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1.  P1 interneurons promote a persistent internal state that enhances inter-male aggression in Drosophila 
eLife  null;4:e11346.
How brains are hardwired to produce aggressive behavior, and how aggression circuits are related to those that mediate courtship, is not well understood. A large-scale screen for aggression-promoting neurons in Drosophila identified several independent hits that enhanced both inter-male aggression and courtship. Genetic intersections revealed that 8-10 P1 interneurons, previously thought to exclusively control male courtship, were sufficient to promote fighting. Optogenetic experiments indicated that P1 activation could promote aggression at a threshold below that required for wing extension. P1 activation in the absence of wing extension triggered persistent aggression via an internal state that could endure for minutes. High-frequency P1 activation promoted wing extension and suppressed aggression during photostimulation, whereas aggression resumed and wing extension was inhibited following photostimulation offset. Thus, P1 neuron activation promotes a latent, internal state that facilitates aggression and courtship, and controls the overt expression of these social behaviors in a threshold-dependent, inverse manner.
DOI: http://dx.doi.org/10.7554/eLife.11346.001
eLife digest
For most animals, mating and fighting are critical for survival and reproduction. These behaviors are also closely related and share similar actions. How are such complex behaviors hard-wired into the brain? A fruit fly called Drosophila melanogaster is an excellent system to investigate this problem, because flies mate and fight, and powerful genetic tools are available to probe the circuits of neurons that control these behaviors.
A great deal has been learned recently about the neural circuits that control mating, but much less was known about how the circuits for aggression are organized. Hoopfer et al. systematically activated different sets of neurons in thousands of male flies to try to find the circuits that trigger aggression. While this identified some neurons that specifically promoted aggression, it also uncovered a cluster – called P1 neurons – that promoted both aggression and courtship. This was unexpected, because P1 neurons were previously thought to only control courtship behavior.
The P1 neurons produced different behaviors at different stimulation thresholds, with the neurons requiring a stronger level of activation to promote courtship instead of aggression. Moreover, the P1 neurons triggered a lasting change in the internal state of the male that increased his tendency to engage in aggression or courtship. These results are reminiscent of observations made in mice, suggesting small groups of neurons that control mating and fighting may represent an evolutionarily conserved neural circuit "motif" for the control of social behavior.
The next step is to figure out how P1 neurons trigger a persistent internal state of arousal or motivation, and to determine whether and how this circuitry participates in the "decision" to engage in mating or fighting.
DOI: http://dx.doi.org/10.7554/eLife.11346.002
doi:10.7554/eLife.11346
PMCID: PMC4749567  PMID: 26714106
aggression; courtship; internal state; persistence; fruitless; social behavior; D. melanogaster
2.  Ventromedial hypothalamic neurons control a defensive emotion state 
eLife  null;4:e06633.
Defensive behaviors reflect underlying emotion states, such as fear. The hypothalamus plays a role in such behaviors, but prevailing textbook views depict it as an effector of upstream emotion centers, such as the amygdala, rather than as an emotion center itself. We used optogenetic manipulations to probe the function of a specific hypothalamic cell type that mediates innate defensive responses. These neurons are sufficient to drive multiple defensive actions, and required for defensive behaviors in diverse contexts. The behavioral consequences of activating these neurons, moreover, exhibit properties characteristic of emotion states in general, including scalability, (negative) valence, generalization and persistence. Importantly, these neurons can also condition learned defensive behavior, further refuting long-standing claims that the hypothalamus is unable to support emotional learning and therefore is not an emotion center. These data indicate that the hypothalamus plays an integral role to instantiate emotion states, and is not simply a passive effector of upstream emotion centers.
DOI: http://dx.doi.org/10.7554/eLife.06633.001
eLife digest
Animals have evolved a large number of ‘defensive behaviors’ to deal with the threat of predators. Examples include reptiles camouflaging themselves to avoid discovery, fish and birds swarming to confuse predators, insects releasing toxic chemicals, and humans readying themselves to fight or flee.
In mammals, defensive behaviors are thought to be mediated by a region of the brain called the amygdala. This structure, which is known as the brain's ‘emotion center’, receives and processes information from the senses about impending threats. It then sends instructions on how to deal with these threats to other regions of the brain including the hypothalamus, which pass them on to the brain regions that control the behavioral, endocrine and involuntary responses of the mammal.
For many years it has been thought that the role of the hypothalamus is to serve simply as a relay for emotion states encoded in the amygdala, rather than as an emotion center itself. However, Kunwar et al. have now challenged this assumption with the aid of a technique called optogenetics, in which light is used to activate specific populations of genetically labeled neurons. When light was used to directly activate neurons within the ventromedial hypothalamus in awake mice, the animals instantly froze and/or fled, just as they would when faced with a predator. Given that the optical stimulation had completely bypassed the amygdala, this suggested that the hypothalamus must be capable of generating this defensive response without any input from the amygdala.
