Homeostasis is a biological principle for regulation of essential physiological parameters within a set range. Behavioural responses due to deviation from homeostasis are critical for survival, but motivational processes engaged by physiological need states are incompletely understood. We examined motivational characteristics and dynamics of two separate neuron populations that regulate energy and fluid homeostasis by using cell type-specific activity manipulations in mice. We found that starvation-sensitive AGRP neurons exhibit properties consistent with a negative-valence teaching signal. Mice avoided activation of AGRP neurons, indicating that AGRP neuron activity has negative valence. AGRP neuron inhibition conditioned preference for flavours and places. Correspondingly, deep-brain calcium imaging revealed that AGRP neuron activity rapidly reduced in response to food-related cues. Complementary experiments activating thirst-promoting neurons also conditioned avoidance. Therefore, these need-sensing neurons condition preference for environmental cues associated with nutrient or water ingestion, which is learned through reduction of negative-valence signals during restoration of homeostasis.

The efficacy of fast synaptic inhibition is critically dependent on the accumulation of GABA A receptors at inhibitory synapses, a process that remains poorly understood. Here, we examined the dynamics of cell surface GABA A receptors using receptor subunits modified with N-terminal extracellular ecliptic pHluorin reporters. In hippocampal neurons, GABA A receptors incorporating pHluorin-tagged subunits were found to be clustered at synaptic sites and also expressed as diffuse extrasynaptic staining. By combining FRAP (fluorescence recovery after photobleaching) measurements with live imaging of FM4-64-labeled active presynaptic terminals, it was evident that clustered synaptic receptors exhibit significantly lower rates of mobility at the cell surface compared with their extrasynaptic counterparts. To examine the basis of this confinement, we used RNAi to inhibit the expression of gephyrin, a protein shown to regulate the accumulation of GABA A receptors at synaptic sites. However, whether gephyrin acts to control the actual formation of receptor clusters, their stability, or is simply a global regulator of receptor cell surface number remains unknown. Inhibiting gephyrin expression did not modify the total number of GABA A receptors expressed on the neuronal cell surface but significantly decreased the number of receptor clusters. Live imaging revealed that clusters that formed in the absence of gephyrin were significantly more mobile compared with those in control neurons. Together, our results demonstrate that synaptic GABA A receptors have lower levels of lateral mobility compared with their extrasynaptic counterparts, and suggest a specific role for gephyrin in reducing the diffusion of GABA A receptors, facilitating their accumulation at inhibitory synapses.

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...

Osteoclasts were incubated on a glass or plastic substrate and the effect of calcitonin (CT) on their behaviour was observed. Before exposure to CT the osteoclasts were actively motile, the cytoplasm advancing behind broad pseudopodial (lamellipodial) processes which showed intense ruffling activity. CT caused cessation of lamellipodial activity within minutes, followed by gradual fragmentation and retraction of lamellipodia. Complete osteoclast quiescence was regularly induced by concentrations of CT above 50 pg/ml, and lesser degrees of quiescence were induced at concentrations down to 10 pg/ml. This quiescent state was reversed on removing CT from the medium, and was abrogated by prior treatment of osteoclasts with trypsin. The quiescent state did not reduce the longevity of the cells in culture, nor did it affect their resistance to removal from glass by trypsin. CT showed no influence on the pseudopodial activity of osteoclasts, peritoneal macrophages or inflammatory giant cells. Osteoclast quiescence seems to be a reversible state induced by the interaction of CT with a trypsin-sensitive CT receptor, present on osteoclasts. The range of concentrations which induce partial osteoclast quiescence are within the physiological range of serum concentrations in man, and this suggests that CT plays a physiological role in the regulation of osteoclasts may help to identify the precursor cell of the osteoclast and may assist investigations into the mechanism of control of osteoclasis.

