Caterpillar-induced nocturnal plant volatiles repel conspecific females

Plants respond to insect herbivory by synthesizing and releasing complex blends of volatile compounds, which provide important host-location cues for insects that are natural enemies of herbivores. The effects of these volatile blends on herbivore behaviour have been investigated to only a limited extent, in part because of the assumption that herbivore-induced volatile emissions occur mainly during the light phase of the photoperiod. Because many moths-whose larvae are some of the most important insect herbivores-are nocturnal, herbivore-induced plant volatiles have not hitherto been considered to be temporally available as host-location cues for ovipositing females. Here we present chemical and behavioural assays showing that tobacco plants (Nicotiana tabacum) release herbivore-induced volatiles during both night and day. Moreover, several volatile compounds are released exclusively at night and are highly repellent to female moths (Heliothis virescens). The demonstration that tobacco plants release temporally different volatile blends and that lepidopteran herbivores use induced plant signals released during the dark phase to choose sites for oviposition adds a new dimension to our understanding of the role of chemical cues in mediating tritrophic interactions.     

Disoriented pathfinding by pioneer neurone growth cones deprived of filopodia by cytochalasin treatment

A major question in developmental neurobiology is how developing nerve cells accurately extend processes to establish connections with their target cells. This problem involves both the nature of cues for growth cone guidance and also the question of how growth cones survey their environment for cues and respond by altering their direction of migration. The filopodia which normally extend from neuronal growth cones have been shown to affect growth cone steering in vitro and it has been proposed that they function in vivo in the detection of and response to guidance cues. This hypothesis could be tested in vivo if growth cones which normally have filopodia could be induced to migrate in their absence. The pair of Ti1 neurones are the first neurones to extend axons through the limb buds of embryonic grasshoppers. We report here an examination of the migration of Ti1 pioneer growth cones deprived of filopodia by culture in agents which disrupt actin microfilaments. Under these conditions, axons continue to extend but a large percentage of growth cones are highly disoriented. Our results indicate that Ti1 filopodia are not necessary for axonal elongation in vivo but that they are important for correctly oriented growth cone steering.     

Trans-sexually grafted antennae alter pheromone-directed behaviour in a moth

When tobacco hornworm moths (Manduca sexta) are tested in a wind tunnel with a source of female pheromones upwind, males but not normal females show pheromone-modulated anemotaxis and a characteristic mate-seeking behavioural sequence. These behaviours are produced by stimulation of sensory neurones found only in male antennae. These neurones project axons only to dendrites of pheromone-specific interneurones in the macroglomerular complex, a region of neuropil in the antennal lobe characteristic of males but not present in normal females. Some interneurones in the antennal lobes of female moths that have received grafts of male antennae (gynandromorphs) respond postsynaptically to stimulation with bombykal, a major component of the pheromone. They branch into a region resembling the macroglomerular complex, like their counterparts in normal males. We show here that gynandromorphic females respond to pheromonal stimulation with anemotaxis. We also find that normal females display a similar sequence in response to the odour of their egg-laying site, the tobacco plant. It is likely that a common motor path is used either by pheromone-specific interneurones in the antennal lobes of males or by tobacco-specific interneurones in females. We assume that the interneurones in gynandromorphic females that branch into the macroglomerular complex induced by a grafted male antenna can activate this pathway.     

Perception of sniff phase in mouse olfaction

Olfactory systems encode odours by which neurons respond and by when they respond. In mammals, every sniff evokes a precise, odour-specific sequence of activity across olfactory neurons. Likewise, in a variety of neural systems, ranging from sensory periphery to cognitive centres, neuronal activity is timed relative to sampling behaviour and/or internally generated oscillations. As in these neural systems, relative timing of activity may represent information in the olfactory system. However, there is no evidence that mammalian olfactory systems read such cues. To test whether mice perceive the timing of olfactory activation relative to the sniff cycle ('sniff phase'), we used optogenetics in gene-targeted mice to generate spatially constant, temporally controllable olfactory input. Here we show that mice can behaviourally report the sniff phase of optogenetically driven activation of olfactory sensory neurons. Furthermore, mice can discriminate between light-evoked inputs that are shifted in the sniff cycle by as little as 10 milliseconds, which is similar to the temporal precision of olfactory bulb odour responses. Electrophysiological recordings in the olfactory bulb of awake mice show that individual cells encode the timing of photoactivation in relation to the sniff in both the timing and the amplitude of their responses. Our work provides evidence that the mammalian olfactory system can read temporal patterns, and suggests that timing of activity relative to sampling behaviour is a potent cue that may enable accurate olfactory percepts to form quickly.     

