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.     

Maturation of odor representation in the honeybee antennal lobe

The antennal lobe (AL) is the first center for processing odors in the insect brain, as is the olfactory bulb (OB) in vertebrates. Both the AL and the OB have a characteristic glomerular structure; odors sensed by olfactory receptor neurons are represented by patterns of glomerular activity. Little is known about when and how an odor begins to be perceived in a developing brain. We address this question by using calcium imaging to monitor odor-evoked neural activity in the ALs of bees of different ages. We find that odor-evoked neural activity already occurs in the ALs of bees as young as 1 or 2 days. In young bees, the responses to odors are relatively weak and restricted to a small number of glomeruli. However, different odors already evoke responses in different combinations of glomeruli. In mature bees, the responses are stronger and are evident in more glomeruli, but continue to have distinct odor-dependent signatures. Our findings indicate that the specific glomerular patterns for odors are conserved during the development, and that odor representations are fully developed in the AL during the first 2 weeks following emergence.     

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection^1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination^4-6, herbivory of food crops⁷, and transmission of disease^8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)^10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed ''GCMR'') from neurons in the antennal lobe (AL; the insect''s primary olfactory center)^13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna''s receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques^14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.     

Six1 is essential for early neurogenesis in the development of olfactory epithelium

The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, “early neurogenesis” occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows “established neurogenesis,” in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1^−/−). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1^−/− at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1^−/−. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5.     

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann^1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor³ for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L''Etoile et al.,⁴ uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC⁴. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC⁵. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC^6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.     

Functional organization of olfactory processing in the accessory lobe of the spiny lobster

An isolated brain preparation was used to characterize neurons innervating the accessory lobe (AL) of the spiny lobster (Panulirus argus). Four distinct classes of neurons responded to electrical stimulation of the olfactory (antennular) nerve. These cells responded to electrical stimulation with a long and variable latency; they also responded to odor stimulation in a nose-brain preparation. Neurons connecting the AL with the olfactory lobe branched in the central AL layer and selectively innervated olfactory lobe glomeruli. These cells had response latencies which were significantly shorter than those of other AL neurons. Intrinsic AL interneurons were heterogeneous as a population, and most arborized in irregular but circumscribed regions of either the lateral or medial layers. The final class of neurons branched ipsilaterally in the deutocerebral neuropil and bilaterally innervated only a few AL glomeruli. The physiology and morphology of these four classes of neurons confirm an olfactory function for the AL and identify the input and output regions of the lobe. Based on these findings, we propose that the AL processes odor information in the context of higher order multimodal input.Abbreviations AL accessory lobe - DCN deutocerebral neuropil - OGT olfactory-globular tract - OGTN olfactory-globular tract neuropil - OL olfactory lobe     

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     

Afferent induction of olfactory glomeruli requires N-cadherin

Drosophila olfactory receptor neurons (ORNs) elaborate a precise internal representation of the external olfactory world in the antennal lobe (AL), a structure analagous to the vertebrate olfactory bulb. ORNs expressing the same odorant receptor innervate common targets in a highly organized neuropilar structure inside the AL, the glomerulus. During normal development, ORNs target to specific regions of the AL and segregate into subclass-specific aggregates called protoglomeruli prior to extensive intermingling with target dendrites to form mature glomeruli. Using a panel of ORN subclass-specific markers, we demonstrate that in the adult AL, N-cadherin (N-cad) mutant ORN terminals remain segregated from dendrites of target neurons. N-cad plays a crucial role in protoglomerulus formation but is largely dispensible for targeting to the appropriate region of the AL. We propose that N-cad, a homophilic cell adhesion molecule, acts in a permissive fashion to promote subclass-specific sorting of ORN axon terminals into protoglomeruli.     

Excitatory interactions between olfactory processing channels in the Drosophila antennal lobe

Each odorant receptor gene defines a unique type of olfactory receptor neuron (ORN) and a corresponding type of second-order neuron. Because each odor can activate multiple ORN types, information must ultimately be integrated across these processing channels to form a unified percept. Here, we show that, in Drosophila, integration begins at the level of second-order projection neurons (PNs). We genetically silence all the ORNs that normally express a particular odorant receptor and find that PNs postsynaptic to the silent glomerulus receive substantial lateral excitatory input from other glomeruli. Genetically confining odor-evoked ORN input to just one glomerulus reveals that most PNs postsynaptic to other glomeruli receive indirect excitatory input from the single ORN type that is active. Lateral connections between identified glomeruli vary in strength, and this pattern of connections is stereotyped across flies. Thus, a dense network of lateral connections distributes odor-evoked excitation between channels in the first brain region of the olfactory processing stream.     