The freezing and fleeing responses resembled the responses to a predator in a number of key ways. Mice chose to avoid areas of their cage in which they had received the stimulation, suggesting that—like a predator—these areas induced an unpleasant emotional state, perhaps akin to anxiety or fear. Freezing and fleeing persisted for several seconds after the stimulation had stopped, just as freezing and fleeing responses to predators do not immediately cease after the threat has gone. And finally, destroying the neurons targeted by the stimulation made mice less likely to avoid one of their main predators, the rat. It also made the animals less anxious.
Overall the results suggest that the hypothalamus may be more than simply a relay for the amygdala, and that ‘amygdala-centric’ views of emotion processing may need to be re-visited.
DOI: http://dx.doi.org/10.7554/eLife.06633.002
doi:10.7554/eLife.06633
PMCID: PMC4379496  PMID: 25748136
fear; emotion; defense; ventromedial hypothalamus; persistance; scalability; mouse
3.  Exposure Classification and Temporal Variability in Urinary Bisphenol A Concentrations among Couples in Utah—The HOPE Study 
Environmental Health Perspectives  2015;124(4):498-506.
Background:
Bisphenol A (BPA) is an endocrine disruptor and potential reproductive toxicant, but results of epidemiologic studies have been mixed and have been criticized for inadequate exposure assessment that often relies on a single measurement.
Objective:
Our goal was to describe the distribution of BPA concentrations in serial urinary specimens, assess temporal variability, and provide estimates of exposure classification when randomly selected samples are used to predict average exposure.
Methods:
We collected and analyzed 2,614 urine specimens from 83 Utah couples beginning in 2012. Female participants collected daily first-morning urine specimens during one to two menstrual cycles and male partners collected specimens during the woman’s fertile window for each cycle. We measured urinary BPA concentrations and calculated geometric means (GM) for each cycle, characterized the distribution of observed values and temporal variability using intraclass correlation coefficients, and performed surrogate category analyses to determine how well repeat samples could classify exposure.
Results:
The GM urine BPA concentration was 2.78 ng/mL among males and 2.44 ng/mL among females. BPA had a high degree of variability among both males (ICC = 0.18; 95% CI: 0.11, 0.26) and females (ICC = 0.11; 95% CI: 0.08, 0.16). Based on our more stringent surrogate category analysis, to reach proportions ≥ 0.80 for sensitivity, specificity, and positive predictive value (PPV) among females, 6 and 10 repeat samples for the high and low tertiles, respectively, were required. For the medium tertile, specificity reached 0.87 with 10 repeat samples, but even with 11 samples, sensitivity and PPV did not exceed 0.36. Five repeat samples, among males, yielded sensitivity and PPV values ≥ 0.75 for the high and low tertiles, but, similar to females, classification for the medium tertile was less accurate.
Conclusion:
Repeated urinary specimens are required to characterize typical BPA exposure.
Citation:
Cox KJ, Porucznik CA, Anderson DJ, Brozek EM, Szczotka KM, Bailey NM, Wilkins DG, Stanford JB. 2016. Exposure classification and temporal variability in urinary bisphenol A concentrations among couples in Utah—the HOPE study. Environ Health Perspect 124:498–506; http://dx.doi.org/10.1289/ehp.1509752
doi:10.1289/ehp.1509752
PMCID: PMC4829981  PMID: 26372668
4.  Identification of the subthalamic nucleus in deep brain stimulation surgery with a novel wavelet-derived measure of neural background activity 
Journal of neurosurgery  2009;111(4):767-774.
Object
A wavelet-based measure was developed to quantitatively assess neural background activity taken during surgical neurophysiological recordings to localize the boundaries of the subthalamic nucleus during target localization for deep brain stimulator implant surgery.
Methods
Neural electrophysiological data was recorded from 14 patients (20 tracks, n = 275 individual recording sites) with dopamine-sensitive idiopathic Parkinson’s disease during the target localization portion of deep brain stimulator implant surgery. During intraoperative recording the STN was identified based upon audio and visual monitoring of neural firing patterns, kinesthetic tests, and comparisons between neural behavior and known characteristics of the target nucleus. The quantitative wavelet-based measure was applied off-line using MATLAB software to measure the magnitude of the neural background activity, and the results of this analysis were compared to the intraoperative conclusions. Wavelet-derived estimates were compared to power spectral density measures.