Decisions about parental care in response to perceived paternity

Evolutionary ecologists are attempting to explain how parents make behavioural decisions about how much care to provide to their young. Theory predicts that when genetic relatedness to young is decreased by cuckoldry, for example, parents should reduce their care in favour of alternative broods that provide greater reproductive success. Experimental manipulation of perceived paternity has been used to test the theory, but such studies have generated mixed results. Some manipulations can fail to alter a parent's perceived paternity, whereas others may directly affect parental behaviour when, for instance, the manipulation involves capturing the parent. No study has demonstrated parental care adjustment in a manner uncomplicated by experimental design or life history correlates. Here I test the theory using the fact that nest-tending parental male bluegill sunfish (Lepomis macrochirus) can assess their paternity using both the visual presence of parasitic cuckolder males during spawning, and olfactory cues released by newly hatched eggs. By manipulating both types of cues I show that parental males dynamically adjust their parental care, favouring broods that are apparently most closely related. These results confirm the importance of genetic relatedness in parental care decision-making.     

Co-release of glutamate and aspartate from cholinergic and GABAergic synaptosomes

Glutamate and aspartate are known to be released in a calcium-dependent fashion by depolarizing stimulation of mammalian brain synaptosomes (isolated nerve endings), an observation which strengthens their claims to be neurotransmitter candidates. The source of these compounds has been interpreted as the exclusively glutamatergic or aspartatergic synaptosome sub-populations assumed to be present in the standard heterogeneous preparations from mammalian brain. Several neurotransmitter-specific synaptosomal surface markers have recently been identified by immunolysis studies and these have allowed separation of subpopulations of synaptosomes by an affinity purification method. These markers appear to be closely related to the biosynthetic enzyme for the principal neurotransmitter released by each sub-category of synaptosome. We have isolated highly purified, metabolically active, GABAergic and cholinergic synaptosomes from cerebral cortex using antisera recognizing either glutamate decarboxylase (GAD) or choline acetyltransferase (ChAT), in conjunction with magnetic microspheres covalently coupled to Protein A (ref. 8), and now report that these synaptosomes release both glutamate and aspartate, in addition to their principal neurotransmitter, when treated with chemical depolarizing agents.     

Functional consequences of a CKIdelta mutation causing familial advanced sleep phase syndrome

Familial advanced sleep phase syndrome (FASPS) is a human behavioural phenotype characterized by early sleep times and early-morning awakening. It was the first human, mendelian circadian rhythm variant to be well-characterized, and was shown to result from a mutation in a phosphorylation site within the casein kinase I (CKI)-binding domain of the human PER2 gene. To gain a deeper understanding of the mechanisms of circadian rhythm regulation in humans, we set out to identify mutations in human subjects leading to FASPS. We report here the identification of a missense mutation (T44A) in the human CKIdelta gene, which results in FASPS. This mutant kinase has decreased enzymatic activity in vitro. Transgenic Drosophila carrying the human CKIdelta-T44A gene showed a phenotype with lengthened circadian period. In contrast, transgenic mice carrying the same mutation have a shorter circadian period, a phenotype mimicking human FASPS. These results show that CKIdelta is a central component in the mammalian clock, and suggest that mammalian and fly clocks might have different regulatory mechanisms despite the highly conserved nature of their individual components.     

