A subcortical feeding circuit linking an interoceptive node to jaw movement.

The brain processes an array of stimuli, enabling the selection of appropriate behavioural responses, but the neural pathways linking interoceptive inputs to outputs for feeding are poorly understood1-3. Here we delineate a subcortical circuit in which brain-derived neurotrophic factor (BDNF)-expressing neurons in the ventromedial hypothalamus (VMH) directly connect interoceptive inputs to motor centres, controlling food consumption and jaw movements. VMHBDNF neuron inhibition increases food intake by gating motor sequences of feeding through projections to premotor areas of the jaw. When food is unavailable, VMHBDNF inhibition elicits consummatory behaviours directed at inanimate objects such as wooden blocks, and inhibition of perimesencephalic trigeminal area (pMe5) projections evokes rhythmic jaw movements. The activity of these neurons is decreased during food consumption and increases when food is in proximity but not consumed. Activity is also increased in obese animals and after leptin treatment. VMHBDNF neurons receive monosynaptic inputs from both agouti-related peptide (AgRP) and proopiomelanocortin neurons in the arcuate nucleus (Arc), and constitutive VMHBDNF activation blocks the orexigenic effect of AgRP activation. These data indicate an Arc → VMHBDNF → pMe5 circuit that senses the energy state of an animal and regulates consummatory behaviours in a state-dependent manner.

Leptin-activated hypothalamic BNC2 neurons acutely suppress food intake.

Leptin is an adipose tissue hormone that maintains homeostatic control of adipose tissue mass by regulating the activity of specific neural populations controlling appetite and metabolism1. Leptin regulates food intake by inhibiting orexigenic agouti-related protein (AGRP) neurons and activating anorexigenic pro-opiomelanocortin (POMC) neurons2. However, whereas AGRP neurons regulate food intake on a rapid time scale, acute activation of POMC neurons has only a minimal effect3-5. This has raised the possibility that there is a heretofore unidentified leptin-regulated neural population that rapidly suppresses appetite. Here we report the discovery of a new population of leptin-target neurons expressing basonuclin 2 (Bnc2) in the arcuate nucleus that acutely suppress appetite by directly inhibiting AGRP neurons. Opposite to the effect of AGRP activation, BNC2 neuronal activation elicited a place preference indicative of positive valence in hungry but not fed mice. The activity of BNC2 neurons is modulated by leptin, sensory food cues and nutritional status. Finally, deleting leptin receptors in BNC2 neurons caused marked hyperphagia and obesity, similar to that observed in a leptin receptor knockout in AGRP neurons. These data indicate that BNC2-expressing neurons are a key component of the neural circuit that maintains energy balance, thus filling an important gap in our understanding of the regulation of food intake and leptin action.

A leptin-BDNF pathway regulating sympathetic innervation of adipose tissue.

Mutations in the leptin gene (ob) result in a metabolic disorder that includes severe obesity1, and defects in thermogenesis2 and lipolysis3, both of which are adipose tissue functions regulated by the sympathetic nervous system. However, the basis of these sympathetic-associated abnormalities remains unclear. Furthermore, chronic leptin administration reverses these abnormalities in adipose tissue, but the underlying mechanism remains to be discovered. Here we report that ob/ob mice, as well as leptin-resistant diet-induced obese mice, show significant reductions of sympathetic innervation of subcutaneous white and brown adipose tissue. Chronic leptin treatment of ob/ob mice restores adipose tissue sympathetic innervation, which in turn is necessary to correct the associated functional defects. The effects of leptin on innervation are mediated via agouti-related peptide and pro-opiomelanocortin neurons in the hypothalamic arcuate nucleus. Deletion of the gene encoding the leptin receptor in either population leads to reduced innervation in fat. These agouti-related peptide and pro-opiomelanocortin neurons act via brain-derived neurotropic factor-expressing neurons in the paraventricular nucleus of the hypothalamus (BDNFPVH). Deletion of BDNFPVH blunts the effects of leptin on innervation. These data show that leptin signalling regulates the plasticity of sympathetic architecture of adipose tissue via a top-down neural pathway that is crucial for energy homeostasis.

Natural hypothalamic circuit dynamics underlying object memorization.

Brain signals that govern memory formation remain incompletely identified. The hypothalamus is implicated in memory disorders, but how its rapidly changing activity shapes memorization is unknown. During encounters with objects, hypothalamic melanin-concentrating hormone (MCH) neurons emit brief signals that reflect object novelty. Here we show that targeted optogenetic silencing of these signals, performed selectively during the initial object encounters (i.e. memory acquisition), prevents future recognition of the objects. We identify an upstream inhibitory microcircuit from hypothalamic GAD65 neurons to MCH neurons, which constrains the memory-promoting MCH cell bursts. Finally, we demonstrate that silencing the GAD65 cells during object memory acquisition improves future object recognition through MCH-receptor-dependent pathways. These results provide causal evidence that object-associated signals in genetically distinct but interconnected hypothalamic neurons differentially control whether the brain forms object memories. This gating of memory formation by hypothalamic activity establishes appropriate behavioral responses to novel and familiar objects.

Fast and Slow Oscillations Recruit Molecularly-Distinct Subnetworks of Lateral Hypothalamic Neurons In Situ.

