Richard D. Palmiter

Deciphering a neuronal circuit that mediates appetite

Hypothalamic neurons that co-express agouti-related protein (AgRP), neuropeptideu2009Y and γ-aminobutyric acid (GABA) are known to promote feeding and weight gain by integration of various nutritional, hormonal, and neuronal signals. Ablation of these neurons in mice leads to cessation of feeding that is accompanied by activation of Fos in most regions where they project. Previous experiments have indicated that the ensuing starvation is due to aberrant activation of the parabrachial nucleus (PBN) and it could be prevented by facilitating GABAA receptor signalling in the PBN within a critical adaptation period. We speculated that loss of GABA signalling from AgRP-expressing neurons (AgRP neurons) within the PBN results in unopposed excitation of the PBN, which in turn inhibits feeding. However, the source of the excitatory inputs to the PBN was unknown. Here we show that glutamatergic neurons in the nucleus tractus solitarius (NTS) and caudal serotonergic neurons control the excitability of PBN neurons and inhibit feeding. Blockade of serotonin (5-HT3) receptor signalling in the NTS by either the chronic administration of ondansetron or the genetic inactivation of Tph2 in caudal serotonergic neurons that project to the NTS protects against starvation when AgRP neurons are ablated. Likewise, genetic inactivation of glutamatergic signalling by the NTS onto N-methyl d-aspartate-type glutamate receptors in the PBN prevents starvation. We also show that suppressing glutamatergic output of the PBN reinstates normal appetite after AgRP neuron ablation, whereas it promotes weight gain without AgRP neuron ablation. Thus we identify the PBN as a hub that integrates signals from several brain regions to bidirectionally modulate feeding and body weight.

Ablation of neurons expressing agouti-related protein, but not melanin concentrating hormone, in leptin-deficient mice restores metabolic functions and fertility.

Leptin-deficient (Lepob/ob) mice are obese, diabetic, and infertile. Ablation of neurons that make agouti-related protein (AgRP) in moderately obese adult Lepob/ob mice caused severe anorexia. The mice stopped eating for 2 wk and then gradually recovered. Their body weight fell to within a normal range for WT mice, at which point food intake and glucose tolerance were restored to that of WT mice. Remarkably, both male and female Lepob/ob mice became fertile. Ablation of neurons that express melanin-concentrating hormone (MCH) in adult Lepob/ob mice had no effect on food intake, body weight, or fertility, but resulted in improved glucose tolerance. We conclude that AgRP-expressing neurons play a critical role in mediating the metabolic syndrome and infertility of Lepob/ob mice, whereas MCH-expressing neurons have only a minor role.

Agouti-related peptide neural circuits mediate adaptive behaviors in the starved state

In the face of starvation, animals will engage in high-risk behaviors that would normally be considered maladaptive. Starving rodents, for example, will forage in areas that are more susceptible to predators and will also modulate aggressive behavior within a territory of limited or depleted nutrients. The neural basis of these adaptive behaviors likely involves circuits that link innate feeding, aggression and fear. Hypothalamic agouti-related peptide (AgRP)-expressing neurons are critically important for driving feeding and project axons to brain regions implicated in aggression and fear. Using circuit-mapping techniques in mice, we define a disynaptic network originating from a subset of AgRP neurons that project to the medial nucleus of the amygdala and then to the principal bed nucleus of the stria terminalis, which suppresses territorial aggression and reduces contextual fear. We propose that AgRP neurons serve as a master switch capable of coordinating behavioral decisions relative to internal state and environmental cues.

Parabrachial CGRP Neurons Control Meal Termination

The lateral parabrachial nucleus is a conduit for visceral signals that cause anorexia. We previously identified a subset of neurons located in the external lateral parabrachial nucleus (PBel) that express calcitonin gene-related peptide (CGRP) and inhibit feeding when activated by illness mimetics. We report here that in otherwise normal mice, functional inactivation of CGRP neurons markedly increases meal size, with meal frequency being reduced in a compensatory manner, and renders mice insensitive to the anorexic effects of meal-related satiety peptides. Furthermore, CGRP neurons are directly innervated by orexigenic hypothalamic AgRP neurons, and photostimulation of AgRP fibers supplying the PBel delays satiation by inhibiting CGRP neurons, thereby contributing toxa0AgRP-driven hyperphagia. By establishing a role for CGRP neurons in the control of meal termination and as a downstream mediator of feeding elicited by AgRP neurons, these findings identify a node in which hunger and satiety circuits interact to control feeding behavior.

Parabrachial Calcitonin Gene-Related Peptide Neurons Mediate Conditioned Taste Aversion

Conditioned taste aversion (CTA) is a phenomenon in which an individual forms an association between a novel tastant and toxin-induced gastrointestinal malaise. Previous studies showed that the parabrachial nucleus (PBN) contains neurons that are necessary for the acquisition of CTA, but the specific neuronal populations involved are unknown. Previously, we identified calcitonin gene-related peptide (CGRP)-expressing neurons in the external lateral subdivision of the PBN (PBel) as being sufficient to suppress appetite and necessary for the anorexigenic effects of appetite-suppressing substances including lithium chloride (LiCl), a compound often used to induce CTA. Here, we test the hypothesis that PBel CGRP neurons are sufficient and necessary for CTA acquisition in mice. We show that optogenetic activation of these neurons is sufficient to induce CTA in the absence of anorexigenic substances, whereas genetically induced silencing of these neurons attenuates acquisition of CTA upon exposure to LiCl. Together, these results demonstrate that PBel CGRP neurons mediate a gastrointestinal distress signal required to establish CTA.