Results
The wavelet-derived background levels were significantly higher in regions encompassed by the clinically estimated boundaries of the STN than in surrounding regions (STN: 225 ± 61 μV vs. ventral to STN: 112 ± 32 μV, and dorsal to STN: 136 ± 66 μV). In every track, the absolute maximum magnitude was found within the clinically identified STN. The wavelet-derived background levels provided a more consistent index with less variability than power spectral density.
Conclusions
The wavelet-derived background activity assessor can be calculated quickly, requires no spike sorting, and can be reliably used to identify the STN with very little subjective interpretation required. This method may facilitate rapid intraoperative identification of subthalamic nucleus borders.
doi:10.3171/2008.11.JNS08392
PMCID: PMC4763615  PMID: 19344225
Subthalamic nucleus; deep brain stimulation; Parkinson’s disease; neurosurgery
5.  Development and validation a LC-MS/MS method for determination of L-type voltage-gated calcium channel and NMDA receptor antagonist NGP1-01 in mouse serum 
NGP1-01 (8-benzylamino-8, 11-oxapentacyclo [5.4.0.02, 6.03, 10.05, 9] undecane) is a heterocyclic cage compound with multifunctional calcium channel blocking activity that has been demonstrated to be neuroprotective in several neurodegenerative models. A sensitive internal standard LC-MS/MS method was developed and validated to quantify NGP1-01 in mouse serum. The internal standard (IS) was 8-phenylethyl-8, 11-oxapentacyclo [5.4.0.0(2, 6).0(3, 10).0(5, 9)] undecane. Sample preparation involved a protein precipitation procedure by addition of acetonitrile. Chromatographic separation was carried out on a Phenomenex Kinetex phenyl-hexyl column (100 x 2.1 mm, 2.6 μm) employing a gradient (45% isocratic 3 min, 45% to 95% linear gradient 6 min, 95% isocratic 3 min) of an elution mobile phase of 5 mM ammonium acetate in 100% acetonitrile mixing with an application mobile phase of 5 mM ammonium acetate in 2% acetonitrile. Detection was achieved by a QTrap 5500 mass spectrometer (AB Sciex) employing electrospray ionization in the positive mode with multiple-reaction-monitoring (MRM) for NGP1-01 (m/z 266 → 91) and IS (m/z 280 → 105). The method validation was carried out in accordance with Food and Drug Administration (FDA) guidelines. The method had a linear range of at least 0.5–50 ng/mL with a correlation coefficient 0.999. The intra-assay and inter-assay precisions (%CV) were equal to or within the range of 1.0 to 4.3% and the accuracies (% relative error) equal to or within −2.5% to 3.4%. The analyte was stable for at least 2 months at −20°C, for at least 8 h at room temperature and for at least three freeze thaw cycles. The extraction recovery was 94.9 to 105.0%, with a %CV ≤ 9.5%. The technique was found to be free of any matrix effects as determined by experiments involving five different lots of mouse serum. Cross-talk interferences were not present. Two different gradient slope chromatography runs were done on dosed mouse serum samples to assess a possible positive error in peak area determination from in-source fragmentation of metabolites generating the same MRM transitions as the parent drug or IS. No such interference was found in the NGP1-01 peak, while a minor interference was identified in the IS peak. The optimized method was applied to the measurement of NGP1-01 in serum of dosed mice.
doi:10.1016/j.jchromb.2014.05.048
PMCID: PMC4744373  PMID: 24950096
NGP1-01; pentacycloundecylamine; neuroprotective agent; multifunctional drug; mouse serum; LC-MS/MS
6.  Independent, reciprocal neuromodulatory control of sweet and bitter taste sensitivity during starvation in Drosophila 
Neuron  2014;84(4):806-820.
SUMMARY
An organism’s behavioral decisions often depend upon the relative strength of appetitive and aversive sensory stimuli, the relative sensitivity to which can be modified by internal states like hunger. However, whether sensitivity to such opposing influences is modulated in a unidirectional or bidirectional manner is not clear. Starved flies exhibit increased sugar and decreased bitter sensitivity. It is widely believed that only sugar sensitivity changes, and that this masks bitter sensitivity. Here we use gene- and circuit-level manipulations to show that sweet- and bitter-sensitivity are independently and reciprocally regulated by starvation in Drosophila. We identify orthogonal neuromodulatory cascades that oppositely control peripheral taste sensitivity for each modality. Moreover, these pathways are recruited at increasing hunger levels, such that low-risk changes (higher sugar sensitivity) precede high-risk changes (lower sensitivity to potentially toxic resources). In this way, state intensity-dependent, reciprocal regulation of appetitive and aversive peripheral gustatory sensitivity permits flexible, adaptive feeding-decisions.
doi:10.1016/j.neuron.2014.09.032
PMCID: PMC4365050  PMID: 25451195
Neuromodulation; Drosophila; gustatory system; decision making; dopamine; neuropeptide
7.  Antagonistic Control of Social Behaviors by Inhibitory and Excitatory Neurons in the Medial Amygdala 
Cell  2014;158(6):1348-1361.
