Phosphorylation of pyruvate dehydrogenase inversely associates with neuronal activity.

For decades, the expression of immediate early genes (IEGs) such as FOS has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity. Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected activity decreases across the brain, which were induced by a wide range of factors including general anesthesia, chemogenetic inhibition, sensory experiences, and natural behaviors. Thus, as an inverse activity marker (IAM) in vivo, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

Vagal sensory neurons mediate the Bezold-Jarisch reflex and induce syncope.

Visceral sensory pathways mediate homeostatic reflexes, the dysfunction of which leads to many neurological disorders1. The Bezold-Jarisch reflex (BJR), first described2,3 in 1867, is a cardioinhibitory reflex that is speculated to be mediated by vagal sensory neurons (VSNs) that also triggers syncope. However, the molecular identity, anatomical organization, physiological characteristics and behavioural influence of cardiac VSNs remain mostly unknown. Here we leveraged single-cell RNA-sequencing data and HYBRiD tissue clearing4 to show that VSNs that express neuropeptide Y receptor Y2 (NPY2R) predominately connect the heart ventricular wall to the area postrema. Optogenetic activation of NPY2R VSNs elicits the classic triad of BJR responses-hypotension, bradycardia and suppressed respiration-and causes an animal to faint. Photostimulation during high-resolution echocardiography and laser Doppler flowmetry with behavioural observation revealed a range of phenotypes reflected in clinical syncope, including reduced cardiac output, cerebral hypoperfusion, pupil dilation and eye-roll. Large-scale Neuropixels brain recordings and machine-learning-based modelling showed that this manipulation causes the suppression of activity across a large distributed neuronal population that is not explained by changes in spontaneous behavioural movements. Additionally, bidirectional manipulation of the periventricular zone had a push-pull effect, with inhibition leading to longer syncope periods and activation inducing arousal. Finally, ablating NPY2R VSNs specifically abolished the BJR. Combined, these results demonstrate a genetically defined cardiac reflex that recapitulates characteristics of human syncope at physiological, behavioural and neural network levels.

Phosphorylation of pyruvate dehydrogenase marks the inhibition of in vivo neuronal activity.

For decades, the expression of immediate early genes (IEGs) such as c- fos has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity (i.e., inhibition). Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected neuronal inhibition across the brain induced by a wide range of factors including general anesthesia, sensory experiences, and natural behaviors. Thus, as an in vivo marker for neuronal inhibition, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

Neural Control and Modulation of Thirst, Sodium Appetite, and Hunger.

The function of central appetite neurons is instructing animals to ingest specific nutrient factors that the body needs. Emerging evidence suggests that individual appetite circuits for major nutrients-water, sodium, and food-operate on unique driving and quenching mechanisms. This review focuses on two aspects of appetite regulation. First, we describe the temporal relationship between appetite neuron activity and consumption behaviors. Second, we summarize ingestion-related satiation signals that differentially quench individual appetite circuits. We further discuss how distinct appetite and satiation systems for each factor may contribute to nutrient homeostasis from the functional and evolutional perspectives.

Temporally and Spatially Distinct Thirst Satiation Signals.

For thirsty animals, fluid intake provides both satiation and pleasure of drinking. How the brain processes these factors is currently unknown. Here, we identified neural circuits underlying thirst satiation and examined their contribution to reward signals. We show that thirst-driving neurons receive temporally distinct satiation signals by liquid-gulping-induced oropharyngeal stimuli and gut osmolality sensing. We demonstrate that individual thirst satiation signals are mediated by anatomically distinct inhibitory neural circuits in the lamina terminalis. Moreover, we used an ultrafast dopamine (DA) sensor to examine whether thirst satiation itself stimulates the reward-related circuits. Interestingly, spontaneous drinking behavior but not thirst drive reduction triggered DA release. Importantly, chemogenetic stimulation of thirst satiation neurons did not activate DA neurons under water-restricted conditions. Together, this study dissected the thirst satiation circuit, the activity of which is functionally separable from reward-related brain activity.

