ROS-Mediated ABA Signaling

In plants, reactive oxygen species (ROS) are short-lived molecules produced through various cellular mechanisms in response to biotic and abiotic stimuli. ROS function as second messengers for hormone signaling, development, oxygen deprivation, programmed cell death, and plant–pathogen interactions. Recent research on ROS-mediated responses has produced stimulating findings such as the specific sources of ROS production, molecular elements that work in ROS-mediated signaling and homeostasis, and a ROS-regulated gene network (Neill et al., Curr Opin Plant Biol 5:388–395, 2002a; Apel and Hirt, Annu Rev Plant Biol 55:373–399, 2004; Mittler et al., Trends Plant Sci 9:490–498, 2004; Mori and Schroeder, Plant Physiol 135:702–708, 2004; Kwak et al., Plant Physiol 141:323–329, 2006; Torres et al., Plant Physiol 141:373–378, 2006; Miller et al., Physiol Plant 133:481–489, 2008). In this review, we highlight new discoveries in ROS-mediated abscisic acid (ABA) signaling. Drs. Daeshik Cho and June M. Kwak are the corresponding authors for this paper.     

Processing of antennal information in extrinsic mushroom body neurons of the bee brain

Summary In the bee brain neural activity of interneurons of the inner antenno-cerebral tract (inputs to the mushroom body) and extrinsic neurons of the-lobe (output cells) was recorded intracellularly. The cells were stained with Lucifer Yellow. The response characteristics of the neurons to light, various antennal stimuli and mechanical stimuli to thorax and abdomen were studied.The cells of the inner antenno-cerebral tract (ACT) have uniglomerular dendritic arborizations in the antennal lobe and send projections into the calyces of the ipsilateral mushroom body and the lateral protocerebral lobe. 93% of the neurons are bi- or multimodal. No responses to light stimuli were found. Tactile stimuli to the antennae are only effective when applied ipsilaterally. Only one neuron showed marked differences in the responses to the qualitative testing of three odors: rose, lavender and isoamyl acetate.The cells can be classified according to their response characteristics; the following response types were found: (1) inhibitory responses to the stimuli, (2) inhibitory responses to olfactory and excitatory responses to mechanical stimuli or vice versa, (3) excitatory responses to mechanical and sugar water stimuli, (4) excitation to olfactory stimuli and to touching the antenna with a drop of water or sugar water, (5) excitation to mechanical stimuli to head, thorax and abdomen and inhibition to sugar water stimuli.The recorded extrinsic-lobe neurons have small dendritic bands perpendicular to the Kenyon cells, their axons project to the contralateral median protocerebrum. These cells have ipsilateral antennal and mostly ipsilateral optic inputs and process information from thoracic and abdominal mechanoreceptors. All responses are excitatory.The recordings suggest that the mushroom bodies are multimodal integration centers, where antennal information is first combined with visual inputs.Abbreviation ACT antenno-cerebral tract     

Learning channels. Cellular physiology of odor processing neurons within the honeybee brain

To understand the cellular mechanisms of olfactory learning in the honeybee brain we study the physiology of identified neurons within the olfactory pathway. Here, we review data on the voltage-sensitive and ligand-gated ionic currents of mushroom body Kenyon cells and antennal lobe neurons in vitro and in situ. Both cell types generate action potentials in vitro, but have different voltage-sensitive K+ currents. They express nicotinic acetylcholine receptors and ionotropic GABA receptors, representing the major transmitter systems in the insect olfactory system. Our data are interpreted with respect to learning-dependent plasticity in the honeybee brain.     

Functionality of divergence and convergence in a model of the insect olfactory system

Recent studies have shown that the insect olfactory system uses a spatio-temporal encoding of odours in the population of projection neurons in the antennal lobe, and suggest that the information thus coded is spread across a large population of Kenyon cells in the mushroom bodies. At this stage, the temporal part of the code might be transformed into a spatial code, especially via the temporally sensitive mechanisms of paired–pulse facilitation and feedback inhibition with its possible associated rebound. We explore here a simple model of the olfactory system using a three–layer network of formal neurons, comprising a fixed number (three) of projection and inhibitory neurons, but a variable number of Kenyon cells. We show how enlarging the divergence of the network (i.e. the ratio between the number of Kenyon cells to the number of input – projection – neurons) alters the number of different output spatial states in response to a fixed set of spatio-temporal inputs, and may therefore improve its effectiveness in discriminating between these inputs. Such enlarged divergence also reduces the variation of this effectiveness among random realisations of the network connectivity. Our model shows that the discriminative effectiveness first increases with the divergence, and then plateaus for a divergence factor of ∼20. The maximal average number of different outputs was 470.2, which was computed from some simulations with random realisations of connectivity and with a set of 512 possible inputs. The discriminative effectiveness of the network is sensitive to paired-pulse facilitation, and especially to inhibition with rebound. Received: 6 April 2001 / Accepted in revised form: 8 April 2002     

