The antennal lobes (ALs) are the primary olfactory centers in the insect brain. In the AL of the honeybee, olfactory glomeruli receive input via four antennal sensory tracts (T1-4). Axons of projection neurons (PNs) leave the AL via several antenno-cerebral tracts (ACTs). To assign the input-output connectivity of all glomeruli, we investigated the spatial relationship of the antennal tracts and two prominent AL output tracts (medial and lateral ACT) mainly formed by uniglomerular (u) PNs using fluorescent tracing, confocal microscopy, and 3D analyses. Furthermore, we investigated the projections of all ACTs in higher olfactory centers, the mushroom-bodies (MB) and lateral horn (LH). The results revealed a clear segregation of glomeruli into two AL hemispheres specifically supplied by PNs of the medial and lateral ACT. PNs of the lateral ACT innervate glomeruli in the ventral-rostral AL and primarily receive input from T1 (plus a few glomeruli from T2 and T3). PNs of the medial ACT innervate glomeruli in the dorsal-caudal hemisphere, and mainly receive input from T3 (plus a few glomeruli from T2 and T4). The PNs of the m- and l-ACT terminate in different areas of the MB calyx and LH and remain largely segregated. Tracing of three mediolateral (ml) ACTs mainly formed by multiglomerular PNs revealed terminals in distinct compartments of the LH and in three olfactory foci within the lateral protocerebrum. The results indicate that olfactory input in the honeybee is processed via two separate, mainly uPN pathways to the MB calyx and LH and several pathways to the lateral protocerebrum.

The anatomical substrates of neural nets are usually composed from reconstructions of neurons that were stained in different preparations. Realistic models of the structural relationships between neurons require a common framework. Here we present 3-D reconstructions of single projection neurons (PN) connecting the antennal lobe (AL) with the mushroom body (MB) and lateral horn, groups of intrinsic mushroom body neurons (type 5 Kenyon cells), and a single mushroom body extrinsic neuron (PE1), aiming to compose components of the olfactory pathway in the honeybee. To do so, we constructed a digital standard atlas of the bee brain. The standard atlas was created as an average-shape atlas of 22 neuropils, calculated from 20 individual immunostained whole-mount bee brains. After correction for global size and positioning differences by repeatedly applying an intensity-based nonrigid registration algorithm, a sequence of average label images was created. The results were qualitatively evaluated by generating average gray-value images corresponding to the average label images and judging the level of detail within the labeled regions. We found that the first affine registration step in the sequence results in a blurred image because of considerable local shape differences. However, already the first nonrigid iteration in the sequence corrected for most of the shape differences among individuals, resulting in images rich in internal detail. A second iteration improved on that somewhat and was selected as the standard. Registering neurons from different preparations into the standard atlas reveals 1) that the m-ACT neuron occupies the entire glomerulus (cortex and core) and overlaps with a local interneuron in the cortical layer; 2) that, in the MB calyces and the lateral horn of the protocerebral lobe, the axon terminals of two identified m-ACT neurons arborize in separate but close areas of the neuropil; and 3) that MB-intrinsic clawed Kenyon cells (type 5), with somata outside the calycal cups, project to the peduncle and lobe output system of the MB and contact (proximate) the dendritic tree of the PE1 neuron at the base of the vertical lobe. Thus the standard atlas and the procedures applied for registration serve the function of creating realistic neuroanatomical models of parts of a neural net. The Honeybee Standard Brain is accessible at www.neurobiologie.fu-berlin.de/beebrain.

Neural connections between the mushroom body (MB) and other protocerebral areas of the honeybee's brain were studied with the help of cobalt chloride and Golgi staining methods. Focal injections of cobalt ions into the alpha-lobe neuropil of the MB reveal seven clusters of somata located in the protocerebrum and deutocerebrum of each brain hemisphere. These neurons connect the mushroom body neuropil with protocerebral areas and number approximately 400. They contact the layered organization of the alpha-lobe at different locations. Some project not only into the alpha-lobe, but also into the beta-lobe and pedunculus neuropils. Fifteen cell types which form intraprotocerebral circuits are morphologically described. They can be divided into three categories: 1) unilateral neurons, with projection fields restricted to the ipsilateral protocerebrum; these neurons connect the alpha-lobe with areas in the protocerebral lobe and ramify with densely layered arborisations arranged perpendicularly to the longitudinal axis of the alpha-lobe; 2) recurrent neurons, which interconnect subcompartments of the MB, forming loops at different levels of the neuropil; their arborisations are mainly restricted to the alpha-lobe, beta-lobe, pedunculus, and calyces of the ipsilateral MB; they also ramify sparsely around the neuropil of the alpha-lobe; and 3) bilateral neurons, which either interconnect both alpha-lobes or connect the ipsilateral alpha-lobe and protocerebral lobe with the dorsolateral protocerebral lobe of the contralateral hemisphere. The connections of different compartments of the MB with other parts of the protocerebrum as revealed in this study are discussed in the context of hypotheses about the functional role of MBs in the honeybee brain.

