Genetic manipulation of the odor-evoked distributed neural activity in the Drosophila mushroom body

Odor-induced neural activity was recorded by Ca2+ imaging in the cell body region of the Drosophila mushroom body (MB), which is the second relay of the olfactory central nervous system. The signals recorded are mainly from the cell layers on the brain surface because of the limited penetration of Ca2+-sensitive dyes. The densely packed cell bodies and their accessibility allow visualization of odor-induced population neural activity. It is revealed that odors evoke diffused neural activities in the MB. Although the signals cannot be attributed to individual neurons, patterns of the population neural activity can be analyzed. The activity pattern, but not the amplitude, of an odor-induced population response is specific for the chemical identity of an odor and its concentration. The distribution pattern of neural activity can be altered specifically by genetic manipulation of an odor binding protein and this alteration is closely associated with a behavioral defect of odor preference. These results suggest that the spatial pattern of the distributed neural activity may contribute to coding of odor information at the second relay of the olfactory system.     

The Role of Dopamine in Drosophila Larval Classical Olfactory Conditioning

Learning and memory is not an attribute of higher animals. Even Drosophila larvae are able to form and recall an association of a given odor with an aversive or appetitive gustatory reinforcer. As the Drosophila larva has turned into a particularly simple model for studying odor processing, a detailed neuronal and functional map of the olfactory pathway is available up to the third order neurons in the mushroom bodies. At this point, a convergence of olfactory processing and gustatory reinforcement is suggested to underlie associative memory formation. The dopaminergic system was shown to be involved in mammalian and insect olfactory conditioning. To analyze the anatomy and function of the larval dopaminergic system, we first characterize dopaminergic neurons immunohistochemically up to the single cell level and subsequent test for the effects of distortions in the dopamine system upon aversive (odor-salt) as well as appetitive (odor-sugar) associative learning. Single cell analysis suggests that dopaminergic neurons do not directly connect gustatory input in the larval suboesophageal ganglion to olfactory information in the mushroom bodies. However, a number of dopaminergic neurons innervate different regions of the brain, including protocerebra, mushroom bodies and suboesophageal ganglion. We found that dopamine receptors are highly enriched in the mushroom bodies and that aversive and appetitive olfactory learning is strongly impaired in dopamine receptor mutants. Genetically interfering with dopaminergic signaling supports this finding, although our data do not exclude on naïve odor and sugar preferences of the larvae. Our data suggest that dopaminergic neurons provide input to different brain regions including protocerebra, suboesophageal ganglion and mushroom bodies by more than one route. We therefore propose that different types of dopaminergic neurons might be involved in different types of signaling necessary for aversive and appetitive olfactory memory formation respectively, or for the retrieval of these memory traces. Future studies of the dopaminergic system need to take into account such cellular dissociations in function in order to be meaningful.     

Olfactory conditioning with single chemicals in the German Cockroach, Blattella germanica (Dictyoptera: Blattellidae)

Many insects find resources by means of the olfactory cues of general odors after learning. To evaluate behavioral responses to the odor of a particular chemical after learning with reward or punishment quantitatively, we developed a standardized odor-training method in the German cockroach, Blattella germanica (Linnaeus), an important urban pest species. A classical olfactory conditioning procedure for a preference test was modified to become applicable to a single odor, by which a (?)-menthol or vanillin odor was independently associated with sucrose (reward) or sodium chloride solution (punishment). The strength of the association with the odor was evaluated with the increase or decrease in visit frequencies to the odor source after olfactory conditioning. The frequency increased after (?)-menthol was presented with a reward, while it did not change with the rewarded vanillin odor. With both odors, the frequency decreased significantly after training with a punishment. These results indicate that cockroaches learn a single compound odor presented as a conditioned stimulus, although the association of the odor with a reward or punishment depends on the chemical. This olfactory conditioning method can not only facilitate the analysis of cockroach behavior elicited by a learned single chemical odor, but also quantify the potential attractiveness or repellency of the chemical after learning.     

Appetitive odor learning does not change olfactory coding in a subpopulation of honeybee antennal lobe neurons

Odors elicit spatio-temporal patterns of activity in the olfactory bulb of vertebrates and the antennal lobe of insects. There have been several reports of changes in these patterns following olfactory learning. These studies pose a conundrum: how can an animal learn to efficiently respond to a particular odor with an adequate response, if its primary representation already changes during this process? In this study, we offer a possible solution for this problem. We measured odor-evoked calcium responses in a subpopulation of uniglomerular AL output neurons in honeybees. We show that their responses to odors are remarkably resistant to plasticity following a variety of appetitive olfactory learning paradigms. There was no significant difference in the changes of odor-evoked activity between single and multiple trial forward or backward conditioning, differential conditioning, or unrewarded successive odor stimulation. In a behavioral learning experiment we show that these neurons are necessary for conditioned odor responses. We conclude that these uniglomerular projection neurons are necessary for reliable odor coding and are not modified by learning in this paradigm. The role that other projection neurons play in olfactory learning remains to be investigated.     

