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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  At–Sea Behavior Varies with Lunar Phase in a Nocturnal Pelagic Seabird, the Swallow-Tailed Gull 
PLoS ONE  2013;8(2):e56889.
Strong and predictable environmental variability can reward flexible behaviors among animals. We used long-term records of activity data that cover several lunar cycles to investigate whether behavior at-sea of swallow-tailed gulls Creagrus furcatus, a nocturnal pelagic seabird, varied with lunar phase in the Galápagos Islands. A Bayesian hierarchical model showed that nighttime at-sea activity of 37 breeding swallow-tailed gulls was clearly associated with changes in moon phase. Proportion of nighttime spent on water was highest during darker periods of the lunar cycle, coinciding with the cycle of the diel vertical migration (DVM) that brings prey to the sea surface at night. Our data show that at-sea behavior of a tropical seabird can vary with environmental changes, including lunar phase.
doi:10.1371/journal.pone.0056889
PMCID: PMC3582633  PMID: 23468889
19.  OPTOGENETICS, SEX AND VIOLENCE IN THE BRAIN: IMPLICATIONS FOR PSYCHIATRY 
Biological psychiatry  2011;71(12):1081-1089.
Pathological aggression, and the inability to control aggressive impulses, takes a tremendous toll on society. Yet aggression is a normal component of the innate behavior repertoire of most vertebrate animal species, as well as of many invertebrates. Progress in understanding the etiology of disorders of aggressive behavior, whether genetic or environmental in nature, therefore requires an understanding of the brain circuitry that controls normal aggression. Efforts to understand this circuitry at the level of specific neuronal populations have been constrained by the limited resolution of classical methodologies, such as electrical stimulation and electrolytic lesion. The availability of new, genetically based tools for mapping and manipulating neural circuits at the level of specific, genetically defined neuronal subtypes provides an opportunity to investigate the functional organization of aggression circuitry with cellular resolution. However these technologies are optimally applied in the mouse, where there has been surprisingly little traditional work on the functional neuroanatomy of aggression. Here we discuss recent, initial efforts to apply optogenetics and other state-of-the-art methods to the dissection of aggression circuitry in the mouse. We find, surprisingly, that neurons necessary and sufficient for inter-male aggression are located within the ventrolateral subdivision of the ventromedial hypothalamic nucleus (VMHvl), a structure traditionally associated with reproductive behavior. These neurons are intermingled with neurons activated during male-female mating, with ~20% overlap between the populations. We discuss the significance of these findings with respect to neuroethological and neuroanatomical perspectives on the functional organization of innate behaviors, and their potential implications for psychiatry.
doi:10.1016/j.biopsych.2011.11.012
PMCID: PMC3380604  PMID: 22209636
aggression; mating; violence; hypothalamus; optogenetics; channelrhodopsin; mouse
20.  Visualizing Neuromodulation In Vivo: TANGO-Mapping of Dopamine Signaling Reveals Appeptite Control of Sugar Sensing 
Cell  2012;148(3):583-595.
SUMMARY
Behavior cannot be predicted from a “connectome,” because the brain contains a chemical “map” of neuromodulation superimposed upon its synaptic connectivity map. Neuromodulation changes how neural circuits process information in different states, such as hunger or arousal. Here we describe a novel, genetically based method to map, in an unbiased and brain-wide manner, sites of neuromodulation under different conditions in the Drosophila brain. This method, and genetic perturbations, reveal that the well-known effect of hunger to enhance behavioral sensitivity to sugar is mediated, at least in part, by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx. These data reinforce the concept that sensory neurons constitute an important locus for state-dependent gain-control of behavior, and introduce a new methodology that can be extended to other neuromodulators and model organisms.
doi:10.1016/j.cell.2011.12.022
PMCID: PMC3295637  PMID: 22304923
21.  A CRE-DEPENDENT, ANTEROGRADE TRANS-SYNAPTIC VIRAL TRACER FOR MAPPING OUTPUT PATHWAYS OF GENETICALLY MARKED NEURONS 
Neuron  2011;72(6):938-950.
SUMMARY
Neurotropic viruses that conditionally infect or replicate in molecularly defined neuronal subpopulations, and then spread trans-synaptically, are powerful tools for mapping neural pathways. Genetically targetable retrograde trans-synaptic tracer viruses are available to map the inputs to specific neuronal subpopulations, but an analogous tool for mapping synaptic outputs is not yet available. Here we describe a Cre recombinase-dependent, anterograde trans-neuronal tracer, based on the H129 strain of herpes simplex virus (HSV). Application of this virus to transgenic or knock-in mice expressing Cre in peripheral neurons of the olfactory epithelium or the retina reveals widespread, polysynaptic labeling of higher-order neurons in the olfactory and visual systems, respectively. Polysynaptic pathways were also labeled from cerebellar Purkinje cells. In each system, the pattern of labeling was consistent with classical circuit-tracing studies, restricted to neurons and anterograde-specific. These data provide proof-of-principle for a conditional, non-diluting anterograde trans-synaptic tracer for mapping synaptic outputs from genetically marked neuronal subpopulations.
doi:10.1016/j.neuron.2011.12.002
PMCID: PMC3275419  PMID: 22196330
22.  Hierarchical chemosensory regulation of male-male social interactions in Drosophila 
Nature neuroscience  2011;14(6):757-762.
