Rapid threat assessment in the Drosophila thermosensory system.

Neurons that participate in sensory processing often display "ON" responses, i.e., fire transiently at the onset of a stimulus. ON transients are widespread, perhaps universal to sensory coding, yet their function is not always well-understood. Here, we show that ON responses in the Drosophila thermosensory system extrapolate the trajectory of temperature change, priming escape behavior if unsafe thermal conditions are imminent. First, we show that second-order thermosensory projection neurons (TPN-IIIs) and their Lateral Horn targets (TLHONs), display ON responses to thermal stimuli, independent of direction of change (heating or cooling) and of absolute temperature. Instead, they track the rate of temperature change, with TLHONs firing exclusively to rapid changes (>0.2 °C/s). Next, we use connectomics to track TLHONs' output to descending neurons that control walking and escape, and modeling and genetic silencing to demonstrate how ON transients can flexibly amplify aversive responses to small thermal change. Our results suggest that, across sensory systems, ON transients may represent a general mechanism to systematically anticipate and respond to salient or dangerous conditions.

A thermometer circuit for hot temperature adjusts Drosophila behavior to persistent heat.

Small poikilotherms such as the fruit fly Drosophila depend on absolute temperature measurements to identify external conditions that are above (hot) or below (cold) their preferred range and to react accordingly. Hot and cold temperatures have a different impact on fly activity and sleep, but the circuits and mechanisms that adjust behavior to specific thermal conditions are not well understood. Here, we use patch-clamp electrophysiology to show that internal thermosensory neurons located within the fly head capsule (the AC neurons1) function as a thermometer active in the hot range. ACs exhibit sustained firing rates that scale with absolute temperature-but only for temperatures above the fly's preferred ∼25°C (i.e., "hot" temperature). We identify ACs in the fly brain connectome and demonstrate that they target a single class of circadian neurons, the LPNs.2 LPNs receive excitatory drive from ACs and respond robustly to hot stimuli, but their responses do not exclusively rely on ACs. Instead, LPNs receive independent drive from thermosensory neurons of the fly antenna via a new class of second-order projection neurons (TPN-IV). Finally, we show that silencing LPNs blocks the restructuring of daytime "siesta" sleep, which normally occurs in response to persistent heat. Our previous work described a distinct thermometer circuit for cold temperature.3 Together, the results demonstrate that the fly nervous system separately encodes and relays absolute hot and cold temperature information, show how patterns of sleep and activity can be adapted to specific temperature conditions, and illustrate how persistent drive from sensory pathways can impact behavior on extended temporal scales.

Recovery from cold-induced reproductive dormancy is regulated by temperature-dependent AstC signaling.

Animals have evolved a variety of behaviors to cope with adverse environmental conditions. Similar to other insects, the fly, Drosophila melanogaster, responds to sustained cold by reducing its metabolic rate and arresting its reproduction. Here, we show that a subset of dorsal neurons (DN3s) that express the neuropeptide allatostatin C (AstC) facilitates recovery from cold-induced reproductive dormancy. The activity of AstC-expressing DN3s, as well as AstC peptide levels, are suppressed by cold. Cold temperature also impacts AstC levels in other Drosophila species and mosquitoes, Aedes aegypti, and Anopheles stephensi. The stimulatory effect of AstC on egg production is mediated by cholinergic AstC-R2 neurons. Our results demonstrate that DN3s coordinate female reproductive capacity with environmental temperature via AstC signaling. AstC/AstC-R2 is conserved across many insect species and their role in regulating female reproductive capacity makes them an ideal target for controlling the population of agricultural pests and human disease vectors.

Sensory biology: The bitter aftertaste.

Bitter taste signals a potentially toxic food that should be avoided. A new study shows that taste neurons in Drosophila produce distinct responses after a bitter sip. A bitter aftertaste may help the fly make wise food choices.

Robustness and plasticity in Drosophila heat avoidance.