SUMMARY
Animals display a range of innate social behaviors that play essential roles in survival and reproduction. While the medial amygdala (MeA) has been implicated in prototypic social behaviors such as aggression, the circuit-level mechanisms controlling such behaviors are not well understood. Using cell-type specific functional manipulations, we find that distinct neuronal populations in the MeA control different social and asocial behaviors. A GABAergic subpopulation promotes aggression and two other social behaviors, while neighboring glutamatergic neurons promote repetitive self-grooming, an asocial behavior. Moreover, this glutamatergic subpopulation inhibits social interactions independently of its effect to promote self-grooming, while the GABAergic subpopulation inhibits self-grooming, even in a non-social context. These data suggest that social vs. repetitive asocial behaviors are controlled in an antagonistic manner by inhibitory vs. excitatory amygdala subpopulations, respectively. These findings provide a framework for understanding circuit-level mechanisms underlying opponency between innate behaviors, with implications for their perturbation in psychiatric disorders.
doi:10.1016/j.cell.2014.07.049
PMCID: PMC4167378  PMID: 25215491
8.  Long-Range Stabilization of Anthrax Protective Antigen upon Binding to CMG2 
Biochemistry  2014;53(38):6084-6091.
Protective antigen (PA) mediates entry of edema factor (EF) and lethal factor (LF) into the cytoplasmic space of the cells through the formation of a membrane-spanning pore. To do this, PA must initially bind to a host cellular receptor. Recent mass spectrometry analysis of PA using histidine hydrogen–deuterium exchange (His-HDX) has shown that binding of the von Willebrand factor A (vWA) domain of the receptor capillary morphogenesis protein-2 (CMG2) lowers the exchange rates of the imidazole C2 hydrogen of several histidines, suggesting that receptor binding decreases the structural flexibility of PA. Here, using His-HDX and fluorescence as a function of denaturant, and protease susceptibility, we show that binding of the vWA domain of CMG2 largely increases the stability of PA and the effect reaches up to 70 Å from the receptor binding interface. We also show that the pKa values and HDX rates of histidines located in separate domains change upon receptor binding. These results indicate that when one end of the protein is anchored, the structure of PA is tightened, noncovalent interactions are strengthened, and the global stability of the protein increases. These findings suggest that CMG2 may be used to stabilize PA in future anthrax vaccines.
doi:10.1021/bi500718g
PMCID: PMC4179592  PMID: 25186975
9.  Combined STAT3 and BCR-ABL1 Inhibition Induces Synthetic Lethality in Therapy-Resistant Chronic Myeloid Leukemia 
Leukemia  2014;29(3):586-597.
Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCRABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays, and hydrogen-deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from CML patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 μM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells (LSCs). Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.
doi:10.1038/leu.2014.245
PMCID: PMC4334758  PMID: 25134459
10.  Decoding Ventromedial Hypothalamic Neural Activity during Male Mouse Aggression 
The Journal of Neuroscience  2014;34(17):5971-5984.
The ventromedial hypothalamus, ventrolateral area (VMHvl) was identified recently as a critical locus for inter-male aggression. Optogenetic stimulation of VMHvl in male mice evokes attack toward conspecifics and inactivation of the region inhibits natural aggression, yet very little is known about its underlying neural activity. To understand its role in promoting aggression, we recorded and analyzed neural activity in the VMHvl in response to a wide range of social and nonsocial stimuli. Although response profiles of VMHvl neurons are complex and heterogeneous, we identified a subpopulation of neurons that respond maximally during investigation and attack of male conspecific mice and during investigation of a source of male mouse urine. These “male responsive” neurons in the VMHvl are tuned to both the inter-male distance and the animal's velocity during attack. Additionally, VMHvl activity predicts several parameters of future aggressive action, including the latency and duration of the next attack. Linear regression analysis further demonstrates that aggression-specific parameters, such as distance, movement velocity, and attack latency, can model ongoing VMHvl activity fluctuation during inter-male encounters. These results represent the first effort to understand the hypothalamic neural activity during social behaviors using quantitative tools and suggest an important role for the VMHvl in encoding movement, sensory, and motivation-related signals.