Improved agonists for chemogenetics Targeting ligand-responsive receptors to specific groups of cells, a strategy known as chemogenetics, is a powerful tool in many neurological applications. There is increasing interest in extending these tools for human treatment. Magnus et al. designed chemogenetic ion channels that improve currently available systems and are activated by the clinically used antismoking drug varenicline. They engineered a ligand-binding domain less responsive to endogenous signals and identified agonists that function at nanomolar concentrations. The combination of drug and introduced channels transiently silenced neurons, with slow but effective washout, and induced behavioral changes in animal models after brain administration. Science , this issue p. eaav5282

Debilitating hearing and balance deficits often arise through damage to the inner ear's hair cells. For humans and other mammals, such deficits are permanent, but non-mammalian vertebrates can quickly recover hearing and balance through their innate capacity to regenerate hair cells. The biological basis for this difference has remained unknown, but recent investigations in wounded balance epithelia have shown that proliferation follows cellular spreading at sites of injury. As mammalian ears mature during the first weeks after birth, the capacity for spreading and proliferation declines sharply. In seeking the basis for those declines, we investigated the circumferential bands of F-actin that bracket the apical junctions between supporting cells in the gravity-sensitive utricle. We found that those bands grow much thicker as mice and humans mature postnatally, while their counterparts in chickens remain thin from hatching through adulthood. When we cultured utricular epithelia from chickens, we found that cellular spreading and proliferation both continued at high levels, even in the epithelia from adults. In contrast, the substantial reinforcement of the circumferential F-actin bands in mammals coincides with the steep declines in cell spreading and production established in earlier experiments. We propose that the presence of thin F-actin bands at the junctions between avian supporting cells may contribute to the lifelong persistence of their capacity for shape change, cell proliferation, and hair cell replacement, while the postnatal reinforcement of the F-actin bands in maturing humans and other mammals may have an important role in limiting hair cell regeneration.

SummaryNeurons are well suited for computations on millisecond timescales, but some neuronal circuits set behavioral states over long time periods, such as those involved in energy homeostasis. We found that multiple types of hypothalamic neurons, including those that oppositely regulate body weight, are specialized as near-perfect synaptic integrators that summate inputs over extended timescales. Excitatory postsynaptic potentials (EPSPs) are greatly prolonged, outlasting the neuronal membrane time-constant up to 10-fold. This is due to the voltage-gated sodium channel Nav1.7 (Scn9a), previously associated with pain-sensation but not synaptic integration. Scn9a deletion in AGRP, POMC, or paraventricular hypothalamic neurons reduced EPSP duration, synaptic integration, and altered body weight in mice. In vivo whole-cell recordings in the hypothalamus confirmed near-perfect synaptic integration. These experiments show that integration of synaptic inputs over time by Nav1.7 is critical for body weight regulation and reveal a mechanism for synaptic control of circuits regulating long term homeostatic functions.

Hair cell losses can produce severe hearing and balance deficits in mammals and nonmammals alike, but nonmammals recover after epithelial supporting cells divide and give rise to replacement hair cells. Here, we describe cellular changes that appear to underlie the permanence of hair cell deficits in mammalian vestibular organs. In sensory epithelia isolated from the utricles of embryonic day 18 (E18) mice, supporting cells readily spread and proliferated, but spreading and proliferation were infrequent in supporting cells from postnatal day 6 (P6) mice. Cellular spreading and proliferation were dependent on alpha6 integrin, which disappeared from lateral cell membranes by P6 and colocalized with beta4 integrin near the basement membrane at both ages. In the many well-spread, proliferating E18 supporting cells, beta4 was localized at cell borders, but it was localized to hemidesmosome-like structures in the columnar, nondividing supporting cells that were prevalent in P6 cultures. We treated cultures with phorbol myristate acetate (PMA) to activate protein kinase C (PKC) in an initial test of the possibility that maturational changes in supporting cell cytoskeletons or their anchorage might restrict the proliferation of these progenitor cells in the developing mammalian inner ear. That treatment triggered the disassembly of the hemidesmosome-like beta4 structures and resulted in significantly increased cellular spreading and S-phase entry in the P6 epithelia. The results suggest that maturational changes in cytoskeletal organization and anchorage restrict proliferation of mammalian supporting cells whose counterparts are the progenitors of replacement hair cells in nonmammals, thereby leaving mammals vulnerable to persistent sensory deficits caused by hair cell loss.