Dopamine receptor 1 neurons in the dorsal striatum regulate food anticipatory circadian activity rhythms in mice

Daily rhythms of food anticipatory activity (FAA) are regulated independently of the suprachiasmatic nucleus, which mediates entrainment of rhythms to light, but the neural circuits that establish FAA remain elusive. In this study, we show that mice lacking the dopamine D1 receptor (D1R KO mice) manifest greatly reduced FAA, whereas mice lacking the dopamine D2 receptor have normal FAA. To determine where dopamine exerts its effect, we limited expression of dopamine signaling to the dorsal striatum of dopamine-deficient mice; these mice developed FAA. Within the dorsal striatum, the daily rhythm of clock gene period2 expression was markedly suppressed in D1R KO mice. Pharmacological activation of D1R at the same time daily was sufficient to establish anticipatory activity in wild-type mice. These results demonstrate that dopamine signaling to D1R-expressing neurons in the dorsal striatum plays an important role in manifestation of FAA, possibly by synchronizing circadian oscillators that modulate motivational processes and behavioral output. DOI: http://dx.doi.org/10.7554/eLife.03781.001

Genetically and functionally defined NTS to PBN brain circuits mediating anorexia.

The central nervous system controls food consumption to maintain metabolic homoeostasis. In response to a meal, visceral signals from the gut activate neurons in the nucleus of the solitary tract (NTS) via the vagus nerve. These NTS neurons then excite brain regions known to mediate feeding behaviour, such as the lateral parabrachial nucleus (PBN). We previously described a neural circuit for appetite suppression involving calcitonin gene-related protein (CGRP)-expressing PBN (CGRPPBN) neurons; however, the molecular identity of the inputs to these neurons was not established. Here we identify cholecystokinin (CCK) and noradrenergic, dopamine β-hydroxylase (DBH)-expressing NTS neurons as two separate populations that directly excite CGRPPBN neurons. When these NTS neurons are activated using optogenetic or chemogenetic methods, food intake decreases and with chronic stimulation mice lose body weight. Our optogenetic results reveal that CCK and DBH neurons in the NTS directly engage CGRPPBN neurons to promote anorexia.

Activity of raphé serotonergic neurons controls emotional behaviors

Despite the well-established role of serotonin signaling in mood regulation, causal relationships between serotonergic neuronal activity and behavior remain poorly understood. Using a pharmacogenetic approach, we find that selectively increasing serotonergic neuronal activity in wild-type mice is anxiogenic and reduces floating in the forced-swim test, whereas inhibition has no effect on the same measures. In a developmental mouse model of altered emotional behavior, increased anxiety and depression-like behaviors correlate with reduced dorsal raphé and increased median raphé serotonergic activity. These mice display blunted responses to serotonergic stimulation and behavioral rescues through serotonergic inhibition. Furthermore, we identify opposing consequences of dorsal versus median raphé serotonergic neuron inhibition on floating behavior, together suggesting that median raphé hyperactivity increases anxiety, whereas a low dorsal/median raphé serotonergic activity ratio increases depression-like behavior. Thus, we find a critical role of serotonergic neuronal activity in emotional regulation and uncover opposing roles of median and dorsal raphé function.

Two Pairs of ON and OFF Retinal Ganglion Cells Are Defined by Intersectional Patterns of Transcription Factor Expression

Visual information is conveyed to the brain by axons of >30 retinal ganglion cell (RGC) types. Characterization of these types is a prerequisite to understanding visual perception. Here, we identify a family of RGCs that we call F-RGCs on the basis of expression of the transcription factor Foxp2. Intersectional expression of Foxp1 and Brn3 transcription factors divides F-RGCs into four types, comprising two pairs, each composed of closely related cells. One pair, F-mini(ON) and F-mini(OFF), shows robust direction selectivity. They are among the smallest RGCs in the mouse retina. The other pair, F-midi(ON) and F-midi(OFF), is larger and not direction selective. Together, F-RGCs comprise >20% of RGCs in the mouse retina, halving the number that remain to be classified and doubling the number of known direction-selective cells. Co-expression of Foxp and Brn3 genes also marks subsets of RGCs in macaques that could be primate homologs of F-RGCs.

Transient activation of specific neurons in mice by selective expression of the capsaicin receptor

The ability to control the electrical activity of a neuronal subtype is a valuable tool in deciphering the role of discreet cell populations in complex neural circuits. Recent techniques that allow remote control of neurons are either labor intensive and invasive or indirectly coupled to neural electrical potential with low temporal resolution. Here we show the rapid, reversible and direct activation of genetically identified neuronal subpopulations by generating two inducible transgenic mouse models. Confined expression of the capsaicin receptor, TRPV1, allows cell-specific activation after peripheral or oral delivery of ligand in freely moving mice. Capsaicin-induced activation of dopaminergic or serotonergic neurons reversibly alters both physiological and behavioural responses within minutes, and lasts ~10u2009min. These models showcase a robust and remotely controllable genetic tool that modulates a distinct cell population without the need for invasive and labour-intensive approaches.