doi:10.1523/JNEUROSCI.5109-13.2014
PMCID: PMC3996217  PMID: 24760856
aggression; hypothalamus; motivation; physiology
11.  The BRAIN Initiative: developing technology to catalyse neuroscience discovery 
The evolution of the field of neuroscience has been propelled by the advent of novel technological capabilities, and the pace at which these capabilities are being developed has accelerated dramatically in the past decade. Capitalizing on this momentum, the United States launched the Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative to develop and apply new tools and technologies for revolutionizing our understanding of the brain. In this article, we review the scientific vision for this initiative set forth by the National Institutes of Health and discuss its implications for the future of neuroscience research. Particular emphasis is given to its potential impact on the mapping and study of neural circuits, and how this knowledge will transform our understanding of the complexity of the human brain and its diverse array of behaviours, perceptions, thoughts and emotions.
doi:10.1098/rstb.2014.0164
PMCID: PMC4387507  PMID: 25823863
BRAIN Initiative; neural circuitry; neurotechnology
12.  A Framework for Studying Emotions Across Phylogeny 
Cell  2014;157(1):187-200.
Since the 19th century, there has been disagreement over the fundamental question of whether “emotions” are cause or consequence of their associated behaviors. This question of causation is most directly addressable in genetically tractable model organisms, including invertebrates such as Drosophila. Yet there is ongoing debate about whether such species even have “emotions,” since emotions are typically defined with reference to human behavior and neuroanatomy. Here we argue that emotional behaviors are a class of behaviors that express internal emotion states. These emotion states exhibit certain general functional and adaptive properties that apply across any specific human emotions like fear or anger, as well as across phylogeny. These general properties, which can be thought of as “emotion primitives”, can be modeled and studied in evolutionarily distant model organisms, allowing functional dissection of their mechanistic bases, and tests of their causal relationships to behavior. More generally, our approach aims not only at better integration of such studies in model organisms with studies of emotion in humans, but also suggests a revision of how emotion should be operationalized within psychology and psychiatry.
doi:10.1016/j.cell.2014.03.003
PMCID: PMC4098837  PMID: 24679535
13.  Biomonitoring method for bisphenol A in human urine by ultra-high-performance liquid chromatography-tandem mass spectrometry 
An ultra-high-performance liquid chromatography-tandem mass spectrometry method for the measurement of total bisphenol A in human urine was developed and validated. The method utilized liquid/liquid extraction with 1-chlorobutane and a human urine aliquot size of 800 µL. Chromatography was performed on an Acquity UPLC® system with a Kinetex® Phenyl-Hexyl column. Mass spectrometric analysis was with negative electrospray ionization on a Quattro Premier XE™. The surrogate matrix method was used for the preparation of calibration standards in synthetic urine due to the presence of BPA in control human urine. The validated calibration range was 0.75 to 20 ng/mL with a limit of detection of 0.1 ng/mL. The internal standard was d16-bisphenol A. Method validation utilized quality control samples at three concentrations in both synthetic urine and human urine. Bisphenol A mono-glucuronide was fortified in synthetic urine in each analytical run to monitor the enzymatic conversion of the glucuronide conjugate to BPA by β-glucuronidase. Validated method parameters included linearity, accuracy, precision, integrity of dilution, selectivity, re-injection reproducibility, recovery/matrix effect, solution stability, and matrix stability in human urine. Acceptance criteria for analytical standards and QCs were ± 20% of nominal concentration. Matrix stability in human urine was validated after 24 hours at ambient temperature, after three freeze/thaw cycles, and after frozen storage at −20 °C and −80 °C for up to 218 days. The method has been applied to the analysis of over 1750 human urine samples from a biomonitoring study. The median and mean urine BPA concentrations were 2.71 ng/mL and 4.75 ng/mL, respectively.
doi:10.1016/j.jchromb.2014.01.039
PMCID: PMC4068800  PMID: 24594944
Bisphenol A; liquid chromatography; tandem mass spectrometry; human urine
14.  Central amygdala PKC-δ+ neurons mediate the influence of multiple anorexigenic signals 
Nature neuroscience  2014;17(9):1240-1248.
