Two intermingled hypothalamic neuron populations, specified by expression of agouti-related peptide (AGRP) or pro-opiomelanocortin (POMC), positively and negatively influence feeding behavior respectively, possibly by reciprocally regulating downstream melanocortin receptors. However, the sufficiency of these neurons to control behavior, and the relationship of their activity to the magnitude and dynamics of feeding are unknown. To measure this, we used channelrhodopsin-2 for cell type-specific photostimulation. Activation of only 800 AGRP neurons in mice evoked voracious feeding within minutes. The behavioral response increased with photoexcitable neuron number, photostimulation frequency, and stimulus duration. Conversely, POMC neuron stimulation reduced food intake and body weight, which required melanocortin receptor signaling. However, AGRP neuron-mediated feeding was not dependent on suppressing this melanocortin pathway, indicating that AGRP neurons directly engage feeding circuits. Furthermore, feeding was evoked selectively over drinking without training or prior photostimulus exposure, which suggests that AGRP neurons serve a dedicated role coordinating this complex behavior.

Hunger is a complex behavioural state that elicits intense food seeking and consumption. These behaviours are rapidly recapitulated by activation of starvation-sensitive AGRP neurons, which present an entry point for reverse-engineering neural circuits for hunger. We mapped synaptic interactions of AGRP neurons with multiple cell populations and probed the contribution of these distinct circuits to feeding behaviour using optogenetic and pharmacogenetic techniques. An inhibitory circuit with paraventricular hypothalamus (PVH) neurons substantially accounted for acute AGRP neuron-evoked eating, whereas two other prominent circuits were insufficient. Within the PVH, we found that AGRP neurons target and inhibit oxytocin neurons, a small population that is selectively lost in Prader-Willi syndrome, a condition involving insatiable hunger. By developing strategies for evaluating molecularly-defined circuits, we show that AGRP neuron suppression of oxytocin neurons is critical for evoked feeding. These experiments reveal a new neural circuit that regulates hunger state and pathways associated with overeating disorders.

Synapses, the junctions between nerve cells through which they communicate, are formed by the coordinated assembly and tight attachment of pre- and postsynaptic specializations. We now show that SynCAM is a brain-specific, immunoglobulin domain-containing protein that binds to intracellular PDZ-domain proteins and functions as a homophilic cell adhesion molecule at the synapse. Expression of the isolated cytoplasmic tail of SynCAM in neurons inhibited synapse assembly. Conversely, expression of full-length SynCAM in nonneuronal cells induced synapse formation by cocultured hippocampal neurons with normal release properties. Glutamatergic synaptic transmission was reconstituted in these nonneuronal cells by coexpressing glutamate receptors with SynCAM, which suggests that a single type of adhesion molecule and glutamate receptor are sufficient for a functional postsynaptic response.

Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. We now show that in cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses, and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits.

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Synaptic plasticity in response to changes in physiologic state is coordinated by hormonal signals across multiple neuronal cell types. Here, we combine cell-type-specific electrophysiological, pharmacological, and optogenetic techniques to dissect neural circuits and molecular pathways controlling synaptic plasticity onto AGRP neurons, a population that regulates feeding. We find that food deprivation elevates excitatory synaptic input, which is mediated by a presynaptic positive feedback loop involving AMP-activated protein kinase. Potentiation of glutamate release was triggered by the orexigenic hormone ghrelin and exhibited hysteresis, persisting for hours after ghrelin removal. Persistent activity was reversed by the anorexigenic hormone leptin, and optogenetic photostimulation demonstrated involvement of opioid release from POMC neurons. Based on these experiments, we propose a memory storage device for physiological state constructed from bistable synapses that are flipped between two sustained activity states by transient exposure to hormones signaling energy levels.

An abundant presynaptic protein, ␣-synuclein, is centrally involved in the pathogenesis of Parkinson's disease. However, conflicting data exist about the normal function of ␣-synuclein, possibly because ␣-synuclein is redundant with the very similar ␤-synuclein. To investigate the functions of synucleins systematically, we have now generated single-and double-knockout (KO) mice that lack ␣-and͞or ␤-synuclein. We find that deletion of synucleins in mice does not impair basic brain functions or survival. We detected no significant changes in the ultrastructure of synuclein-deficient synapses, in short-or long-term synaptic plasticity, or in the pool size or replenishment of recycling synaptic vesicles. However, protein quantitations revealed that KO of synucleins caused selective changes in two small synaptic signaling proteins, complexins and 14-3-3 proteins. Moreover, we found that dopamine levels in the brains of double-KO but not single-KO mice were decreased by Ϸ20%. In contrast, serotonin levels were unchanged, and dopamine uptake and release from isolated nerve terminals were normal. These results show that synucleins are not essential components of the basic machinery for neurotransmitter release but may contribute to the long-term regulation and͞or maintenance of presynaptic function.

Ionic flux in defined cell populations mediates essential physiological and behavioral functions. Cell type-specific activators of diverse ionic conductances are needed for probing these relationships. We combined chemistry and protein engineering to enable systematic creation of a toolbox of ligand-gated ion channels (LGICs) with orthogonal pharmacologic selectivity and divergent functional properties. The LGICs and their small molecule effectors can activate a range of ionic conductances in genetically-specified cell types.LGICs constructed for neuronal perturbation can be used to selectively manipulate neuron activity in mammalian brains in vivo.The diversity of ion channel tools accessible from this approach will be useful for examining the relationship between neuronal activity and animal behavior, as well as for cell biological and physiological applications requiring chemical control of ion conductance.Ion channels are complex molecular machines with critical cell biological functions. Ligandgated ion channels (LGICs) provide rapid, remote control over conductances for different ions. In neurons, LGICs can be exploited for stimulation or silencing to examine causal relationships between electrical activity and animal behavior. Several neuron manipulation tools have been derived fromLGICs and G-protein coupled receptors (1-4) that can be genetically targeted and are reported to be orthogonal to endogenous systems. These tools are useful (5-7) but also face limitations such as ligand instability and lack of brain access (2), slow pharmacokinetics (6), the need to knockout endogenous alleles (3), or reliance on complex intracellular signaling pathways (4). Optogenetic tools (8-10) activate conductances with millisecond precision, but optimization of ion conductance properties has been limited and light targeting is invasive.To overcome these limitations, we have developed a strategy to create chimeric LGICs with distinct conductance properties derived from modular combinations of pharmacologicallyselective ligand binding domains (LBDs) and functionally diverse ion pore domains (IPDs). Within the Cys-loop receptor superfamily, the LBD of the α7 nicotinic acetylcholine receptor (nAChR) behaves as an independent actuator module that can be transplanted onto the IPDs of other Cys-loop receptors (11,12). These include at least 43 ion channel subunits in vertebrates (13), and many additional invertebrate (14) and prokaryotic (15) subunits. Distinct IPDs confer selectivity for chloride or calcium as well as nonspecific cations. For example, splicing the α7 nAChR LBD to the IPDs of the serotonin receptor 3a or the glycine receptor produces chimeric channels (α7-5HT3 or α7-GlyR) with α7 nAChR pharmacology and cation or chloride conductance properties, respectively (11,12). This modular property is a strong foundation for tailoring functional characteristics. However, the † To whom correspondence should be addressed. sternsons@janelia.hhmi.org (S.M.S.), HHMI Author ManuscriptHHMI Author Manuscript HHMI Auth...