Neural populations for maintaining body fluid balance.

Fine balance between loss-of water and gain-of water is essential for maintaining body fluid homeostasis. The development of neural manipulation and mapping tools has opened up new avenues to dissect the neural circuits underlying body fluid regulation. Recent studies have identified several nodes in the brain that positively and negatively regulate thirst. The next step forward would be to elucidate how neural populations interact with each other to control drinking behavior.

Chemosensory modulation of neural circuits for sodium appetite.

Sodium is the main cation in the extracellular fluid and it regulates various physiological functions. Depletion of sodium in the body increases the hedonic value of sodium taste, which drives animals towards sodium consumption1,2. By contrast, oral sodium detection rapidly quenches sodium appetite3,4, suggesting that taste signals have a central role in sodium appetite and its satiation. Nevertheless, the neural mechanisms of chemosensory-based appetite regulation remain poorly understood. Here we identify genetically defined neural circuits in mice that control sodium intake by integrating chemosensory and internal depletion signals. We show that a subset of excitatory neurons in the pre-locus coeruleus express prodynorphin, and that these neurons are a critical neural substrate for sodium-intake behaviour. Acute stimulation of this population triggered robust ingestion of sodium even from rock salt, while evoking aversive signals. Inhibition of the same neurons reduced sodium consumption selectively. We further demonstrate that the oral detection of sodium rapidly suppresses these sodium-appetite neurons. Simultaneous in vivo optical recording and gastric infusion revealed that sodium taste-but not sodium ingestion per se-is required for the acute modulation of neurons in the pre-locus coeruleus that express prodynorphin, and for satiation of sodium appetite. Moreover, retrograde-virus tracing showed that sensory modulation is in part mediated by specific GABA (γ-aminobutyric acid)-producing neurons in the bed nucleus of the stria terminalis. This inhibitory neural population is activated by sodium ingestion, and sends rapid inhibitory signals to sodium-appetite neurons. Together, this study reveals a neural architecture that integrates chemosensory signals and the internal need to maintain sodium balance.

Peripheral and Central Nutrient Sensing Underlying Appetite Regulation.

The precise regulation of fluid and energy homeostasis is essential for survival. It is well appreciated that ingestive behaviors are tightly regulated by both peripheral sensory inputs and central appetite signals. With recent neurogenetic technologies, considerable progress has been made in our understanding of basic taste qualities, the molecular and/or cellular basis of taste sensing, and the central circuits for thirst and hunger. In this review, we first highlight the functional similarities and differences between mammalian and invertebrate taste processing. We then discuss how central thirst and hunger signals interact with peripheral sensory signals to regulate ingestive behaviors. We finally indicate some of the directions for future research.

Hierarchical neural architecture underlying thirst regulation.

Neural circuits for appetites are regulated by both homeostatic perturbations and ingestive behaviour. However, the circuit organization that integrates these internal and external stimuli is unclear. Here we show in mice that excitatory neural populations in the lamina terminalis form a hierarchical circuit architecture to regulate thirst. Among them, nitric oxide synthase-expressing neurons in the median preoptic nucleus (MnPO) are essential for the integration of signals from the thirst-driving neurons of the subfornical organ (SFO). Conversely, a distinct inhibitory circuit, involving MnPO GABAergic neurons that express glucagon-like peptide 1 receptor (GLP1R), is activated immediately upon drinking and monosynaptically inhibits SFO thirst neurons. These responses are induced by the ingestion of fluids but not solids, and are time-locked to the onset and offset of drinking. Furthermore, loss-of-function manipulations of GLP1R-expressing MnPO neurons lead to a polydipsic, overdrinking phenotype. These neurons therefore facilitate rapid satiety of thirst by monitoring real-time fluid ingestion. Our study reveals dynamic thirst circuits that integrate the homeostatic-instinctive requirement for fluids and the consequent drinking behaviour to maintain internal water balance.