Behavioral functions of the insect mushroom bodies

New methods of intervention in Drosophila and other insect species reveal that the mushroom bodies are involved in a diverse set of behavioral functions. The intrinsic Kenyon cells (those neurons with projections within the mushroom bodies) house part of the short-term memory trace for odors and are required for courtship conditioning memory. A pair of extrinsic mushroom body neurons (neurons with projections both inside and outside the mushroom bodies) provides a neuropeptide important for 1-hour olfactory memory. In addition, the mushroom bodies are necessary for context generalization in visual learning and for regulating the transition from walking to rest.     

Hydroxyurea‐induced partial mushroom body ablation does not affect acquisition and retention of olfactory differential conditioning in honeybees

The mushroom bodies (MBs), a paired structure in the insect brain, play a major role in storing and retrieving olfactory memories. We tested whether olfactory learning and odor processing is impaired in honeybees in which MB subunits were partially ablated. Using hydroxyurea (HU) to selectively kill proliferating cells, we created honeybees with varying degrees of MB lesions. Three‐dimensional reconstructions of brains were generated to analyze the drug‐induced morphological changes. These reconstructions show that, with few exceptions, only the MBs were affected by the drug, while other brain areas remained morphometrically intact. Typically, lesions affected only the MB in one hemisphere of the brain. To preclude HU‐induced physiologic deficits in the antennal lobe (AL) affecting olfactory learning, we measured the responses to odors in the AL using an in vivo calcium imaging approach. The response patterns did not differ between the AL of intact versus ablated brain sides within respective specimens. We, therefore, carried out side‐specific classical discriminative olfactory conditioning of the proboscis extension reflex (PER) with control bees and with HU‐treated bees with or without MB ablations. All experimental groups learned equally to discriminate and respond to a rewarded (CS+) but not to an unrewarded (CS?) conditioned stimulus during acquisition and retention tests. Thus, our results indicate that partial MB lesions do not affect this form of elemental olfactory learning. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 343–360, 2002     

Olfactory learning

The olfactory nervous systems of insects and mammals exhibit many similarities, suggesting that the mechanisms for olfactory learning may be shared. Neural correlates of olfactory memory are distributed among many neurons within the olfactory nervous system. Perceptual olfactory learning may be mediated by alterations in the odorant receptive fields of second and/or third order olfactory neurons, and by increases in the coherency of activity among ensembles of second order neurons. Operant olfactory conditioning is associated with an increase in the coherent population activity of these neurons. Olfactory classical conditioning increases the odor responsiveness and synaptic activity of second and perhaps third order neurons. Operant and classical conditioning both produce an increased responsiveness to conditioned odors in neurons of the basolateral amygdala. Molecular genetic studies of olfactory learning in Drosophila have revealed numerous molecules that function within the third order olfactory neurons for normal olfactory learning.     

Testing odor response stereotypy in the Drosophila mushroom body

The mushroom body is an insect brain structure required for olfactory learning. Its principal neurons, the Kenyon cells (KCs), form a large cell population. The neuronal populations from which their olfactory input derives (olfactory sensory and projection neurons) can be identified individually by genetic, anatomical, and physiological criteria. We ask whether KCs are similarly identifiable individually, using genetic markers and whole-cell patch-clamp in vivo. We find that across-animal responses are as diverse within the genetically labeled subset as across all KCs in a larger sample. These results combined with those from a simple model, using projection neuron odor responses as inputs, suggest that the precise circuit specification seen at earlier stages of odor processing is likely absent among the mushroom body KCs.     

A neural network model of general olfactory coding in the insect antennal lobe.