Feeding can be inhibited by multiple cues, including those associated with satiety, sickness or unpalatable food. How such anorexigenic signals inhibit feeding at the neural circuit level is incompletely understood. While some inhibitory circuits have been identified, it is not yet clear whether distinct anorexigenic influences are processed in a convergent or parallel manner. The amygdala central nucleus (CEA) has been implicated in feeding control, but its role is controversial. The lateral subdivision of CEA (CEl) contains a subpopulation of GABAergic neurons, marked by protein kinase C-δ. Here we show that CEl PKC-δ+ neurons in mice are activated by diverse anorexigenic signals in vivo, required for the inhibition of feeding by such signals, and strongly suppress food intake when activated. They receive pre-synaptic inputs from anatomically distributed neurons activated by different anorexigenic agents. These data suggest that CEl PKC-δ+ neurons constitute an important node that mediates the influence of multiple anorexigenic signals.
doi:10.1038/nn.3767
PMCID: PMC4146747  PMID: 25064852
15.  Control of stress-induced persistent anxiety by an extra-amygdala septohypothalamic circuit 
Cell  2014;156(3):522-536.
The extended amygdala has dominated research on the neural circuitry of fear and anxiety, but the septo-hippocampal axis plays an important role as well. The lateral septum (LS) is thought to suppress fear and anxiety, through its outputs to the hypothalamus. However, this structure has not yet been dissected using modern tools. The type 2 CRF receptor (Crfr2) marks a subset of LS neurons, whose functional connectivity we have investigated using optogenetics. Crfr2+ cells include GABAergic projection neurons that connect with the anterior hypothalamus. Surprisingly, we find that these LS outputs enhance stress-induced behavioral measures of anxiety. Furthermore, transient activation of Crfr2+ neurons promotes, while inhibition suppresses, persistent anxious behaviors. LS Crfr2+ outputs also positively regulate circulating corticosteroid levels. These data identify a subset of LS projection neurons that promote, rather than suppress, stress-induced behavioral and endocrinological dimensions of persistent anxiety states, and provide a cellular point-of-entry to LS circuitry.
doi:10.1016/j.cell.2013.12.040
PMCID: PMC3982923  PMID: 24485458
17.  Scalable Control of Mounting and Attack by ESR1+ Neurons in the Ventromedial Hypothalamus 
Nature  2014;509(7502):627-632.
Social behaviors, such as aggression or mating, proceed through a series of appetitive and consummatory phases1 that are associated with increasing levels of arousal2. How such escalation is encoded in the brain, and linked to behavioral action selection, remains an important unsolved problem in neuroscience. The ventrolateral subdivision of the murine ventromedial hypothalamus (VMHvl) contains neurons whose activity increases during male-male and male-female social encounters. Non-cell type-specific optogenetic activation of this region elicited attack behavior, but not mounting3. We have identified a subset of VMHvl neurons marked by the estrogen receptor 1 (Esr1), and investigated their role in male social behavior. Optogenetic manipulations indicated that Esr1+ (but not Esr1-) neurons are sufficient to initiate attack, and that their activity is continuously required during ongoing agonistic behavior. Surprisingly, weaker optogenetic activation of these neurons promoted mounting behavior, rather than attack, towards both males and females, as well as sniffing and close investigation (CI). Increasing photostimulation intensity could promote a transition from CI and mounting to attack, within a single social encounter. Importantly, time-resolved optogenetic inhibition experiments revealed requirements for Esr1+ neurons in both the appetitive (investigative) and the consummatory phases of social interactions. Combined optogenetic activation and calcium imaging experiments in vitro, as well as c-Fos analysis in vivo, indicated that increasing photostimulation intensity increases both the number of active neurons and the average level of activity per neuron. These data suggest that Esr1+ neurons in VMHvl control the progression of a social encounter from its appetitive through its consummatory phases, in a scalable manner that reflects the number or type of active neurons in the population.
doi:10.1038/nature13169
PMCID: PMC4098836  PMID: 24739975
18.  Evidence for Asymmetrical Divergence-Gene Flow of Nuclear Loci, but Not Mitochondrial Loci, between Seabird Sister Species: Blue-Footed (Sula nebouxii) and Peruvian (S. variegata) Boobies 
PLoS ONE  2013;8(4):e62256.
Understanding the process of speciation requires understanding how gene flow influences divergence. Recent analyses indicate that divergence can take place despite gene flow and that the sex chromosomes can exhibit different levels of gene flow than autosomes and mitochondrial DNA. Using an eight marker dataset including autosomal, z-linked, and mitochondrial loci we tested the hypothesis that blue-footed (Sula nebouxii) and Peruvian (S. variegata) boobies diverged from their common ancestor with gene flow, paying specific attention to the differences in gene flow estimates from nuclear and mitochondrial markers. We found no gene flow at mitochondrial markers, but found evidence from the combined autosomal and z-linked dataset that blue-footed and Peruvian boobies experienced asymmetrical gene flow during or after their initial divergence, predominantly from Peruvian boobies into blue-footed boobies. This gene exchange may have occurred either sporadically between periods of allopatry, or regularly throughout the divergence process. Our results add to growing evidence that diverging species can remain distinct but exchange genes.
doi:10.1371/journal.pone.0062256
PMCID: PMC3629132  PMID: 23614045
19.  Optogenetic control of freely behaving adult Drosophila using a red-shifted channelrhodopsin 
Nature methods  2013;11(3):325-332.