Dopamine neurons modulate pheromone responses in Drosophila courtship learning

Learning through trial-and-error interactions allows animals to adapt innate behavioural ‘rules of thumb’ to the local environment, improving their prospects for survival and reproduction. Naive Drosophila melanogaster males, for example, court both virgin and mated females, but learn through experience to selectively suppress futile courtship towards females that have already mated. Here we show that courtship learning reflects an enhanced response to the male pheromone cis-vaccenyl acetate (cVA), which is deposited on females during mating and thus distinguishes mated females from virgins. Dissociation experiments suggest a simple learning rule in which unsuccessful courtship enhances sensitivity to cVA. The learning experience can be mimicked by artificial activation of dopaminergic neurons, and we identify a specific class of dopaminergic neuron that is critical for courtship learning. These neurons provide input to the mushroom body (MB) γ lobe, and the DopR1 dopamine receptor is required in MBγ neurons for both natural and artificial courtship learning. Our work thus reveals critical behavioural, cellular and molecular components of the learning rule by which Drosophila adjusts its innate mating strategy according to experience.     

Modulation of TRPA1 thermal sensitivity enables sensory discrimination in Drosophila

Discriminating among sensory stimuli is critical for animal survival. This discrimination is particularly essential when evaluating whether a stimulus is noxious or innocuous. From insects to humans, transient receptor potential (TRP) channels are key transducers of thermal, chemical and other sensory cues. Many TRPs are multimodal receptors that respond to diverse stimuli, but how animals distinguish sensory inputs activating the same TRP is largely unknown. Here we determine how stimuli activating Drosophila TRPA1 are discriminated. Although Drosophila TRPA1 responds to both noxious chemicals and innocuous warming, we find that TRPA1-expressing chemosensory neurons respond to chemicals but not warmth, a specificity conferred by a chemosensory-specific TRPA1 isoform with reduced thermosensitivity compared to the previously described isoform. At the molecular level, this reduction results from a unique region that robustly reduces the channel's thermosensitivity. Cell-type segregation of TRPA1 activity is critical: when the thermosensory isoform is expressed in chemosensors, flies respond to innocuous warming with regurgitation, a nocifensive response. TRPA1 isoform diversity is conserved in malaria mosquitoes, indicating that similar mechanisms may allow discrimination of host-derived warmth--an attractant--from chemical repellents. These findings indicate that reducing thermosensitivity can be critical for TRP channel functional diversification, facilitating their use in contexts in which thermal sensitivity can be maladaptive.     

Expression of a mammalian Na-H exchanger in muscle fibres of the giant barnacle

It is now well established that the internal pH (pHi) of mammalian cells is regulated by means of a plasma membrane transport system that exchanges extracellular Na+ for intracellular H+ (ref. 1). Furthermore, modulation of the activity of the Na-H exchanger seems to have a crucial role in the action of various mitogens and growth factors. The possibility that such a mammalian Na-H exchanger might be efficiently expressed in a giant invertebrate cell was suggested to us by recent results of Barnard and Miledi and colleagues, who demonstrated in frog oocytes the expression of various plasma membrane channels that presumably were encoded by the mammalian messenger RNA wih which the oocytes had been injected. We used muscle fibres of the giant barnacle, which normally have no demonstrable Na-H exchanger activity, and report here that, when injected with poly(A)+ RNA isolated from rabbit liver, the muscle fibres express a Na-H exchanger. No such expression is observed, however, when the injected material is pretreated with ribonuclease A. As hepatocytes are known to possess a Na-H exchanger, the most straightforward interpretation of our data is that a mammalian Na-H exchanger has been expressed in the muscle fibre of an invertebrate.     

Protein methylation in behavioural control mechanisms and in signal transduction.

In both prokaryotes and eukaryotes methyl groups can be added to and removed from the carboxyl groups of proteins. Recent work has revealed that these reactions have a role in several behavioural phenomena. The nature of this role has been uncovered in one case--that of bacterial chemotaxis.     

A receptor that mediates the post-mating switch in Drosophila reproductive behaviour

Mating in many species induces a dramatic switch in female reproductive behaviour. In most insects, this switch is triggered by factors present in the male's seminal fluid. How these factors exert such profound effects in females is unknown. Here we identify a receptor for the Drosophila melanogaster sex peptide (SP, also known as Acp70A), the primary trigger of post-mating responses in this species. Females that lack the sex peptide receptor (SPR, also known as CG16752), either entirely or only in the nervous system, fail to respond to SP and continue to show virgin behaviours even after mating. SPR is expressed in the female's reproductive tract and central nervous system. The behavioural functions of SPR map to the subset of neurons that also express the fruitless gene, a key determinant of sex-specific reproductive behaviour. SPR is highly conserved across insects, opening up the prospect of new strategies to control the reproductive and host-seeking behaviours of agricultural pests and human disease vectors.     

