Scanning Laser Optical Tomography Resolves Structural Plasticity during Regeneration in an Insect Brain

Motherhood induces a drastic, sometimes long-lasting, change in internal state and behavior in many female animals. How a change in reproductive state or the discrete event of mating modulates specific female behaviors is still incompletely understood. Using calcium imaging of the whole brain of Drosophila females, we find that mating does not induce a global change in brain activity. Instead, mating modulates the pheromone response of dopaminergic neurons innervating the fly’s learning and memory center, the mushroom body (MB). Using the mating-induced increased attraction to the odor of important nutrients, polyamines, we show that disruption of the female fly’s ability to smell, for instance the pheromone cVA, during mating leads to a reduction in polyamine preference for days later indicating that the odor environment at mating lastingly influences female perception and choice behavior. Moreover, dopaminergic neurons including innervation of the β’1 compartment are sufficient to induce the lasting behavioral increase in polyamine preference. We further show that MB output neurons (MBON) of the β’1 compartment are activated by pheromone odor and their activity during mating bidirectionally modulates preference behavior in mated and virgin females. Their activity is not required, however, for the expression of polyamine attraction. Instead, inhibition of another type of MBON innervating the β’2 compartment enables expression of high odor attraction. In addition, the response of a lateral horn (LH) neuron, AD1b2, which output is required for the expression of polyamine attraction, shows a modulated polyamine response after mating. Taken together, our data in the fly suggests that mating-related sensory experience regulates female odor perception and expression of choice behavior through a dopamine-gated learning circuit.

Olfactory receptor neurons (ORNs) provide an animal with information about odors in its environment. In mammals and insects, individual ORNs mostly express a single olfactory receptor (OR). ORNs expressing the same OR gene form subpopulations, converging on one or a few spatially invariant glomeruli. Glomeruli are spherical subunits of the olfactory bulb (vertebrates) or antennal lobe (AL, insects), the first olfactory processing center in the brain. Within a glomerulus ORNs make synaptic contacts with inhibitory GABAergic neurons local to the OB/AL and 2nd order neurons relaying the olfactory information to higher brain centers. The basis for understanding how odors are coded within olfactory systems is knowledge about its input. In order to describe the input to olfactory systems we need to know the odors activating or inactivating all subpopulations of ORNs, defined as their molecular receptive ranges (MRR). To this end I characterized the MRR of an exemplary ORN subpopulation in great detail using the model organism Drosophila melanogaster. Employing the UAS-Gal4 system, I expressed Cameleon2.1, a Ca2+ sensitive fluorescent dye, under control of the OR gene promotor Or22a. This allowed me to monitor selectively the odor-evoked responses of the ORNs expressing this OR in vivo by using optical imaging. I tested a panel of 104 odors from a variety of chemical classes. Odor evoked responses were measured from the primary dendrites and somata of the ORNs on the antenna and from their axonal terminals in the glomeruli of the AL. The results showed graded responses to 39 stimuli for which I subsequently established dose-response curves. The most potent odors were ethyl and methyl hexanoate. These odor molecules elicited responses at dilutions as low as 1:100,000,000, thereby showing that Or22a has a broad yet selective MRR challenging the distinction between specialist and generalist ORNs. By systematically testing another 15 structurally very similar odors I was able to determine some molecular determinants of the MRR. These observations were corroborated by a modeling approach in collaboration with Daniel Baum from the Zuse Institute Berlin (ZIB) who developed an algorithm for semi-flexible superpositioning of 3D molecular structures. Comparing the MRR measured in the dendrites and somata on the antenna with the MRR obtained at the axonal endings within the AL, showed that both were identical. This finding further substantiated the notion that the MRR of a glomerulus reflects the odor specificity of the OR expressed in the ORNs innervating it. However, a detailed analysis of antennal and AL response kinetics revealed differences e.g. in fall time and response duration. In order to test whether these temporal differences were due to a shaping role of inhibitory AL local neurons, I applied the ionotropic GABA agonist muscimol and the Cl- channel blocker picrotoxin. I found evidence for odor-specific presynaptic inhibition of ORN responses. While presynaptic inhibition changed the amplitude of the responses, fall time and response duration remained stable. Thus the differences in kinetics of antennal and AL responses could be attributed to different Ca2+ sources contributing to either signal. Finally, I started the characterization of another OR, namely Or47b. This OR is in many regards different from Or22a and of special interest because to date hardly anything is known about the physiology of ORNs expressing Or47b. I identified several ligands the majority of which was inactivating. These results show the coding complexity of the olfactory system already at the input level. This study represents a further step towards understanding odor coding in the periphery of the Drosophila olfactory system. Furthermore, it provides a sound basis for further exploring the role of the AL network.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions. Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP and Cameleon, the bioluminescent calcium indicator GFP-aequorin, or a reporter of synaptic transmission, synapto-pHluorin has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS, LexA/LexAop, Q system) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed. Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Comparative Development of the Ant Chemosensory System