A central problem in olfaction is understanding how the quality of olfactory stimuli is encoded in the insect antennal lobe (or in the analogously structured vertebrate olfactory bulb) for perceptual processing in the mushroom bodies of the insect protocerebrum (or in the vertebrate olfactory cortex). In the study reported here, a relatively simple neural network model, inspired by our current knowledge of the insect antennal lobes, is used to investigate how each of several features and elements of the network, such as synapse strengths, feedback circuits and the steepness of neural activation functions, influences the formation of an olfactory code in neurons that project from the antennal lobes to the mushroom bodies (or from mitral cells to olfactory cortex). An optimal code in these projection neurons (PNs) should minimize potential errors by the mushroom bodies in misidentifying the quality of an odor across a range of concentrations while maximizing the ability of the mushroom bodies to resolve odors of different quality. Simulation studies demonstrate that the network is able to produce codes independent or virtually independent of concentration over a given range. The extent of this range is moderately dependent on a parameter that characterizes how long it takes for the voltage in an activated neuron to decay back to its resting potential, strongly dependent on the strength of excitatory feedback by the PNs onto antennal lobe intrinsic neurons (INs), and overwhelmingly dependent on the slope of the activation function that transforms the voltage of depolarized neurons into the rate at which spikes are produced. Although the code in the PNs is degraded by large variations in the concentration of odor stimuli, good performance levels are maintained when the complexity of stimuli, as measured by the number of component odorants, is doubled. When excitatory feedback from the PNs to the INs is strong, the activity in the PNs undergoes transitions from initial states to stimulus-specific equilibrium states that are maintained once the stimulus is removed. When this PN-IN feedback is weak the PNs are more likely to relax back to a stimulus-independent equilibrium state, in which case the code is not maintained beyond the application of the stimulus. Thus, for the architecture simulated here, strong feedback from the PNs onto the INs, together with step-like neuronal activation functions, could well be important in producing easily discriminable odor quality codes that are invariant over several orders of magnitude in stimulus concentration.     

Classical Olfactory Conditioning in the Oriental Fruit Fly,Bactrocera dorsalis

The oriental fruit fly, Bactrocera dorsalis, is a serious pest of fruits and vegetables. Methyl eugenol (ME), a male attractant, is used to against this fly by mass trapping. Control effect may be influenced by learning, which could modify the olfactory response of the fly to this attractant. To collect the behavioral evidence, studies on the capability of this fly for olfactory learning are necessary. We investigated olfactory learning in male flies with a classical olfactory conditioning procedure using restrained individuals under laboratory conditions. The acquisition of the proboscis extension reflex was used as the criterion for conditioning. A high conditioned response level was found in oriental fruit flies when an odor was presented in paired association with a sucrose reward but not when the odor and sucrose were presented unpaired. We also found that the conditioning performance was influenced by the odor concentration, intertrial interval, and starvation time. A slight sensitization elicited by imbibing sucrose was observed. These results indicate that oriental fruit flies have a high capacity to form an olfactory memory as a result of classical conditioning.     

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.     

Go contributes to olfactory reception in Drosophila melanogaster

Background  

Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology and function as ligand-gated cation channels. Consequently, the involvement of cyclic nucleotides and G proteins in insect odor reception is controversial. Since the heterotrimeric G_oα subunit is expressed in Drosophila olfactory receptor neurons, we reasoned that G_o acts together with insect odorant receptor cation channels to mediate odor-induced physiological responses.     

Hydroxyurea-induced partial mushroom body ablation does not affect acquisition and retention of olfactory differential conditioning in honeybees

The mushroom bodies (MBs), a paired structure in the insect brain, play a major role in storing and retrieving olfactory memories. We tested whether olfactory learning and odor processing is impaired in honeybees in which MB subunits were partially ablated. Using hydroxyurea (HU) to selectively kill proliferating cells, we created honeybees with varying degrees of MB lesions. Three-dimensional reconstructions of brains were generated to analyze the drug-induced morphological changes. These reconstructions show that, with few exceptions, only the MBs were affected by the drug, while other brain areas remained morphometrically intact. Typically, lesions affected only the MB in one hemisphere of the brain. To preclude HU-induced physiologic deficits in the antennal lobe (AL) affecting olfactory learning, we measured the responses to odors in the AL using an in vivo calcium imaging approach. The response patterns did not differ between the AL of intact versus ablated brain sides within respective specimens. We, therefore, carried out side-specific classical discriminative olfactory conditioning of the proboscis extension reflex (PER) with control bees and with HU-treated bees with or without MB ablations. All experimental groups learned equally to discriminate and respond to a rewarded (CS+) but not to an unrewarded (CS-) conditioned stimulus during acquisition and retention tests. Thus, our results indicate that partial MB lesions do not affect this form of elemental olfactory learning.     