Simple innate behavior is often described as hard-wired and largely inflexible. Here, we show that the avoidance of hot temperature, a simple innate behavior, contains unexpected plasticity in Drosophila. First, we demonstrate that hot receptor neurons of the antenna and their molecular heat sensor, Gr28B.d, are essential for flies to produce escape turns away from heat. High-resolution fly tracking combined with a 3D simulation of the thermal environment shows that, in steep thermal gradients, the direction of escape turns is determined by minute temperature differences between the antennae (0.1°-1 °C). In parallel, live calcium imaging confirms that such small stimuli reliably activate both peripheral thermosensory neurons and central circuits. Next, based on our measurements, we evolve a fly/vehicle model with two symmetrical sensors and motors (a "Braitenberg vehicle") which closely approximates basic fly thermotaxis. Critical differences between real flies and the hard-wired vehicle reveal that fly heat avoidance involves decision-making, relies on rapid learning, and is robust to new conditions, features generally associated with more complex behavior.

Descending Dopaminergic Inputs to Reticulospinal Neurons Promote Locomotor Movements.

Meso-diencephalic dopaminergic neurons are known to modulate locomotor behaviors through their ascending projections to the basal ganglia, which in turn project to the mesencephalic locomotor region, known to control locomotion in vertebrates. In addition to their ascending projections, dopaminergic neurons were found to increase locomotor movements through direct descending projections to the mesencephalic locomotor region and spinal cord. Intriguingly, fibers expressing tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis, were also observed around reticulospinal neurons of lampreys. We now examined the origin and the role of this innervation. Using immunofluorescence and tracing experiments, we found that fibers positive for dopamine innervate reticulospinal neurons in the four reticular nuclei of lampreys. We identified the dopaminergic source using tracer injections in reticular nuclei, which retrogradely labeled dopaminergic neurons in a caudal diencephalic nucleus (posterior tuberculum [PT]). Using voltammetry in brain preparations isolated in vitro, we found that PT stimulation evoked dopamine release in all four reticular nuclei, but not in the spinal cord. In semi-intact preparations where the brain is accessible and the body moves, PT stimulation evoked swimming, and injection of a D1 receptor antagonist within the middle rhombencephalic reticular nucleus was sufficient to decrease reticulospinal activity and PT-evoked swimming. Our study reveals that dopaminergic neurons have access to command neurons that integrate sensory and descending inputs to activate spinal locomotor neurons. As such, our findings strengthen the idea that dopamine can modulate locomotor behavior both via ascending projections to the basal ganglia and through descending projections to brainstem motor circuits.SIGNIFICANCE STATEMENT Meso-diencephalic dopaminergic neurons play a key role in modulating locomotion by releasing dopamine in the basal ganglia, spinal networks, and the mesencephalic locomotor region, a brainstem region that controls locomotion in a graded fashion. Here, we report in lampreys that dopaminergic neurons release dopamine in the four reticular nuclei where reticulospinal neurons are located. Reticulospinal neurons integrate sensory and descending suprareticular inputs to control spinal interneurons and motoneurons. By directly modulating the activity of reticulospinal neurons, meso-diencephalic dopaminergic neurons control the very last instructions sent by the brain to spinal locomotor circuits. Our study reports on a new direct descending dopaminergic projection to reticulospinal neurons that modulates locomotor behavior.

A Circuit Encoding Absolute Cold Temperature in Drosophila.

Animals react to environmental changes over timescales ranging from seconds to days and weeks. An important question is how sensory stimuli are parsed into neural signals operating over such diverse temporal scales. Here, we uncover a specialized circuit, from sensory neurons to higher brain centers, that processes information about long-lasting, absolute cold temperature in Drosophila. We identify second-order thermosensory projection neurons (TPN-IIs) exhibiting sustained firing that scales with absolute temperature. Strikingly, this activity only appears below the species-specific, preferred temperature for D. melanogaster (∼25°C). We trace the inputs and outputs of TPN-IIs and find that they are embedded in a cold "thermometer" circuit that provides powerful and persistent inhibition to brain centers involved in regulating sleep and activity. Our results demonstrate that the fly nervous system selectively encodes and relays absolute temperature information and illustrate a sensory mechanism that allows animals to adapt behavior specifically to cold conditions on the timescale of hours to days.

A descending dopamine pathway conserved from basal vertebrates to mammals.