Pheromones regulate male social behaviors in Drosophila, but the identities and behavioral role(s) of these chemosensory signals, and how they interact, are incompletely understood. Here we show that (Z)-7-tricosene (7-T), a male-enriched cuticular hydrocarbon (CH) previously shown to inhibit male-male courtship, is also essential for normal levels of aggression. The opposite influences of 7-T on aggression and courtship are independent, but both require the gustatory receptor Gr32a. Surprisingly, sensitivity to 7-T is required for the aggression-promoting effect of 11-cis-vaccenyl acetate (cVA), an olfactory pheromone, but 7-T sensitivity is independent of cVA. 7-T and cVA therefore regulate aggression in a hierarchical manner. Furthermore, the increased courtship caused by depletion of male CHs is suppressed by a mutation in the olfactory receptor Or47b. Thus, male social behaviors are controlled by gustatory pheromones that promote and suppress aggression and courtship, respectively, and whose influences are dominant to olfactory pheromones that enhance these behaviors.
doi:10.1038/nn.2800
PMCID: PMC3102769  PMID: 21516101
23.  The ABL switch control inhibitor DCC-2036 is active against the chronic myeloid leukemia mutant BCR-ABLT315I and exhibits a narrow resistance profile 
Cancer research  2011;71(9):3189-3195.
Acquired point mutations within the BCR-ABL kinase domain represent a common mechanism of resistance to ABL inhibitor therapy in patients with chronic myeloid leukemia (CML). The BCR-ABLT315I mutant is highly resistant to imatinib, nilotinib, and dasatinib and is frequently detected in relapsed patients. This critical gap in resistance coverage drove development of DCC-2036, an ABL inhibitor which binds the switch control pocket involved in conformational regulation of the kinase domain. We evaluated the efficacy of DCC-2036 against BCR-ABLT315I and other mutants in cellular and biochemical assays and conducted cell-based mutagenesis screens. DCC-2036 inhibited autophosphorylation of ABL and ABLT315I enzymes, and this activity was consistent with selective efficacy against Ba/F3 cells expressing BCR-ABL (IC50: 19 nmol/L), BCR-ABLT315I (IC50: 63 nmol/L), and most kinase domain mutants. Ex vivo exposure of CML cells from patients harboring BCR-ABL or BCR-ABLT315I to DCC-2036 revealed marked inhibition of colony formation and reduced phosphorylation of the direct BCR-ABL target CrkL. Cell-based mutagenesis screens identified a resistance profile for DCC-2036 centered around select P-loop mutations (G250E, Q252H, Y253H, E255K/V), although a concentration of 750 nmol/L DCC-2036 suppressed the emergence of all resistant clones. A decreased concentration of DCC-2036 (160 nmol/L) in dual-combination with either nilotinib or dasatinib achieved the same zero outgrowth result. Further screens for resistance due to BCR-ABL compound mutations (two mutations in the same clone) identified BCR-ABLE255V / T315I as the most resistant mutant. Taken together, these findings support continued evaluation of DCC-2036 as an important new agent for treatment-refractory CML.
doi:10.1158/0008-5472.CAN-10-3224
PMCID: PMC3206627  PMID: 21505103
BCR-ABL; imatinib resistance; DCC-2036
24.  Pain behavior in the formalin test persists after ablation of the great majority of C-fiber nociceptors 
Pain  2010;151(2):422-429.
Although the formalin test is a widely used model of persistent pain, the primary afferent fiber types that underlie the cellular and behavioral responses to formalin injection are largely unknown. Here we used a combined genetic and pharmacological approach to investigate the effect of ablating subsets of primary afferent nociceptors on formalin-induced nocifensive behaviors and spinal cord Fos protein expression. Intrathecal capsaicin-induced ablation of the central terminals of TRPV1+ neurons greatly reduced the behavioral responses and Fos elicited by low-dose (0.5%) formalin. In contrast, genetic ablation of the MrgprD-expressing subset of nonpeptidergic unmyelinated afferents, which constitute a largely non-overlapping population, altered neither the behavior nor the Fos induced by low-dose formalin. Remarkably, nocifensive behavior following high-dose (2%) formalin was unchanged in mice lacking either afferent population, or even in mice lacking both populations, which together make up the great majority of C-fiber nociceptors. Thus, at high doses, which are routinely used in the formalin test, formalin-induced “pain” behavior persists in the absence of the vast majority of C-fiber nociceptors, which points to a contribution of a large spectrum of afferents secondary to non-specific formalin-induced tissue and nerve damage.
doi:10.1016/j.pain.2010.08.001
PMCID: PMC2955806  PMID: 20832171
25.  Functional identification of an aggression locus in the mouse hypothalamus 
Nature  2011;470(7333):221-226.
Electrical stimulation of certain hypothalamic regions in cats and rodents can elicit attack behavior, but the exact location of relevant cells within these regions, their requirement for naturally occurring aggression and their relationship to mating circuits have not been clear. Genetic methods for neural circuit manipulation in mice provide a potentially powerful approach to this problem, but brain stimulation-evoked aggression has never been demonstrated in this species. Here we show that optogenetic, but not electrical, stimulation of neurons in the ventromedial hypothalamus, ventrolateral subdivision (VMHvl) causes male mice to attack both females and inanimate objects, as well as males. Pharmacogenetic silencing of VMHvl reversibly inhibits inter-male aggression. Immediate early gene analysis and single unit recordings from VMHvl during social interactions reveal overlapping but distinct neuronal subpopulations involved in fighting and mating. Neurons activated during attack are inhibited during mating, suggesting a potential neural substrate for competition between these behaviors.
doi:10.1038/nature09736
PMCID: PMC3075820  PMID: 21307935

Results 1-25 (48)