Metamorphosis and adult development of the mushroom bodies of the red flour beetle, Tribolium castaneum

The insect mushroom bodies play important roles in a number of higher processing functions such as sensory integration, higher level olfactory processing, and spatial and associative learning and memory. These functions have been established through studies in a handful of tractable model systems, of which only the fruit fly Drosophila melanogaster has been readily amenable to genetic manipulations. The red flour beetle Tribolium castaneum has a sequenced genome and has been subject to the development of molecular tools for the ready manipulation of gene expression; however, little is known about the development and organization of the mushroom bodies of this insect. The present account bridges this gap by demonstrating that the organization of the Tribolium mushroom bodies is strikingly like that of the fruit fly, with the significant exception that the timeline of neurogenesis is shifted so that the last population of Kenyon cells is born entirely after adult eclosion. Tribolium Kenyon cells are generated by two large neuroblasts per hemisphere and segregate into an early-born delta lobe subpopulation followed by clear homologs of the Drosophila gamma, alpha'/beta' and alpha/beta lobe subpopulations, with the larval-born cohorts undergoing dendritic reorganization during metamorphosis. BrdU labeling and immunohistochemical staining also reveal that a proportion of individual Tribolium have variable numbers of mushroom body neuroblasts. If heritable, this variation represents a unique opportunity for further studies of the genetic control of brain region size through the control of neuroblast number and cell cycle dynamics.     

Decoding Temporal Information Through Slow Lateral Excitation in the Olfactory System of Insects

Sensory information is represented in a spatio-temporal code in the antennal lobe, the first processing stage of the olfactory system of insects. We propose a novel mechanism for decoding this information in the next processing stage, the mushroom body. The Kenyon cells in the mushroom body of insects exhibit lateral excitatory connections at their axons. We demonstrate that slow lateral excitation between Kenyon cells allows one to decode sequences of activity in the antennal lobe. We are thus able to clarify the role of the existing connections as well as to demonstrate a novel mechanism for decoding temporal information in neuronal systems. This mechanism complements the variety of existing temporal decoding schemes. It seems that neuronal systems not only have a rich variety of code types but also quite a diversity of algorithms for transforming different codes into each other.     

Neurons with dopamine-like immunoreactivity target mushroom body Kenyon cell somata in the brain of some hymenopteran insects

The mushroom bodies of the insect brain are centers for olfactory and multimodal information processing and they are involved in associative olfactory learning. They are comprised of numerous (340,000 in the bee brain), small (3–8 μm soma diameter) local interneurons, the Kenyon cells. In the brain of honeybees (Apis mellifera) of all castes (worker bees, drones and queens), wasps (Vespula germanica) and hornets (Vespa crabro) immunostaining revealed fibers with dopamine-like immunoreactivity projecting from the pedunculus and the lip neuropil of the mushroom bodies into the Kenyon cell perikaryal layer. These fibers terminate with numerous varicosities, mainly around the border between medial and lateral Kenyon cell soma groups. Visualization of immunostained terminals in the transmission electron microscope showed that they directly contact the somata of the Kenyon cells and contain presynaptic elements. The somata of the Kenyon cells are clearly non-immunoreactive. Synaptic contacts at the somata are unusual for the central nervous systems of insects and other arthropods. This finding suggests that the somata of the Kenyon cells of Hymenoptera may serve an integrative role, and not merely a supportive function.     

Chaperone receptors: guiding proteins to intracellular compartments

Despite mitochondria and chloroplasts having their own genome, 99% of mitochondrial proteins (Rehling et al., Nat Rev Mol Cell Biol 5:519–530, 2004) and more than 95% of chloroplast proteins (Soll, Curr Opin Plant Biol 5:529–535, 2002) are encoded by nuclear DNA, synthesised in the cytosol and imported post-translationally. Protein targeting to these organelles depends on cytosolic targeting factors, which bind to the precursor, and then interact with membrane receptors to deliver the precursor into a translocase. The molecular chaperones Hsp70 and Hsp90 have been widely implicated in protein targeting to mitochondria and chloroplasts, and receptors capable of recognising these chaperones have been identified at the surface of both these organelles (Schlegel et al., Mol Biol Evol 24:2763–2774, 2007). The role of these chaperone receptors is not fully understood, but they have been shown to increase the efficiency of protein targeting (Young et al., Cell 112:41–50, 2003; Qbadou et al., EMBO J 25:1836–1847, 2006). Whether these receptors contribute to the specificity of targeting is less clear. A class of chaperone receptors bearing tetratricopeptide repeat domains is able to specifically bind the highly conserved C terminus of Hsp70 and/or Hsp90. Interestingly, at least of one these chaperone receptors can be found on each organelle (Schlegel et al., Mol Biol Evol 24:2763–2774, 2007), which suggests a universal role in protein targeting for these chaperone receptors. This review will investigate the role that chaperone receptors play in targeting efficiency and specificity, as well as examining recent in silico approaches to find novel chaperone receptors.     