New insights into an ancient insect nose: The olfactory pathway of Lepismachilis y-signata (Archaeognatha: Machilidae)

We examined the development of olfactory neuropils in the hemimetabolous insect Locusta migratoria with an emphasis on the mushroom bodies, protocerebral integration centers implicated in memory formation. Using a marker of the cyclic adenosine monophosphate (cAMP) signaling cascade and lipophilic dye labeling, we obtained new insights into mushroom body organization by resolving previously unrecognized accessory lobelets arising from Class III Kenyon cells. We utilized antibodies against axonal guidance cues, such as the cell surface glycoproteins Semaphorin 1a (Sema 1a) and Fasciclin I (Fas I), as embryonic markers to compile a comprehensive atlas of mushroom body development. During embryogenesis, all neuropils of the olfactory pathway transiently expressed Sema 1a. The immunoreactivity was particularly strong in developing mushroom bodies. During late embryonic stages, Sema 1a expression in the mushroom bodies became restricted to a subset of Kenyon cells in the core region of the peduncle. Sema 1a was differentially sorted to the Kenyon cell axons and absent in the dendrites. In contrast to Drosophila, locust mushroom bodies and antennal lobes expressed Fas I, but not Fas II. While Fas I immunoreactivity was widely distributed in the midbrain during embryogenesis, labeling persisted into adulthood only in the mushroom bodies and antennal lobes. Kenyon cells proliferated throughout the larval stages. Their neurites retained the embryonic expression pattern of Sema 1a and Fas I, suggesting a role for these molecules in developmental mushroom body plasticity. Our study serves as an initial step toward functional analyses of Sema 1a and Fas I expression during locust mushroom body formation.

In vertebrates and many invertebrates, olfactory signals detected by peripheral olfactory receptor neurons (ORNs) are conveyed to a primary olfactory center with glomerular organization in which odor-specific activity patterns are generated. In the cockroach, Periplaneta americana, ORNs in antennal olfactory sensilla project to 205 unambiguously identifiable antennal lobe (AL) glomeruli that are classified into 10 glomerular clusters (T1-T10 glomeruli) innervated by distinct sensory tracts. In this study we employed single sensillum staining techniques and investigated the topographic projection patterns of individual ORNs to elucidate the relationship between sensillum types and glomerular organization in the AL. Axons of almost all ORNs projected to individual glomeruli. Axons of ORNs in perforated basiconic sensilla selectively innervated the anterodorsal T1-T4 glomeruli, whereas those in trichoid and grooved basiconic sensilla innervated the posteroventral T5-T9 glomeruli. About 90% of stained ORNs in trichoid sensilla sent axons to the T5 glomeruli and more than 90% of ORNs in grooved basiconic sensilla innervated the T6, T8, and T9 glomeruli. The T5 and T9 glomeruli exclusively receive sensory inputs from the trichoid and grooved basiconic sensilla, respectively. All investigated glomeruli received convergent input from a single type of sensillum except F11 glomerulus in the T6 glomeruli, which was innervated from both trichoid and grooved basiconic sensilla. These results suggest that ORNs in distinct sensillum types project to glomeruli in distinct glomerular clusters. Since ORNs in distinct sensillum types are each tuned to distinct subsets of odorant molecules, the AL is functionally compartmentalized into groups of glomeruli.