Eph Receptor Effector Ephexin Mediates Olfactory Dendrite Targeting in Drosophila

Deciphering the mechanisms of sensory neural map formation is a central aim in neurosciences. Failure to form a correct map frequently leads to defects in sensory processing and perception. The olfactory map develops in subsequent steps initially forming a rough and later a precise map of glomeruli in the antennal lobe (AL), mainly consisting of olfactory receptor neuron (ORN) axons and projection neuron (PN) dendrites. The mechanisms underpinning the later stage of class‐specific glomerulus formation are not understood. Recent studies have shown that the important guidance molecule Eph and its ligand ephrin play a role in class‐specific PN targeting. Here, we reveal aspects of the mechanism downstream of Eph signaling during olfactory map formation. We show that the Eph‐specific RhoGEF Ephexin (Exn) is required to fine tune PN dendrite patterning within specific glomeruli. We provide the first report showing an in vivo neurite guidance defect in an exn mutant. Interestingly, the quality of the phenotypes is different between eph and exn mutants; while loss of Eph leads to strong misprojections of DM3/Or47a neurons along the medial–lateral axis of the antennal lobe (AL), loss of Exn induces ventral ectopic innervation of a neighboring glomerulus. Genetic interaction experiments suggest that differential signaling of the small GTPases Rac1 and Cdc42 mediated by Exn‐dependent and ‐independent Eph signaling fine tunes spatial targeting of PN dendrites within the olfactory map. We propose that their distinct activities on the actin cytoskeleton are required for precise navigation of PN dendrites within the olfactory map. Taken together, our results suggest that the precise connectivity of an individual neuron can depend on different modes of signaling downstream of a single guidance receptor. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000–000, 2018     

Measuring activity in olfactory receptor neurons in Drosophila: Focus on spike amplitude

Olfactory responses at the receptor level have been thoroughly described in Drosophila melanogaster by electrophysiological methods. Single sensilla recordings (SSRs) measure neuronal activity in intact individuals in response to odors. For sensilla that contain more than one olfactory receptor neuron (ORN), their different spontaneous spike amplitudes can distinguish each signal under resting conditions. However, activity is mainly described by spike frequency.Some reports on ORN response dynamics studied two components in the olfactory responses of ORNs: a fast component that is reflected by the spike frequency and a slow component that is observed in the LFP (local field potential, the single sensillum counterpart of the electroantennogram, EAG). However, no apparent correlation was found between the two elements.In this report, we show that odorant stimulation produces two different effects in the fast component, affecting spike frequency and spike amplitude. Spike amplitude clearly diminishes at the beginning of a response, but it recovers more slowly than spike frequency after stimulus cessation, suggesting that ORNs return to resting conditions long after they recover a normal spontaneous spike frequency. Moreover, spike amplitude recovery follows the same kinetics as the slow voltage component measured by the LFP, suggesting that both measures are connected.These results were obtained in ab2 and ab3 sensilla in response to two odors at different concentrations. Both spike amplitude and LFP kinetics depend on odorant, concentration and neuron, suggesting that like the EAG they may reflect olfactory information.     

A major role for intracortical circuits in the strength and tuning of odor-evoked excitation in olfactory cortex

In primary sensory cortices, there are two main sources of excitation: afferent sensory input relayed from the periphery and recurrent intracortical input. Untangling the functional roles of these two excitatory pathways is fundamental for understanding how cortical neurons process sensory stimuli. Odor representations in the primary olfactory (piriform) cortex depend on excitatory sensory afferents from the olfactory bulb. However, piriform cortex pyramidal cells also receive dense intracortical excitatory connections, and the relative contribution of these two pathways to odor responses is unclear. Using a combination of in vivo whole-cell voltage-clamp recording and selective synaptic silencing, we show that the recruitment of intracortical input, rather than olfactory bulb input, largely determines the strength of odor-evoked excitatory synaptic transmission in rat piriform cortical neurons. Furthermore, we find that intracortical synapses dominate odor-evoked excitatory transmission in broadly tuned neurons, whereas bulbar synapses dominate excitatory synaptic responses in more narrowly tuned neurons.     