Evolution of the techniques used in studying associative olfactory learning and memory in adult Drosophila in vivo: a historical and technical perspective

Drosophila melanogaster behavioral mutants have been isolated in which the ability to form associative olfactory memories has been disrupted primarily by altering cyclic adenosine monophosphate signal transduction. Unfortunately, the small size of the fruit fly and its neurons has made the application of neurobiological techniques typically used to investigate the physiology underlying these behaviors daunting. However, the realization that adult fruit flies could tolerate a window in the head capsule allowing access to the central structures thought to be involved plus the development of genetically expressed reporters of neuronal function has allowed a meteoric expansion of this field over the last decade. This review attempts to summarize the evolution of the techniques involved from the first use of a window to access these brain areas thought to be involved in associative olfactory learning and memory, the mushroom bodies and antennal lobes, to the current refinements which allow both high-resolution multiphoton imaging and patch clamping of identified neurons while applying the stimuli used in the behavioral protocols. This area of research now appears poised to reveal some very exciting mechanisms underlying behavior.     

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time¹. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)^2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning^4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs^6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode³. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings⁸. We provide details of our experimental setup and present representative recording traces for each of these techniques.     

Physiological properties and response modulations of mushroom body feedback neurons during olfactory learning in the honeybee, Apis mellifera

Mushroom bodies are central brain structures and essentially involved in insect olfactory learning. Within the mushroom bodies γ-aminobutyric acid (GABA)-immunoreactive feedback neurons are the most prominent neuron group. The plasticity of inhibitory neural activity within the mushroom body was investigated by analyzing modulations of odor responses of feedback neurons during olfactory learning in vivo. In the honeybee, Apis mellifera, feedback neurons were intracellularly recorded at their neurites. They produced complex patterns of action potentials without experimental stimulation. Summating postsynaptic potentials indicate that their synaptic input region lies within the lobes. Odor and antennal sucrose stimuli evoked excitatory phasic-tonic responses. Individual neurons responded to various odors; responses of different neurons to the same odor were highly variable. Response modulations were determined by comparing odor responses of feedback neurons before and after one-trial olfactory conditioning or sensitisation. Shortly after pairing an odor stimulus with a sucrose reward, odor-induced spike activity of feedback neurons decreased. Repeated odor stimulations alone, equally spaced as in the conditioning experiment, did not affect the odor-induced excitation. A single sensitisation trial also did not alter odor responses. These findings indicate that the level of odor-induced inhibition within the mushroom bodies is specifically modulated by experience. Accepted: 9 September 1999     

Olfactory coding in the insect brain: molecular receptive ranges, spatial and temporal coding

Odor information is coded in the insect brain in a sequence of steps, ranging from the receptor cells, via the neural network in the antennal lobe, to higher order brain centers, among which the mushroom bodies and the lateral horn are the most prominent. Across all of these processing steps, coding logic is combinatorial, in the sense that information is represented as patterns of activity across a population of neurons, rather than in individual neurons. Because different neurons are located in different places, such a coding logic is often termed spatial, and can be visualized with optical imaging techniques. We employ in vivo calcium imaging in order to record odor‐evoked activity patterns in olfactory receptor neurons, different populations of local neurons in the antennal lobes, projection neurons linking antennal lobes to the mushroom bodies, and the intrinsic cells of the mushroom bodies themselves, the Kenyon cells. These studies confirm the combinatorial nature of coding at all of these stages. However, the transmission of odor‐evoked activity patterns from projection neuron dendrites via their axon terminals onto Kenyon cells is accompanied by a progressive sparsening of the population code. Activity patterns also show characteristic temporal properties. While a part of the temporal response properties reflect the physical sequence of odor filaments, another part is generated by local neuron networks. In honeybees, γ‐aminobutyric acid (GABA)‐ergic and histaminergic neurons both contribute inhibitory networks to the antennal lobe. Interestingly, temporal properties differ markedly in different brain areas. In particular, in the antennal lobe odor‐evoked activity develops over slow time courses, while responses in Kenyon cells are phasic and transient. The termination of an odor stimulus is reflected by a decrease in activity within most glomeruli of the antennal lobe and an off‐response in some glomeruli, while in the mushroom bodies about half of the odor‐activated Kenyon cells also exhibit off‐responses.     