Global inhibition is a fundamental physiological mechanism that has been proposed to shape odor representation in higher-order olfactory centers. A pair of mushroom bodies (MBs) in insect brains, an analog of the mammalian olfactory cortex, are implicated in multisensory integration and associative memory formation. With the use of single/multiple intracellular recording and staining in the cockroach Periplaneta americana, we succeeded in unambiguous identification of four tightly bundled GABA-immunoreactive giant interneurons that are presumably involved in global inhibitory control of the MB. These neurons, including three spiking neurons and one nonspiking neuron, possess dendrites in termination fields of MB output neurons and send axon terminals back to MB input sites, calyces, suggesting feedback roles onto the MB. The largest spiking neuron innervates almost exclusively the basal region of calyces, while the two smaller spiking neurons and the second-largest nonspiking neuron innervate more profusely the peripheral (lip) region of the calyces than the basal region. This subdivision corresponds well to the calycal zonation made by axon terminals of two populations of uniglomerular projection neurons with dendrites in distinct glomerular groups in the antennal lobe. The four giant neurons exhibited excitatory responses to every odor tested in a neuron-specific fashion, and two of the neurons also exhibited inhibitory responses in some recording sessions. Our results suggest that two parallel olfactory inputs to the MB undergo different forms of inhibitory control by the giant neurons, which may, in turn, be involved in different aspects of odor discrimination, plasticity, and state-dependent gain control. J. Comp. Neurol. 525:204-230, 2017. © 2016 Wiley Periodicals, Inc.

Olfactory sensory neurons (OSNs) detect odours at a wide range of intensities. In Drosophila, volatile compounds bind to specific odorant receptors (ORs), which tune the sensitivity of chemoreception. To test whether additional mechanisms underlie odour-specific neuronal processing, we analysed the spatial distribution of ORs in dendrites and investigated OSN synapses in the antennal lobe, the first relay station of the olfactory pathway. Here, we studied the molecular structure and plasticity of the presynaptic active zone (AZ), the specialized site of neurotransmitter release. We focused on a highly sensitive OSN type that expresses the receptor Or56a and is exclusively activated by geosmin, an odorant signalling ecologically harmful microorganisms. Our results uncover a differential arrangement of dendritic ORs and core AZ proteins in alarm odour-detecting Or56a compared to conventional food-odour detecting OSNs. Interestingly, the data also show that Or56a OSNs display a limited capacity for homeostatic plasticity in response to a genetic reduction of presynaptic release probability. We hypothesise that this feature reflects the basal tuning of geosmin-sensing neurons towards maximum levels of performance.

The function of the olfactory receptor organs of insects is to send the central nervous system (CNS), on a split-second basis, a rendering of the relative abundance of single odorants that together comprise odour blends. The organs’ olfactory receptor neurons (ORNs) generate action potentials (spikes) that are transmitted to the antennal lobe (AL) (Chaps. 4 and 5), which then sends the processed signal on to higher integrative centers of the brain (Chap. 9). When an ORN is hit by molecules of an active stimulus, the molecules are thought to interact with receptor proteins situated in the dendritic membrane (Chap. 2). These interactions form a receptor potential that travels down the dendrite to a site where action potentials are triggered when the receptor potential reaches a threshold level. The action potentials travel down the length of the ORN axon into the AL (Chap. 5). The action potentials of particular ORNs have characteristic sizes and waveforms (Kaissling 1974) that often differ greatly enough among ORNs that researchers can discriminate them and assess their activities separately. Spike sizes may be related to the size of the ORNs generating them (Hansson et al. 1994b), but the size of the spike is not known to carry any extra information to the AL. It is the frequency of spikes rather than their size that is important for generating behavioural responses.

The mosquito Aedes aegypti is an important vector of diseases including dengue, Zika, chikungunya, and yellow fever. Olfaction is a critical modality for mosquitoes enabling them to locate hosts, sources of nectar, and sites for oviposition. GABA is an essential neurotransmitter in olfactory processing in the insect brain, including the primary olfactory center, the antennal lobe. Previous work with Ae. aegypti has suggested that antennal lobe inhibition via GABA may be involved in the processing of odors. However, little is known about GABA receptor expression in the mosquito brain, or how they may be involved in odor attraction. In this context, generating mutants that target the mosquito’s olfactory responses, and particularly the GABAergic system, is essential to achieve a better understanding of these diverse processes and olfactory coding in these disease vectors. Here we demonstrate the potential of a transgenic line using the QF2 transcription factor, GABA-B1QF2−ECFP, as a new neurogenetic tool to investigate the neural basis of olfaction in Ae. aegypti. Our results show that the gene insertion has a moderate impact on mosquito fitness. Moreover, the line presented here was crossed with a QUAS reporter line expressing the green fluorescent protein and used to determine the location of the metabotropic GABA-B1 receptor expression. We find high receptor expression in the antennal lobes, especially the cell bodies surrounding the antennal lobes. In the mushroom bodies, receptor expression was high in the Kenyon cells, but had low expression in the mushroom body lobes. Behavioral experiments testing the fruit odor attractants showed that the mutants lost their behavioral attraction. Together, these results show that the GABA-B1QF2−ECFP line provides a new tool to characterize GABAergic systems in the mosquito nervous system.