Functional Roles for Synaptic-Depression within a Model of the Fly Antennal Lobe

Several experiments indicate that there exists substantial synaptic-depression at the synapses between olfactory receptor neurons (ORNs) and neurons within the drosophila antenna lobe (AL). This synaptic-depression may be partly caused by vesicle-depletion, and partly caused by presynaptic-inhibition due to the activity of inhibitory local neurons within the AL. While it has been proposed that this synaptic-depression contributes to the nonlinear relationship between ORN and projection neuron (PN) firing-rates, the precise functional role of synaptic-depression at the ORN synapses is not yet fully understood. In this paper we propose two hypotheses linking the information-coding properties of the fly AL with the network mechanisms responsible for ORN[Formula: see text]AL synaptic-depression. Our first hypothesis is related to variance coding of ORN firing-rate information - once stimulation to the ORNs is sufficiently high to saturate glomerular responses, further stimulation of the ORNs increases the regularity of PN spiking activity while maintaining PN firing-rates. The second hypothesis proposes a tradeoff between spike-time reliability and coding-capacity governed by the relative contribution of vesicle-depletion and presynaptic-inhibition to ORN[Formula: see text]AL synaptic-depression. Synaptic-depression caused primarily by vesicle-depletion will give rise to a very reliable system, whereas an equivalent amount of synaptic-depression caused primarily by presynaptic-inhibition will give rise to a less reliable system that is more sensitive to small shifts in odor stimulation. These two hypotheses are substantiated by several small analyzable toy models of the fly AL, as well as a more physiologically realistic large-scale computational model of the fly AL involving [Formula: see text] glomerular channels.     

Olfactory neuron responsiveness and pheromone blend preference in hybrids between Ostrinia furnacalis and Ostrinia nubilalis (Lepidoptera: Crambidae)

The olfactory receptor neuron (ORN) and behavioral responses of hybrids between the Asian corn borer (ACB), Ostrinia furnacalis, and the E-strain European corn borer (ECB(E)), Ostrinia nubilalis were examined and compared to the parental populations. In hybrids and both parents, the large-spike-size ORN was capable of responding to all four pheromone components of ACB and ECB, despite differences in which compounds elicited the greatest spike frequency in each population. There was a small-spiking ORN more narrowly tuned to the minor pheromone components in both ACB and ECB(E). In hybrids the homologous small-spiking ORN was tuned primarily to the ECB(E) minor pheromone component, with some responsiveness to the ACB minor component. Both species and all the hybrids had an intermediate spike-size ORN tuned primarily to their common behavioral antagonist. Dominance of responsiveness to the ECB(E) versus the ACB minor pheromone component on the small-spiking ORN may explain the greater tendency of hybrids to fly upwind to the ECB(E) pheromone blend than the ACB blend. This finding points toward a distinct evolutionary role for this ORN in allowing a pheromone shift.     

The effect of some drugs on the mitral cell odor-evoked responses in the gecko olfactory bulb

The activity of odor-evoked olfactory mitral cell response of the gecko was recorded extracellularly by glass microelectrodes. The activities of the mitral cell observed during the presentation of the odor (n-amyl acetate) could be described as excitation, suppression or zero. The present experiments were undertaken to study the neural activities of the mitral cell in the olfactory bulb by perfusion application of some drugs (cobalt chloride, carnosine, norepinephrine, GABA and D-L-homocysteate) on the olfactory bulb surface or iontophoretic application of some drugs (carnosine, norepinephrine, GABA and D-L-homocysteate) to the glomerulus and the external plexiform layer to change the physiological environment. The effect of the drugs suggested that the synaptic neurons on the mitral cell have different chemical characteristics.     

Internalization of G Protein-Coupled Receptors in Single Olfactory Receptor Neurons

Abstract : Desensitization of many G protein-coupled receptors after ligand binding generally involves phosphorylation of the receptors and internalization of the ligandbound, phosphorylated receptors by a clathrin-mediated endocytic pathway. Olfactory receptor neurons from the channel catfish ( Ictalurus punctatus ) express the G protein-coupled odorant receptors and metabotropic glutamate receptors. To determine whether a clathrin-dependent receptor internalization pathway exists in olfactory receptor neurons, western blotting and immunocytochemistry were used to identify and localize clathrin and dynamin in isolated olfactory neurons. Clathrin and dynamin immunoreactivity was found in the cell bodies, dendrites, and dendritic knobs of the neurons. Using the activity-dependent fluorescent dye FM1-43 to monitor receptor internalization, we show that single olfactory neurons stimulated with the odorant amino acid l -glumate internalized the dye. Odorant-stimulated neurons showed a consistent pattern of internalized FM1-43 fluorescence localized in the cell bodies and dendritic knobs. Odorant-stimulated internalization was unaffected by the caveolae activator okadaic acid and was significantly decreased by a metabotropic glutamate receptor antagonist, suggesting that a functional, clathrindependent, receptor-mediated internalization pathway exists in olfactory receptor neurons.     