Dopamine neurons are classically known to modulate locomotion indirectly through ascending projections to the basal ganglia that project down to brainstem locomotor networks. Their loss in Parkinson's disease is devastating. In lampreys, we recently showed that brainstem networks also receive direct descending dopaminergic inputs that potentiate locomotor output. Here, we provide evidence that this descending dopaminergic pathway is conserved to higher vertebrates, including mammals. In salamanders, dopamine neurons projecting to the striatum or brainstem locomotor networks were partly intermingled. Stimulation of the dopaminergic region evoked dopamine release in brainstem locomotor networks and concurrent reticulospinal activity. In rats, some dopamine neurons projecting to the striatum also innervated the pedunculopontine nucleus, a known locomotor center, and stimulation of the dopaminergic region evoked pedunculopontine dopamine release in vivo. Finally, we found dopaminergic fibers in the human pedunculopontine nucleus. The conservation of a descending dopaminergic pathway across vertebrates warrants re-evaluating dopamine's role in locomotion.

Dynamic labelling of neural connections in multiple colours by trans-synaptic fluorescence complementation.

Determining the pattern of activity of individual connections within a neural circuit could provide insights into the computational processes that underlie brain function. Here, we develop new strategies to label active synapses by trans-synaptic fluorescence complementation in Drosophila. First, we demonstrate that a synaptobrevin-GRASP chimera functions as a powerful activity-dependent marker for synapses in vivo. Next, we create cyan and yellow variants, achieving activity-dependent, multi-colour fluorescence reconstitution across synapses (X-RASP). Our system allows for the first time retrospective labelling of synapses (rather than whole neurons) based on their activity, in multiple colours, in the same animal. As individual synapses often act as computational units in the brain, our method will promote the design of experiments that are not possible using existing techniques. Moreover, our strategies are easily adaptable to circuit mapping in any genetic system.

A synaptic mechanism for network synchrony.

Within neural networks, synchronization of activity is dependent upon the synaptic connectivity of embedded microcircuits and the intrinsic membrane properties of their constituent neurons. Synaptic integration, dendritic Ca(2+) signaling, and non-linear interactions are crucial cellular attributes that dictate single neuron computation, but their roles promoting synchrony and the generation of network oscillations are not well understood, especially within the context of a defined behavior. In this regard, the lamprey spinal central pattern generator (CPG) stands out as a well-characterized, conserved vertebrate model of a neural network (Smith et al., 2013a), which produces synchronized oscillations in which neural elements from the systems to cellular level that control rhythmic locomotion have been determined. We review the current evidence for the synaptic basis of oscillation generation with a particular emphasis on the linkage between synaptic communication and its cellular coupling to membrane processes that control oscillatory behavior of neurons within the locomotor network. We seek to relate dendritic function found in many vertebrate systems to the accessible lamprey central nervous system in which the relationship between neural network activity and behavior is well understood. This enables us to address how Ca(2+) signaling in spinal neuron dendrites orchestrate oscillations that drive network behavior.

Forebrain dopamine neurons project down to a brainstem region controlling locomotion.

The contribution of dopamine (DA) to locomotor control is traditionally attributed to ascending dopaminergic projections from the substantia nigra pars compacta and the ventral tegmental area to the basal ganglia, which in turn project down to the mesencephalic locomotor region (MLR), a brainstem region controlling locomotion in vertebrates. However, a dopaminergic innervation of the pedunculopontine nucleus, considered part of the MLR, was recently identified in the monkey. The origin and role of this dopaminergic input are unknown. We addressed these questions in a basal vertebrate, the lamprey. Here we report a functional descending dopaminergic pathway from the posterior tuberculum (PT; homologous to the substantia nigra pars compacta and/or ventral tegmental area of mammals) to the MLR. By using triple labeling, we found that dopaminergic cells from the PT not only project an ascending pathway to the striatum, but send a descending projection to the MLR. In an isolated brain preparation, PT stimulation elicited excitatory synaptic inputs into patch-clamped MLR cells, accompanied by activity in reticulospinal cells. By using voltammetry coupled with electrophysiological recordings, we demonstrate that PT stimulation evoked DA release in the MLR, together with the activation of reticulospinal cells. In a semi-intact preparation, stimulation of the PT elicited reticulospinal activity together with locomotor movements. Microinjections of a D1 antagonist in the MLR decreased the locomotor output elicited by PT stimulation, whereas injection of DA had an opposite effect. It appears that this descending dopaminergic pathway has a modulatory role on MLR cells that are known to receive glutamatergic projections and promotes locomotor output.

Synaptic NMDA receptor-dependent Ca²âº entry drives membrane potential and Ca²âº oscillations in spinal ventral horn neurons.