Drosophila senses diverse kinds of olfactory stimuli in the external environment. These stimuli are often not pure chemicals, but rather mixtures of many odorants. The fly olfactory system, however, needs to extract the requisite information from this complex stimulus. This is done by converting odor representations into meaningful, discrete categories in the perceptual space. The first olfactory relay system in the Drosophila brain is the antennal lobe (AL) which consists of olfactory sensory neurons (OSNs), projection neurons (PNs) and local interneurons (LNs), and these three populations of neurons interact and process the odor stimuli. To observe any transformations of odor representations in terms of odor classification, linearly varying odor stimuli were presented. This was achieved using binary mixtures such that one odor was "morphed" into another. Three binary mixtures were made from combinations of three distinct odorants ,i.e., 4-methylcyclohexanol (MCH), 3-Octanol (3-Oct) and Pentyl acetate (PA). In the first step, I determined and confirmed that each single odor-evoked distinct odor representations across its different concentrations in the AL, especially in the OSNs. A second requirement was to determine the degree of similarity between the three odors in both OSNs and PNs. From their odor representations, it was found that PA was more similar to 3-Oct compared to MCH in OSNs. Moreover, the similarity between PA and 3-Oct became even stronger in PNs. After determining the prerequisites of using PA, 3-Oct and MCH for binary mixtures, odor-evoked Ca2+ activity patterns for the three different types of binary mixtures were determined in both OSNs and PNs. Each binary odor mixture showed a different kind of odor classification. The MCH/3-Oct binary mixtures showed no odor classification in OSNs, but in PNs two distinct categories were observed. However, the same could not be found in either of the other two odor mixtures. For MCH/PA binary mixtures, no odor classification was found in OSNs. But in PNs, all mixtures having greater than 0% PA got categorized and distinctly separated from the 100% MCH-0% PA. This was referred to as odor dominance of PA. The last odor mixture was of the similar odor pair, 3-Oct/PA, in which all the intermediate mixtures formed one cluster in both OSNs and PNs. This was due to the high degree of similarity already present between the individual odors in OSNs and PNs. The glomeruli in the AL are interconnected by a population of GABAergic LNs and they are known from previous studies to be involved in odor processing. GABA binds to two kinds of receptors: GABAA and GABAB. GABAA receptors are known to be involved in the early phase of the odor processing and they can be pharmacologically blocked by picrotoxin. Therefore, fly brains were subjected to picrotoxin solution and its effects were observed on the three types of odor classification found with the combination of the selected odors. GABAergic inhibition mediated via GABAA receptors was found to be required for the odor classification of binary mixtures of MCH/3-Oct. However, no changes were found due to picrotoxin on MCH/PA and 3-Oct/PA binary mixtures. In conclusion, different kinds of odor classification could be observed for different odors in the AL of the fly. GABAergic inhibition has been found to play a role in mediating odor classification of different stimuli. However, not every odor classification requires GABAergic inhibition, as was observed in the case of MCH/PA binary mixtures, thereby also suggesting an existence of other possible mechanisms for odor classification.

The mouse olfactory system employs ~1100 G‐protein‐coupled odorant receptors (ORs). Each mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and the expressed OR determines the odorant response properties of the OSN. The broadest odorant response profile thus far demonstrated in native mouse OSNs is for OSNs that express the OR gene SR1 (also known as Olfr124 and MOR256‐3). Here we showed that the odorant responsiveness of native mouse OSNs expressing the OR gene MOR256‐17 (also known as Olfr15 and OR3) is even broader than that of OSNs expressing SR1. We investigated the electrophysiological properties of green fluorescent protein (GFP)+ OSNs in a MOR256‐17‐IRES‐tauGFP gene‐targeted mouse strain, in parallel with GFP+ OSNs in the SR1‐IRES‐tauGFP gene‐targeted mouse strain that we previously reported. Of 35 single chemical compounds belonging to distinct structural classes, MOR256‐17+ OSNs responded to 31 chemicals, compared with 10 for SR1+ OSNs. The 10 compounds that activated SR1+ OSNs also activated MOR256‐17+ OSNs. Interestingly, MOR256‐17+ OSNs were activated by three amines (cyclohexylamine, isopenthylamine, and phenylethylamine) that are typically viewed as ligands for chemosensory neurons in the main olfactory epithelium that express trace amine‐associated receptor genes, a family of 15 genes encoding G‐protein‐coupled receptors unrelated in sequence to ORs. We did not observe differences in membrane properties, indicating that the differences in odorant response profiles between the two OSN populations were due to the expressed OR. MOR256‐17+ OSNs appear to be at one extreme of odorant responsiveness among populations of OSNs expressing distinct OR genes in the mouse.