A large-scale model of the locust antennal lobe

The antennal lobe (AL) is the primary structure within the locust’s brain that receives information from olfactory receptor neurons (ORNs) within the antennae. Different odors activate distinct subsets of ORNs, implying that neuronal signals at the level of the antennae encode odors combinatorially. Within the AL, however, different odors produce signals with long-lasting dynamic transients carried by overlapping neural ensembles, suggesting a more complex coding scheme. In this work we use a large-scale point neuron model of the locust AL to investigate this shift in stimulus encoding and potential consequences for odor discrimination. Consistent with experiment, our model produces stimulus-sensitive, dynamically evolving populations of active AL neurons. Our model relies critically on the persistence time-scale associated with ORN input to the AL, sparse connectivity among projection neurons, and a synaptic slow inhibitory mechanism. Collectively, these architectural features can generate network odor representations of considerably higher dimension than would be generated by a direct feed-forward representation of stimulus space.     

Renewing olfactory receptor neurons in goldfish do not require contact with the olfactory bulb to develop normal chemical responsiveness

This study investigated whether contact with the olfactory bulb was necessary for developing and renewing olfactory receptor neurons (ORNs) to attain normal odorant responsiveness, and whether the anatomical and functional recoveries of the olfactory epithelium were similar in both bulbectomized (BE) and bilaterally axotomized (AX) preparations. In vivo electrophysiological recordings were obtained in response to amino acids, a bile acid [taurolithocholic acid sulfate(TLCS)] and a pheromonal odorant [17α, 20β,-dihydroxy-4-pregnen-3-one (17,20P)] from sexually immature goldfish. Both transmission and scanning electron microscopy indicated that the olfactory epithelium degenerated in BE and AX goldfish. Within 1–2 weeks subsequent to the respective surgeries, responses to high concentrations (>0.1 mmol · l⁻¹) of the more stimulatory amino acids remained, whereas responses were no longer obtainable to TLCS and 17,20P. At 4 weeks, responses to amino acid stimuli recovered to control levels, while responses to TLCS and 17,20P were minimal. By 7 weeks post bilateral axotomy, the olfactory epithelium recovered to a condition similar to control sensory epithelium; however, the rate of degeneration and proliferation of receptor neurons in BE preparations appeared to remain in balance, thus blocking further recovery of the olfactory epithelium. At 7 weeks post surgery, odorant responses of AX and BE goldfish to TLCS and 17,20P were still recovering. Accepted: 14 June 1997     

The role of <Emphasis Type="Italic">Dlx</Emphasis> homeogenes in early development of the olfactory pathway

Development of the olfactory pathway requires interaction between cells and signals of different origin. Olfactory receptor neurons (ORN) in the olfactory placodes (OP) extend axons towards the forebrain (FB); with innervation taking place at a later time following degradation of the basement membrane. Cells from the OP migrate along ORN axons and differentiate into various elements, including ensheathing and Gonadotropin Releasing Hormone (GnRH)+ cells. The importance of the olfactory connection and migration is highlighted by the severe endocrine phenotype in Kallmann’s patients who lack this migratory pathway. Little is known about the genetic control of intrinsic ORN properties. Inactivation of the distalless-related Dlx5 prevents connections between ORNs and FB. Using a grafting approach we show that misguidance and lack of connectivity is due to intrinsic defects in ORN neurites and migratory cells (MgC), and not to environmental factors. These data point to a cell-autonomous function of Dlx5 in providing ORN axons with their connectivity properties. Dlx5 also marks a population of early MgC that partly overlaps with the GnRH+ population. In the absence of Dlx5 MgCs of the Dlx5+ lineage migrate, associated with PSA-NCAM+ axons, but fail to reach the FB as a consequence of the lack of axonal connection and not an inability to migrate. These data suggests that Dlx5 is not required to initiate migration and differentiation of MgCs. An erratum to this article can be found at