Optogenetics allows the manipulation of neural activity in freely moving animals with millisecond precision, but its application in Drosophila has been limited. Here we show that a recently described Red activatable Channelrhodopsin (ReaChR) permits control of complex behavior in freely moving adult flies, at wavelengths that are not thought to interfere with normal visual function. This tool affords the opportunity to control neural activity over a broad dynamic range of stimulation intensities. Using time-resolved activation, we show that the neural control of male courtship song can be separated into probabilistic, persistent and deterministic, command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, supporting the idea that they constitute a locus of state-dependent influence. This separation is not evident using thermogenetic tools, underscoring the importance of temporally precise control of neuronal activation in the functional dissection of neural circuits in Drosophila.
doi:10.1038/nmeth.2765
PMCID: PMC4151318  PMID: 24363022
20.  How Food Controls Aggression in Drosophila 
PLoS ONE  2014;9(8):e105626.
How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. For instance, it is unclear whether food increases aggression directly, or as a secondary consequence of increased social interactions caused by attraction to food. Here we use the vinegar fly, Drosophila melanogaster, to investigate the manner by which food influences aggression. We show that food promotes aggression in flies, and that it does so independently of any effect on frequency of contact between males, increase in locomotor activity or general enhancement of social interactions. Importantly, the level of aggression depends on the absolute amount of food, rather than on its surface area or concentration. When food resources exceed a certain level, aggression is diminished, suggestive of reduced competition. Finally, we show that detection of sugar via Gr5a+ gustatory receptor neurons (GRNs) is necessary for food-promoted aggression. These data demonstrate that food exerts a specific effect to promote aggression in male flies, and that this effect is mediated, at least in part, by sweet-sensing GRNs.
doi:10.1371/journal.pone.0105626
PMCID: PMC4146546  PMID: 25162609
21.  Novel multi-sided, microelectrode arrays for implantable neural applications 
Biomedical microdevices  2011;13(3):441-451.
A new parylene-based microfabrication process is presented for neural recording and drug delivery applications. We introduce a large design space for electrode placement and structural flexibility with a six mask process. By using chemical mechanical polishing, electrode sites may be created top-side, back-side, or on the edge of the device having three exposed sides. Added surface area was achieved on the exposed edge through electroplating. Poly(3,4-ethylenedioxythiophene) (PEDOT) modified edge electrodes having an 85-μm2 footprint resulted in an impedance of 200 kΩ at 1 kHz. Edge electrodes were able to successfully record single unit activity in acute animal studies. A finite element model of planar and edge electrodes relative to neuron position reveals that edge electrodes should be beneficial for increasing the volume of tissue being sampled in recording applications.
doi:10.1007/s10544-011-9512-z
PMCID: PMC4013149  PMID: 21301965
Neural recording; Microelectrode array; Parylene; Neural prostheses; Drug delivery; Chemical mechanical polishing
22.  Quantitative Measurement of Solvent Accessibility of Histidine Imidazole Groups in Proteins 
Biochemistry  2012;51(36):7202-7208.
We report a method to express the solvent accessibility of histidine imidazole groups in proteins. The method is based on measuring the rate of hydrogen exchange (HX) reaction of the imidazole Cε1-hydrogen. The rate profile of the HX reaction as a function of pH gives a sigmoidal curve, which reaches the maximum rate constant (kmax) on the alkaline side of the sigmoidal curve. To quantitatively describe the solvent accessibility of imidazole groups in proteins, it is necessary to compare the kmax of the imidazole groups with their intrinsic kmax (ikmax), the maximum rate constants for the given imidazole groups when they are fully exposed to the bulk solvent. However, the mechanism of HX reaction suggests that the ikmax of an imidazole group differs depending on its pKa, and no systematic study has been conducted to clarify how the ikmax is affected by pKa. We therefore investigated the relationship between ikmax and pKa using four imidazole derivatives at three different temperatures. The experimentally determined pKa-specific ikmax values allowed us to derive a general formula to estimate the ikmax value of any given imidazole group exhibiting a specific pKa at a specific temperature. Using the formula, the protection factors (PF), the ratio of ikmax to kmax, of five imidazole groups in dihydrofolate reductase were obtained and used to express the magnitude of their solvent accessibility. In this definition, the smaller the PF value, the higher the solvent accessibility, and a value of 1 indicates full exposure to the bulk solvent. The solvent accessibility expressed by the PF values agreed well with the solvent accessible surface areas (ASA) obtained from the X-ray diffraction data.
doi:10.1021/bi300911d
PMCID: PMC3462353  PMID: 22901083
23.  Genetic identification of C-fibers that detect massage-like stroking of hairy skin in vivo 
Nature  2013;493(7434):669-673.