The mushroom bodies are regions in the insect brain involved in processing complex multimodal information. They are composed of many parallel sets of intrinsic neurons that receive input from and transfer output to extrinsic neurons that connect the mushroom bodies with the surrounding neuropils. Mushroom bodies are particularly large in social Hymenoptera and are thought to be involved in the control of conspicuous orientation, learning, and memory capabilities of these insects. The present account compares the organization of sensory input to the mushroom body's calyx in different Hymenoptera. Tracer and conventional neuronal staining procedures reveal the following anatomic characteristics: The calyx comprises three subdivisions, the lip, collar, and basal ring. The lip receives antennal lobe afferents, and these olfactory input neurons can terminate in two or more segregated zones within the lip. The collar receives visual afferents that are bilateral with equal representation of both eyes in each calyx. Visual inputs provide two to three layers of processes in the collar subdivision. The basal ring is subdivided into two modality-specific zones, one receiving visual, the other antennal lobe input. Some overlap of modality exists between calycal subdivisions and within the basal ring, and the degree of segregation of sensory input within the calyx is species-specific. The data suggest that the many parallel channels of intrinsic neurons may each process different aspects of sensory input information.

Chemosensory perception involves the detection of chemical compounds. In animals, there are 2 chemical senses: taste, and olfaction. The two are related in that they utilize ligand-gated receptors, expressed in primary sensory neurons, to detect chemical stimuli from the surrounding environment. However, the processing of these inputs is quite different in the two systems, leading to divergent roles for olfaction and taste in sensory perception. This dissertation highlights some of these differences, by looking at processing of ethologically relevant stimuli at the very peripheral receptor neurons. The work is divided into 2 parts: water sensing by the mammalian taste system, and CO₂ sensing by the Drosophila olfactory system. In Chapter 1, I talk about water sensing in the mammalian taste system. Initiation of drinking behavior relies on peripheral water detection. It is likely that this detection is mediated, at least in part, by the taste system. Here, I have shown that acid-sensing taste receptor cells (TRCs) that were previously suggested as the sensors for sour taste, also respond to water. This response is mediated by a bicarbonate-dependent molecular mechanism, likely involving the Carbonic Anhydrase enzyme family. Furthermore, optogenetic stimulation of the acid-sensing TRCs in thirsty animals induces robust licking responses towards the light source, even in the absence of water. Conversely, thirsty animals lacking functional acid-sensing TRCs show compromised discrimination between water and non-aqueous fluids. Taken together, this work reveals the cellular mechanism of water detection by the mammalian taste system. In chapter 2, I talk about CO₂ sensing in the fruit fly. The Drosophila olfactory system responds to most odors with the activation of a large subset of its olfactory receptors (ORs). This broad activation is a consequence of the ORs having affinity to multiple chemical compounds. In contrast, a small number of odors, like CO₂, elicit responses in only single ORs. These ORs are, in contrast to most ORs, very narrowly tuned, generally responding only to that one odor. It has been assumed up until now that the specificity of these unique ORs is inherited by the olfactory receptor neurons (ORNs) they are expressed in, and even in the projection neurons (PNs), that the ORNs synapse onto. I show here that CO₂, though it activates only a single OR, the GR63a/GR21a hetero-dimer complex, actually activates multiple ORN axon terminals. This activation is due to lateral excitatory connections between axon terminals of the GR63a/GR21a expressing ORNs, and at least 4 other ORN types. Focusing on one of these ORNs, Ab1B, I show the lateral connections bypass the ORN cell bodies, only driving responses at the axon terminals. Consequently, Ab1B ORN axon terminals receive 2 sources of excitatory input, a feed-forward excitation from its endogenous OR, and a lateral excitation from GR63a/GR21a. This effectively divides the ORN into 2 compartments, distinct in their odor tuning. Finally, I show that lateral excitation is a general feature of the ORN circuit by silencing the feed-forward input of another ORN class, Ab1A. The Ab1A cell body is completely silent, but the axon terminals retain odor responses from lateral excitatory inputs. Thus, there is a lateral flow of odor information between multiple ORNs of the Drosophila olfactory system.