Dopamine modulation of the In vivo acetylcholine response in the Drosophila mushroom body

Olfactory sensory information in Drosophila is transmitted through antennal lobe projections to Mushroom Body neurons (Kenyon cells) by means of cholinergic synapses. Application of acetylcholine (ACh) and odors produce significant increases in intracellular calcium ([Ca²⁺]_i) in these neurons. Behavioral studies show that Kenyon cell activity is modulated by dopaminergic inputs and this modulation is thought to be the basis for an olfactory conditioned response. However, quantitative assessment of the synaptic inputs to Kenyon cells is currently lacking. To assess neuronal activity under in vivo conditions, we have used the endogenously‐expressed camgaroo reporter to measure [Ca²⁺]_i in these neurons. We report here the dose‐response relationship of Kenyon cells for ACh and dopamine (DA). Importantly, we also show that simultaneous application of ACh and DA results in a significant decrease in the response to ACh alone. In addition, we show inhibition of the ACh response by cyclic adenosine monophosphate. This is the first quantitative assessment of the effects of these two important transmitters in this system, and it provides an important basis for future analysis of the cellular mechanisms of this well established model for associative olfactory learning. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Physiology and morphology of olfactory neurons associating with the protocerebral lobe of the honeybee brain

Physiology and morphology of olfactory neurons associated with the protocerebral lobe around the alpha-lobe of the mushroom body were studied in the brain of the honeybee Apis mellifera using intracellular recording and staining techniques. The responses of neurons to behaviorally relevant odorants (a blend, and components of the Nasonov pheromone, and some other non-pheromonal odors) were recorded. Different response patterns were observed within different neurons, and often within the same neuron, in response to different stimuli. All the neurons stained had innervations in the protocerebral lobe. The cell profiles varied from cells connecting the antennal lobe with both the protocerebral and lateral protocerebral lobes (projection neurons), cells linking the pedunculus of the mushroom body with both the protocerebral and lateral protocerebral lobes (PE1 neurons), cells linking the alpha-lobe and protocerebral lobe with the calyces of the mushroom body (feedback neurons), and cells linking the alpha-lobe and protocerebral lobe with the antennal lobe (recurrent neurons), to cells connecting the protocerebral lobe with the contralateral protocerebrum (bilateral neurons). These findings suggest that the protocerebral lobe acts as an olfactory center associating with other centers, and provides multi-layered recurrent networks within the protocerebrum and between the deutocerebrum and the protocerebrum in honeybee olfactory pathways.     

Genetically expressed cameleon in Drosophila melanogaster is used to visualize olfactory information in projection neurons

Complex external stimuli such as odorants are believed to be internally represented in the brain by spatiotemporal activity patterns of extensive neuronal ensembles. These activity patterns can be recorded by optical imaging techniques. However, optical imaging with conventional fluorescence dyes usually does not allow for resolving the activity of biologically defined groups of neurons. Therefore, specifically targeting reporter molecules to neuron populations of common genetic identity is an important goal. We report the use of the genetically encoded calcium-sensitive fluorescence protein cameleon 2.1 in the Drosophila brain. We visualized odorant-evoked intracellular calcium concentration changes in selectively labeled olfactory projection neurons both postsynaptically in the antennal lobe, the primary olfactory neuropil, and presynaptically in the mushroom body calyx, a structure involved in olfactory learning and memory. As a technical achievement, we show that calcium imaging with a genetically encoded fluorescence probe is feasible in a brain in vivo. This will allow one to combine Drosophila's advanced genetic tools with the physiological analysis of brain function. Moreover, we report for the first time optical imaging recordings in synaptic regions of the Drosophila mushroom body calyx and antennal lobe. This provides an important step for the use of Drosophila as a model system in olfaction.     

Discrimination training with multimodal stimuli changes activity in the mushroom body of the hawkmoth Manduca sexta

Background

The mushroom bodies of the insect brain play an important role in olfactory processing, associative learning and memory. The mushroom bodies show odor-specific spatial patterns of activity and are also influenced by visual stimuli.