During vertebrate locomotion, spinal neurons act as oscillators when initiated by glutamate release from descending systems. Activation of NMDA receptors initiates Ca²âº-mediated intrinsic membrane potential oscillations in central pattern generator (CPG) neurons. NMDA receptor-dependent intrinsic oscillations require Ca²âº-dependent K⁺ (K(Ca)2) channels for burst termination. However, the location of Ca²âº entry mediating K(Ca)2 channel activation, and type of Ca²âº channel--which includes NMDA receptors and voltage-gated Ca²âº channels (VGCCs)--remains elusive. NMDA receptor-dependent Ca²âº entry necessitates presynaptic release of glutamate, implying a location at active synapses within dendrites, whereas VGCC-dependent Ca²âº entry is not similarly constrained. Where Ca²âº enters relative to K(Ca)2 channels is crucial to information processing of synaptic inputs necessary to coordinate locomotion. We demonstrate that Ca²âº permeating NMDA receptors is the dominant source of Ca²âº during NMDA-dependent oscillations in lamprey spinal neurons. This Ca²âº entry is synaptically located, NMDA receptor-dependent, and sufficient to activate K(Ca)2 channels at excitatory interneuron synapses onto other CPG neurons. Selective blockade of VGCCs reduces whole-cell Ca²âº entry but leaves membrane potential and Ca²âº oscillations unaffected. Furthermore, repetitive oscillations are prevented by fast, but not slow, Ca²âº chelation. Taken together, these results demonstrate that K(Ca)2 channels are closely located to NMDA receptor-dependent Ca²âº entry. The close spatial relationship between NMDA receptors and K(Ca)2 channels provides an intrinsic mechanism whereby synaptic excitation both excites and subsequently inhibits ventral horn neurons of the spinal motor system. This places the components necessary for oscillation generation, and hence locomotion, at glutamatergic synapses.

Differential regulation of synaptic transmission by pre- and postsynaptic SK channels in the spinal locomotor network.

The generation of activity in the central nervous system requires precise tuning of cellular properties and synaptic transmission. Neural networks in the spinal cord produce coordinated locomotor movements. Synapses in these networks need to be equipped with multiple mechanisms that regulate their operation over varying regimes to produce locomotor activity at different frequencies. Using the in vitro lamprey spinal cord, we explored whether Ca(2+) influx via different routes in postsynaptic soma and dendrites and in presynaptic terminals can activate apamin-sensitive Ca(2+)-activated K(+) (SK) channels and thereby shape synaptic transmission. We show that postsynaptic SK channels are tightly coupled to Ca(2+) influx via NMDA receptors. Activation of these channels by synaptically induced NMDA-dependent Ca(2+) transients restrains the time course of the synaptic current and the amplitude of the synaptic potential. In addition, presynaptic SK channels are activated by Ca(2+) influx via voltage-gated channels and control the waveform of the action potential and the resulting Ca(2+) dynamics in the axon terminals. The coupling of SK channels to different Ca(2+) sources, pre- and postsynaptically, acts as a negative feedback mechanism to shape synaptic transmission. Thus SK channels can play a pivotal role in setting the dynamic range of synapses and enabling short-term plasticity in the spinal locomotor network.

Nitric oxide modulation of the electrically excitable skin of Xenopus laevis frog tadpoles.

Nitric oxide (NO) is a highly diffusible signalling molecule with widespread effects on the integrative electrical properties of a variety of neuronal and muscle cells. We have explored the effects of NO on the cardiac-like impulse generated by skin cells of the hatchling Xenopus tadpole. Skin cell impulses propagate from cell to cell via gap junctions and form an unusual sensory system, which triggers escape behaviour at early stages of amphibian development. We show that the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increases the duration of the skin impulse and slows the rate of impulse propagation across the skin, and also produces a significant depolarization of the membrane potential of skin cells. Each of these effects of SNAP is significantly reversed by the NO scavenger, C-PTIO. Possible sources of NO have been investigated using both NADPH-diaphorase histochemistry and nNOS immunocytochemistry to label the enzyme nitric oxide synthase (NOS), and DAF-2 to label NO itself. In each case a punctate distribution of skin cells is labelled, indicating that the endogenous production of NO may regulate the properties of the skin impulse.