Current source-density analysis in the mushroom bodies of the honeybee (Apis mellifera carnica)

Summary Information processing in the mushroom bodies which are an important part of most invertebrate central nervous systems was analysed by extracellular electrophysiological techniques. The mushroom bodies consist of layers of parallel intrinsic neurons which make synaptic contact with extrinsic input and output neurons. The intrinsic neurons (approximately 170,000/mushroom body) have very small axon diameters (0.1–1 m) which makes it difficult to record their activity intracellularly. In order to analyse the functional properties of this neuropil field potentials were measured extracellularly.Series of averaged evoked potentials (AEPs) were recorded along electrode tracks at consecutive depth intervals in different parts of the mushroom bodies of the bee. These potentials were elicited by olfactory, mechanical and visual stimuli.In order to locate the synaptic areas generating these potentials, current source-densities (CSD) were calculated using the consecutively measured evoked potentials. The conductivities of the extracellular space along the electrode tracks in the pedunculus and calyx and in part of the alpha-lobe of the mushroom bodies were found to be constant.The CSD analysis reveals a complex pattern of source-sink distributions in the mushroom bodies. There is a high degree of correlation between current sinks and sources detected by CSD analysis and the morphological distribution of neurons.The CSD analysis shows that the inputs and outputs of the mushroom bodies involve multimodal synaptic interactions, whereas information processing in the intrinsic Kenyon-cells is limited to sensory inputs from the antenna.Comparison of the electrophysiological with the histological results shows that the intrinsic cells of the mushroom bodies are physiologically not a homogeneous group as is often proposed. Among the intrinsic neurons clearly defined areas of current sources and sinks can be identified and attributed to Kenyon-cells in different layers.Abbreviations AEP averaged evoked potentials - AGT antennoglomerular tract - CSD current source-density - PCT antennoglomerular tract     

Punishment prediction by dopaminergic neurons in Drosophila

The temporal pairing of a neutral stimulus with a reinforcer (reward or punishment) can lead to classical conditioning, a simple form of learning in which the animal assigns a value (positive or negative) to the formerly neutral stimulus. Olfactory classical conditioning in Drosophila is a prime model for the analysis of the molecular and neuronal substrate of this type of learning and memory. Neuronal correlates of associative plasticity have been identified in several regions of the insect brain. In particular, the mushroom bodies have been shown to be necessary for aversive olfactory memory formation. However, little is known about which neurons mediate the reinforcing stimulus. Using functional optical imaging, we now show that dopaminergic projections to the mushroom-body lobes are weakly activated by odor stimuli but respond strongly to electric shocks. However, after one of two odors is paired several times with an electric shock, odor-evoked activity is significantly prolonged only for the "punished" odor. Whereas dopaminergic neurons mediate rewarding reinforcement in mammals, our data suggest a role for aversive reinforcement in Drosophila. However, the dopaminergic neurons' capability of mediating and predicting a reinforcing stimulus appears to be conserved between Drosophila and mammals.     

Olfactory conditioning of proboscis activity in Drosophila melanogaster

Olfactory learning and memory processes in Drosophila have been well investigated with aversive conditioning, but appetitive conditioning has rarely been documented. Here, we report for the first time individual olfactory conditioning of proboscis activity in restrained Drosophila melanogaster. The protocol was adapted from those developed for proboscis extension conditioning in the honeybee Apis mellifera. After establishing a scale of small proboscis movements necessary to characterize responses to olfactory stimulation, we applied Pavlovian conditioning, with five trials consisting of paired presentation of a banana odour and a sucrose reward. Drosophila showed conditioned proboscis activity to the odour, with a twofold increase of percentage of responses after the first trial. No change occurred in flies experiencing unpaired presentations of the stimuli, confirming an associative basis for this form of olfactory learning. The adenylyl cyclase mutant rutabaga did not exhibit learning in this paradigm. This protocol generated at least a short-term memory of 15 min, but no significant associative memory was detected at 1 h. We also showed that learning performance was dependent on food motivation, by comparing flies subjected to different starvation regimes.     

Voltage-activated and odor-modulated conductances in olfactory neurons of Drosophila melanogaster

Voltage-activated currents and odor-modulated conductances were studied in cells in semi-intact Drosophila third antennal segments (the main olfactory organ) using patch-clamp techniques. All neurons expressed outward currents, and most expressed labile fast transient inward currents with kinetics similar to Na⁺ currents in other systems. Action potentials were detected as bipolar capacitative current transients in cell-attached or loose patches from the soma of both odor-sensitive (97%) and insensitive neurons. A mixture of odorants from five chemical classes caused an increase (∼70%), decrease (∼10%), or no effect on firing frequency in pharate adult neurons. The development of chemosensitivity was examined and odor-induced changes in action potential firing frequency were recorded in pupal antennal neurons as early as P8, a stage after completion of sensillar development. The character of odor-induced responses was more profound and complex later in development; small, tonic increases in firing frequency were observed at pupal stages P8 through P11(ii), while in older pupae and young adults ∼25% of the increased responses were phasic-tonic. The apical dendrite was the site of odor modulation in ∼90% and 100% of responsive adult and early pupal neurons, respectively. Whole-cell recordings revealed that apparent nonselective cation and chloride conductances were modulated by a mixture of odorants in separate antennal neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 123–137, 1997.     