Methodology/Principal Findings

Functional imaging was used to investigate changes in the in vivo responses of the mushroom body of the hawkmoth Manduca sexta during multimodal discrimination training. A visual and an odour stimulus were presented either together or individually. Initially, mushroom body activation patterns were identical to the odour stimulus and the multimodal stimulus. After training, however, the mushroom body response to the rewarded multimodal stimulus was significantly lower than the response to the unrewarded unimodal odour stimulus, indicating that the coding of the stimuli had changed as a result of training. The opposite pattern was seen when only the unimodal odour stimulus was rewarded. In this case, the mushroom body was more strongly activated by the multimodal stimuli after training. When no stimuli were rewarded, the mushroom body activity decreased for both the multimodal and unimodal odour stimuli. There was no measurable response to the unimodal visual stimulus in any of the experiments. These results can be explained using a connectionist model where the mushroom body is assumed to be excited by olfactory stimulus components, and suppressed by multimodal configurations.

Conclusions

Discrimination training with multimodal stimuli consisting of visual and odour cues leads to stimulus specific changes in the in vivo responses of the mushroom body of the hawkmoth.     

Drosophila olfaction: the end of stereotypy?

Recent work has demonstrated substantial wiring and functional stereotypy in the fly olfactory system. In this issue of Neuron, Murthy et al. demonstrate that in the mushroom body, a site of olfactory associative learning, this initial peripheral stereotypy gives way to functionally nonstereotyped circuits.     

Morphology and physiology of the serotonin-immunoreactive putative antennal lobe feedback neuron in the male silkmoth Bombyx mori

In the male silkmoth Bombyx mori, olfactory information is relayed from olfactory receptor neurons in the antennae to the antennal lobe, and then to a variety of protocerebral neuropils. Currently, very little is known about neuromodulators that may affect the dynamics of this olfactory neural network. Immunocytochemical studies have revealed the presence of a serotonin-immunoreactive (SI) neuron that, in several insect species, is thought to provide feedback to the antennal lobe. To date, no studies have revealed details of this neuron's physiology. Using intracellular recording and staining, the silkmoth SI neuron (in two individuals) was first characterized physiologically and then stained with Lucifer Yellow to reveal morphological details. Immunocytochemical methods were also used to confirm the presence of serotonin. The silkmoth SI neuron branched in many important brain neuropils such as the mushroom body, central body, lateral accessory lobe and antennal lobe. The SI neuron in both individuals fired spontaneous, long duration action potentials, and responded to mechanosensory stimuli to the antennae.     

Neuroblast lineage-specific origin of the neurons of the Drosophila larval olfactory system

The complete neuronal repertoire of the central brain of Drosophila originates from only approximately 100 pairs of neural stem cells, or neuroblasts. Each neuroblast produces a highly stereotyped lineage of neurons which innervate specific compartments of the brain. Neuroblasts undergo two rounds of mitotic activity: embryonic divisions produce lineages of primary neurons that build the larval nervous system; after a brief quiescence, the neuroblasts go through a second round of divisions in larval stage to produce secondary neurons which are integrated into the adult nervous system. Here we investigate the lineages that are associated with the larval antennal lobe, one of the most widely studied neuronal systems in fly. We find that the same five neuroblasts responsible for the adult antennal lobe also produce the antennal lobe of the larval brain. However, there are notable differences in the composition of larval (primary) lineages and their adult (secondary) counterparts. Significantly, in the adult, two lineages (lNB/BAlc and adNB/BAmv3) produce uniglomerular projection neurons connecting the antennal lobe with the mushroom body and lateral horn; another lineage, vNB/BAla1, generates multiglomerular neurons reaching the lateral horn directly. lNB/BAlc, as well as a fourth lineage, vlNB/BAla2, generate a diversity of local interneurons. We describe a fifth, previously unknown lineage, BAlp4, which connects the posterior part of the antennal lobe and the neighboring tritocerebrum (gustatory center) with a higher brain center located adjacent to the mushroom body. In the larva, only one of these lineages, adNB/BAmv3, generates all uniglomerular projection neurons. Also as in the adult, lNB/BAlc and vlNB/BAla2 produce local interneurons which, in terms of diversity in architecture and transmitter expression, resemble their adult counterparts. In addition, lineages lNB/BAlc and vNB/BAla1, as well as the newly described BAlp4, form numerous types of projection neurons which along the same major axon pathways (antennal tracts) used by the antennal projection neurons, but which form connections that include regions outside the “classical” olfactory circuit triad antennal lobe-mushroom body-lateral horn. Our work will benefit functional studies of the larval olfactory circuit, and shed light on the relationship between larval and adult neurons.