Neuropeptides modulate the beta-adrenergic response of purified astrocytes in vitro

Neuropeptides may have functions in the central nervous system (CNS) other than altering neuronal excitability. For example, they may act as regulators of brain metabolism by affecting glycogenolysis. Since it has been suggested that glial cells might provide metabolic support for neuronal activity, they may well be one of the targets for neuropeptide regulation of metabolism. Consistent with this view are reports that peptide-containing nerve terminals have been seen apposed to astrocytes, but it is also quite possible that peptides could act at sites lacking morphological specialization. Primary cultures containing CNS glial cells have been shown to respond to beta-adrenergic agonists with an increase in cyclic AMP and, as a result, with an increase in glycogenolysis and have also been shown to respond to a variety of peptides with changes in cyclic AMP. In the study reported here, we have examined the effects of several peptides on relatively pure cultures of rat astrocytes. We demonstrate that the increase in intracellular cyclic AMP induced by noradrenaline is markedly enhanced by somatostatin and substance P and is inhibited by enkephalin, even though these peptides on their own have little or no effect on the basal levels of cyclic AMP. Vasoactive intestinal peptide (VIP) on the other hand increases cyclic AMP in the absence of noradrenaline. These results suggest that neuropeptides influence glial cells as well as neurones in the CNS and, in the case of somatostatin and substance P, provide further examples of neuropeptides modulating the response to another chemical signal without having a detectable action on their own.     

Forebrain peptides modulate sexually polymorphic vocal circuitry

The peptide arginine-vasopressin (mammals) and its evolutionary precursor arginine-vasotocin (non-mammals) modulate reproductive physiology and numerous related social behaviours, as do oxytocin (mammals) and its homologues mesotocin and isotocin (fish). The distributions in the brain and/or the behavioural functions of these peptides often differ between the sexes, and between species with divergent social structures. Here we present neurophysiological evidence that males with vocal characteristics typical of females share a pattern of neuropeptide function with females rather than conspecific males. The plainfin midshipman fish (Porichthys notatus) has two male morphs with different reproductive tactics and vocalizations (a key species-typical behaviour which varies in its physical attributes and contextual usage, depending on the morph's social strategy). Forebrain-evoked, rhythmic vocal-motor activity that precisely mimics natural vocalizations was modulated by arginine-vasotocin, isotocin and their antagonists delivered to the preoptic area-anterior hypothalamus, a primary site for behavioural integration in all vertebrates. Peptides had different effects in males that acoustically court females (arginine-vasotocin-sensitive) than in females and sneak-spawning males (isotocin-sensitive), showing that the neuromodulatory mechanisms that establish reproduction-related behaviour can be dissociated from gonadal sex.     

Cryptochrome mediates light-dependent magnetosensitivity in Drosophila

Although many animals use the Earth's magnetic field for orientation and navigation, the precise biophysical mechanisms underlying magnetic sensing have been elusive. One theoretical model proposes that geomagnetic fields are perceived by chemical reactions involving specialized photoreceptors. However, the specific photoreceptor involved in such magnetoreception has not been demonstrated conclusively in any animal. Here we show that the ultraviolet-A/blue-light photoreceptor cryptochrome (Cry) is necessary for light-dependent magnetosensitive responses in Drosophila melanogaster. In a binary-choice behavioural assay for magnetosensitivity, wild-type flies show significant naive and trained responses to a magnetic field under full-spectrum light ( approximately 300-700 nm) but do not respond to the field when wavelengths in the Cry-sensitive, ultraviolet-A/blue-light part of the spectrum (<420 nm) are blocked. Notably, Cry-deficient cry(0) and cry(b) flies do not show either naive or trained responses to a magnetic field under full-spectrum light. Moreover, Cry-dependent magnetosensitivity does not require a functioning circadian clock. Our work provides, to our knowledge, the first genetic evidence for a Cry-based magnetosensitive system in any animal.     