Multimodal interneurons in the cricket brain: properties of identified extrinsic mushroom body cells

Summary 322 neurons were recorded intracellularly within the central part of the insect brain and 150 of them were stained with Lucifer Yellow or cobaltous sulphide. Responses to mechanical, olfactory, visual and acoustical stimulation were determined and compared between morphologically different cell types in different regions of the central brain. Almost all neurons responded to multimodal stimulation and showed complex responses. It was not possible to divide the cells into different groups using physiological criteria alone.Extrinsic neurons with projections to the calyces connect the mushroom bodies with the deutocerebrum and also with parts of the diffuse protocerebrum. These cells probably give input to the mushroom body system. The majority are multimodal and they often show olfactory responses. Among those cells that extend from the antennal neuropil are neurons that respond to non-antennal stimulation (Figs. 1, 2).Extrinsic neurons with projections in the lobes of the mushroom bodies often project to the lateral protocerebrum. Anatomical and physiological evidence suggest that they form an output system of the mushroom bodies. They are also multimodal and often exhibit long lasting after discharges and changes in sensitivity and activity level, which can be related to specific stimuli or stimulus combinations (Figs. 3, 4).Extrinsic neurons, especially those projecting to the region where both lobes bifurcate, exhibit stronger responses to multimodal stimuli than other local brain neurons. Intensity coding for antennal stimulation is not different from other areas of the central protocerebrum, but the signal-tonoise ratio is increased (Fig. 5).Abbreviation AGT antenno-glomerular tract     

Response properties of higher level neurons in the central olfactory pathway of the crayfish

We have examined the electrical activity of interneurons within the higher levels of the crayfish olfactory system. In unstimulated isolated crayfish head preparations, local protocerebral interneurons (LPI) of the hemiellipsoid bodies generate periodic, low-frequency membrane depolarizations. The most reasonable explanation for these baseline fluctuations, which were exhibited by all of the LPIs examined and which were reversibly abolished by either tetrodotoxin or low-calcium saline solution, is that they reflect periodic synaptic drive from the axon terminals of olfactory projection neurons. One-third of tested LPIs generated impulses in response to the odor stimuli we applied to the antennules. Those cells that did respond exhibited a brief excitatory postsynaptic potential and one or two action potentials, even during prolonged odor pulses. Many of the responding neurons also exhibited a delayed impulse burst 1 or 2 s following the stimulus pulse. Most of the responding cells recovered their sensitivity to odors very slowly, exhibiting disadaptation periods of several minutes. The apparent refractory nature of individual LPIs to olfactory stimulation is attributed in part to a hypothesized selectivity of connections between projection neurons and protocerebral targets and in part to the electrical isolation of the recording electrode from many regions of the extensive LPI dendritic tree. Accepted: 20 March 1997     

Effects of olfactory peduncle sectioning on the single unit responses of olfactory bulb neurons to odor presentation in awake rabbits

The influences of centrifugal inputs to the olfactory bulb werestudied by recording singlecell responses evoked by olfactorystimuli in intact and peduncle-sectioned bulbs of awake freebreathingrabbits. Responses of intact animals were mainly characterizedby a temporal reorganization of the single unit discharge -responsive second order neurons increased their firing activityduring inspirations and were silent during expirations. Thissynchronization of firing discharge with respiration occurredin the absence of any significant change in the overall firingactivity measured over intervals which included both the inspiratoryand expiratory phases of the respiratory cycle. By contrast,neurons recorded in isolated olfactory bulbs exhibited eithera significant increase or a decrease in firing activity duringodor presentation, and, furthermore, the synchronization ofthese units to the respiratory cycle was markedly reduced comparedwith that in intact animals. Comparison of cell responsivenessbetween intact and isolated olfactory bulbs indicated that thelesion increased the number of odors which induced a response,but did not change the percentage of cells which failed to respondto any of the 5 odorants used in this study. The cell responsivenessincreased for camphor and isoamyl acetate, and to a lesser extentfor food odor. The results indicate that high order nervousstructures exert a powerful inhibitory influence on the responsesof olfactory bulb second-order neurons to odor stimuli. Theyalso suggest that, in intact rabbits, centrifugal inputs playa role in the odor-induced synchronization of the single unitactivity with respiration.