions at synapses, revealing a broad role for MEF2 in synapse development. Several of the MEF2 targets are mutated in human neurological disorders including epilepsy and autism-spectrum disorders, suggesting that these disorders may be caused by disruption of an activity-dependent gene program that controls synapse development. Our analyses also reveal that neuronal activity promotes alternative polyadenylation site usage at many of the MEF2 target genes, leading to the production of truncated mRNAs that may have different functions than their full-length counterparts. Taken together, these analyses suggest that the ubiquitously expressed transcription factor MEF2 regulates an intricate transcriptional program in neurons that controls synapse development.