Stroking of the skin produces pleasant sensations that can occur during social interactions with conspecifics, such as grooming1. Despite numerous physiological studies (reviewed in ref. 2), molecularly defined sensory neurons that detect pleasant stroking of hairy skin3,4 in vivo have not been reported. Previously, we identified a rare population of unmyelinated sensory neurons that express the G protein-coupled receptor (GPCR) MrgprB45,6. These neurons exclusively innervate hairy skin with large terminal arborizations7 that resemble the receptive fields of C-tactile (CT) afferents in humans8. Unlike other molecularly defined mechanosensory C-fiber subtypes9,10, MrgprB4+ neurons could not be detectably activated by sensory stimulation of the skin ex vivo. Therefore, we developed a preparation for calcium imaging in their spinal projections during stimulation of the periphery in intact animals. MrgprB4+ neurons were activated by massage-like stroking of hairy skin, but not by noxious punctate mechanical stimulation. By contrast, a different population of C-fibers expressing MrgprD11 was activated by pinching but not by stroking, consistent with previous physiological and behavioral data10,12. Pharmacogenetic activation of MrgprB4- expressing neurons in freely behaving animals promoted conditioned place preference13, suggesting that such activation is positively reinforcing and/or anxiolytic. These data open the way to understanding the function of MrgprB4 neurons during natural behaviors, and provide a general approach to functionally characterizing genetically identified subsets of somatosensory neurons in vivo.
doi:10.1038/nature11810
PMCID: PMC3563425  PMID: 23364746
24.  DISCOVERY OF NOVEL GLUCOSE-REGULATED PROTEINS IN ISOLATED HUMAN PANCREATIC ISLETS USING LC-MS/MS-BASED PROTEOMICS 
Journal of Proteome Research  2012;11(7):3520-3532.
The prevalence of diabetes mellitus is increasing dramatically throughout the world, and the disease has become a major public health issue. The most common form of the disease, type 2 diabetes, is characterized by insulin resistance and insufficient insulin production from the pancreatic beta-cell. Since glucose is the most potent regulator of beta-cell function under physiological conditions, identification of the insulin secretory defect underlying type 2 diabetes requires a better understanding of glucose regulation of human beta-cell function. To this aim, a bottom-up LC-MS/MS-based proteomics approach was used to profile pooled islets from multiple donors under basal (5 mM) or high (15 mM) glucose conditions. Our analysis discovered 256 differentially abundant proteins (~p<0.05) after 24 h of high glucose exposure from more than 4500 identified in total. Several novel glucose-regulated proteins were elevated under high glucose conditions, including regulators of mRNA splicing (Pleiotropic regulator 1), processing (Retinoblastoma binding protein 6), and function (Nuclear RNA export factor 1), in addition to Neuron navigator 1 and Plasminogen activator inhibitor 1. Proteins whose abundances markedly decreased during incubation at 15 mM glucose included Bax inhibitor 1 and Synaptotagmin-17. Up-regulation of Dicer 1 and SLC27A2 and down-regulation of Phospholipase Cβ4 were confirmed by Western blots. Many proteins found to be differentially abundant after high glucose stimulation are annotated as uncharacterized or hypothetical. These findings expand our knowledge of glucose regulation of the human islet proteome and suggest many hitherto unknown responses to glucose that require additional studies to explore novel functional roles.
doi:10.1021/pr3002996
PMCID: PMC3391329  PMID: 22578083
human; pancreatic islet; glucose; type 2 diabetes; proteomics; mass spectrometry; LC-MS/MS
25.  Genetic dissection of an amygdala microcircuit that gates conditioned fear 
Nature  2010;468(7321):270-276.
The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. We used molecular genetic approaches to map the functional connectivity of a subpopulation of GABAergic neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-delta (PKCδ). Channelrhodopsin-2 assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKCδ+ neurons inhibit output neurons in the medial CE (CEm), and also make reciprocal inhibitory synapses with PKCδ− neurons in CEl. Electrical silencing of PKCδ+ neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus (CS), called CEloff units (Ciocchi et al, this issue). This correspondence, together with behavioral data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing.
doi:10.1038/nature09553
PMCID: PMC3597095  PMID: 21068836

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