Inhibition of shoot branching by new terpenoid plant hormones

Shoot branching is a major determinant of plant architecture and is highly regulated by endogenous and environmental cues. Two classes of hormones, auxin and cytokinin, have long been known to have an important involvement in controlling shoot branching. Previous studies using a series of mutants with enhanced shoot branching suggested the existence of a third class of hormone(s) that is derived from carotenoids, but its chemical identity has been unknown. Here we show that levels of strigolactones, a group of terpenoid lactones, are significantly reduced in some of the branching mutants. Furthermore, application of strigolactones inhibits shoot branching in these mutants. Strigolactones were previously found in root exudates acting as communication chemicals with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Thus, we propose that strigolactones act as a new hormone class-or their biosynthetic precursors-in regulating above-ground plant architecture, and also have a function in underground communication with other neighbouring organisms.     

A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone

Insects, like many other animals, use sex pheromones to coordinate their reproductive behaviours. Volatile pheromones are detected by odorant receptors expressed in olfactory receptor neurons (ORNs). Whereas fruit odours typically activate multiple ORN classes, pheromones are thought to act through single dedicated classes of ORN. This model predicts that activation of such an ORN class should be sufficient to trigger the appropriate behavioural response. Here we show that the Drosophila melanogaster male-specific pheromone 11-cis-vaccenyl acetate (cVA) acts through the receptor Or67d to regulate both male and female mating behaviour. Mutant males that lack Or67d inappropriately court other males, whereas mutant females are less receptive to courting males. These data suggest that cVA has opposite effects in the two sexes: inhibiting mating behaviour in males but promoting mating behaviour in females. Replacing Or67d with moth pheromone receptors renders these ORNs sensitive to the corresponding moth pheromones. In such flies, moth pheromones elicit behavioural responses that mimic the normal response to cVA. Thus, activation of a single ORN class is both necessary and sufficient to mediate behavioural responses to the Drosophila sex pheromone cVA.     

Subsecond dopamine release promotes cocaine seeking

The dopamine-containing projection from the ventral tegmental area of the midbrain to the nucleus accumbens is critically involved in mediating the reinforcing properties of cocaine. Although neurons in this area respond to rewards on a subsecond timescale, neurochemical studies have only addressed the role of dopamine in drug addiction by examining changes in the tonic (minute-to-minute) levels of extracellular dopamine. To investigate the role of phasic (subsecond) dopamine signalling, we measured dopamine every 100 ms in the nucleus accumbens using electrochemical technology. Rapid changes in extracellular dopamine concentration were observed at key aspects of drug-taking behaviour in rats. Before lever presses for cocaine, there was an increase in dopamine that coincided with the initiation of drug-seeking behaviours. Notably, these behaviours could be reproduced by electrically evoking dopamine release on this timescale. After lever presses, there were further increases in dopamine concentration at the concurrent presentation of cocaine-related cues. These cues alone also elicited similar, rapid dopamine signalling, but only in animals where they had previously been paired to cocaine delivery. These findings reveal an unprecedented role for dopamine in the regulation of drug taking in real time.     

An amino-acid taste receptor

The sense of taste provides animals with valuable information about the nature and quality of food. Mammals can recognize and respond to a diverse repertoire of chemical entities, including sugars, salts, acids and a wide range of toxic substances. Several amino acids taste sweet or delicious (umami) to humans, and are attractive to rodents and other animals. This is noteworthy because L-amino acids function as the building blocks of proteins, as biosynthetic precursors of many biologically relevant small molecules, and as metabolic fuel. Thus, having a taste pathway dedicated to their detection probably had significant evolutionary implications. Here we identify and characterize a mammalian amino-acid taste receptor. This receptor, T1R1+3, is a heteromer of the taste-specific T1R1 and T1R3 G-protein-coupled receptors. We demonstrate that T1R1 and T1R3 combine to function as a broadly tuned L-amino-acid sensor responding to most of the 20 standard amino acids, but not to their D-enantiomers or other compounds. We also show that sequence differences in T1R receptors within and between species (human and mouse) can significantly influence the selectivity and specificity of taste responses.