Electrical signals generated by molecularly-distinct classes of lateral hypothalamus (LH) neurons have distinct physiological consequences. For example, LH orexin neurons promote net body energy expenditure, while LH non-orexin neurons [VGAT, melanin-concentrating hormone (MCH)] drive net energy conservation. Appropriate switching between such physiologically-opposing LH outputs is traditionally thought to require cell-type-specific chemical modulation of LH firing. However, it was recently found that, in vivo, the LH neurons are also physiologically exposed to electrical oscillations of different frequency bands. The role of the different physiological oscillation frequencies in firing of orexin vs non-orexin LH neurons remains unknown. Here, we used brain-slice whole-cell patch-clamp technology to target precisely-defined oscillation waveforms to individual molecularly-defined classes LH cells (orexin, VGAT, MCH, GAD65), while measuring the action potential output of the cells. By modulating the frequency of sinusoidal oscillatory input, we found that high-frequency oscillations (γ, ≈30-200 Hz) preferentially silenced the action potential output orexinLH cells. In contrast, low frequencies (δ-θ, ≈0.5-7 Hz) similarly permitted outputs from different LH cell types. This differential control of orexin and non-orexin cells by oscillation frequency was mediated by cell-specific, impedance-unrelated resonance mechanisms. These results substantiate electrical oscillations as a novel input modality for cell-type-specific control of LH firing, which offers an unforeseen way to control specific cell ensembles within this highly heterogeneous neuronal cluster.

Orexin-driven GAD65 network of the lateral hypothalamus sets physical activity in mice.

Damage to the lateral hypothalamus (LH) causes profound physical inactivity in mammals. Several molecularly distinct types of LH neurons have been identified, including orexin cells and glutamic acid decarboxylase 65 (GAD65) cells, but their interplay in orchestrating physical activity is not fully understood. Here, using optogenetic circuit analysis and cell type-specific deep-brain recordings in behaving mice, we show that orexin cell activation rapidly recruits GAD65LH neurons. We demonstrate that internally initiated GAD65LH cell bursts precede and accompany spontaneous running bouts, that selective chemogenetic silencing of natural GAD65LH cell activity depresses voluntary locomotion, and that GAD65LH cell overactivation leads to hyperlocomotion. These results thus identify a molecularly distinct, orexin-activated LH submodule that governs physical activity in mice.

Gamma oscillations organize top-down signalling to hypothalamus and enable food seeking.

Both humans and animals seek primary rewards in the environment, even when such rewards do not correspond to current physiological needs. An example of this is a dissociation between food-seeking behaviour and metabolic needs, a notoriously difficult-to-treat symptom of eating disorders. Feeding relies on distinct cell groups in the hypothalamus, the activity of which also changes in anticipation of feeding onset. The hypothalamus receives strong descending inputs from the lateral septum, which is connected, in turn, with cortical networks, but cognitive regulation of feeding-related behaviours is not yet understood. Cortical cognitive processing involves gamma oscillations, which support memory, attention, cognitive flexibility and sensory responses. These functions contribute crucially to feeding behaviour by unknown neural mechanisms. Here we show that coordinated gamma (30-90 Hz) oscillations in the lateral hypothalamus and upstream brain regions organize food-seeking behaviour in mice. Gamma-rhythmic input to the lateral hypothalamus from somatostatin-positive lateral septum cells evokes food approach without affecting food intake. Inhibitory inputs from the lateral septum enable separate signalling by lateral hypothalamus neurons according to their feeding-related activity, making them fire at distinct phases of the gamma oscillation. Upstream, medial prefrontal cortical projections provide gamma-rhythmic inputs to the lateral septum; these inputs are causally associated with improved performance in a food-rewarded learning task. Overall, our work identifies a top-down pathway that uses gamma synchronization to guide the activity of subcortical networks and to regulate feeding behaviour by dynamic reorganization of functional cell groups in the hypothalamus.

A unifying computational framework for stability and flexibility of arousal.

Arousal and consciousness flexibly adjust to salient cues, but remain stable despite noise and disturbance. Diverse, highly interconnected neural networks govern the underlying transitions of behavioral state; these networks are robust but very complex. Frameworks from systems engineering provide powerful tools for understanding functional logic behind component complexity. From a general systems viewpoint, a minimum of three communicating control modules may enable flexibility and stability to coexist. Comparators would subtract current arousal from desired arousal, producing an error signal. Regulators would compute control signals from this error. Generators would convert control signals into arousal, which is fed back to comparators, to make the system noise-proof through self-correction. Can specific neurons correspond to these control elements? To explore this, here we consider the brain-wide orexin/hypocretin network, which is experimentally established to be vital for flexible and stable arousal. We discuss whether orexin neurons may act as comparators, and their target neurons as regulators and generators. Experiments are proposed for testing such predictions, based on computational simulations showing that comparators, regulators, and generators have distinct temporal signatures of activity. If some regulators integrate orexin-communicated errors, robust arousal control may be achieved via integral feedback (a basic engineering strategy for tracking a set-point despite noise). An integral feedback view also suggests functional roles for specific molecular aspects, such as differing life-spans of orexin peptides. The proposed framework offers a unifying logic for molecular, cellular, and network details of arousal systems, and provides insight into behavioral state transitions, complex behavior, and bases for disease.

Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti.

Inward-rectifying K(+) (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K(+) currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na(+). Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl(+) > K(+) > Rb(+) > NH(4)(+)) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K(+).