To analyze morphologic and physiological properties of olfactory interneurons in the honeybee, Apis mellifera, antennal lobe (AL) neurons were intracellularly recorded and subsequently labeled with Neurobiotin. Additional focal injections were carried out with cobalt hexamine chloride and dextran fluorescent markers. Olfactory interneurons (projection neurons, PNs) project by means of five tracts, the lateral, the median, and three mediolateral antennocerebral tracts (l-, m-, and ml-ACT, respectively) to the mushroom bodies (MBs) and the protocerebral lobe (PL) of the ipsilateral protocerebrum. Uniglomerular PNs of the m- and l-ACT receiving input from a single glomerulus of the AL also arborize in different regions of the AL. The vast majority of l-ACT innervate the T1 region, whereas m-ACT neurons arborize exclusively in the T2, T3, and T4 regions (T1-4 : AL projection area of sensory cells from the antennae). In the calyces of the MB, uniglomerular PNs form varicosities in the basal ring and the lip region. Individual neurons of both types exhibit unequal innervation within and between the two calyces. In addition, m-ACT fibers ramify more densely within the lip neuropil and show a higher incidence of spine-like processes than l-ACTs. In the PL, l-ACTs arborize exclusively within the lateral horn, whereas some m-ACT neurons innervate a broader region. Multiglomerular neurons of the ml-ACT leave the AL by means of three subtracts (ml-ACT 1-3). Two different types can be distinguished according to their protocerebral target areas: ml-ACTs projecting to the lateral PL (LPL) and to the neuropil around the alpha-lobe (tracts 2 and 3) and neurons projecting only to the LPL (tract 1). Intracellular recordings indicate that both l- and m-ACT neurons respond to general odors but with different response properties, indicating that odor information is processed in parallel pathways with different functional characteristics. Just like m-ACT neurons, ml-ACT neurons respond to odors with complex activity patterns. Bilateral interneurons, originating in the suboesophageal ganglion, connect glomeruli of both AL, and send an axon through the m-ACT in each hemisphere of the brain, terminating in the lip region of the calyces. These neurons respond to contact chemical stimuli.

To internally reflect the sensory environment, animals create neural maps encoding the external stimulus space. From that primary neural code relevant information has to be extracted for accurate navigation. We analyzed how different odor features such as hedonic valence and intensity are functionally integrated in the lateral horn (LH) of the vinegar fly, Drosophila melanogaster. We characterized an olfactory-processing pathway, comprised of inhibitory projection neurons (iPNs) that target the LH exclusively, at morphological, functional and behavioral levels. We demonstrate that iPNs are subdivided into two morphological groups encoding positive hedonic valence or intensity information and conveying these features into separate domains in the LH. Silencing iPNs severely diminished flies' attraction behavior. Moreover, functional imaging disclosed a LH region tuned to repulsive odors comprised exclusively of third-order neurons. We provide evidence for a feature-based map in the LH, and elucidate its role as the center for integrating behaviorally relevant olfactory information.DOI: http://dx.doi.org/10.7554/eLife.04147.001

Although the neural circuit of the Drosophila AL has been intensively studied at both the input and the output level, the internal circuit is not yet well understood. An unambiguous characterization of LNs is essential to remedy this lack of knowledge. We used whole cell patch-clamp recordings and characterized four classes of LNs in detail using electrophysiological and morphological properties at the single neuron level. Each class of LN displayed unique characteristics in intrinsic electrophysiological properties, showing differences in firing patterns, degree of spike adaptation, and amplitude of spike afterhyperpolarization. Notably, one class of LNs had characteristic burst firing properties, whereas the others were tonically active. Morphologically, neurons from three classes innervated almost all glomeruli, while LNs from one class innervated a specific subpopulation of glomeruli. Three-dimensional reconstruction analyses revealed general characteristics of LN morphology and further differences in dendritic density and distribution within specific glomeruli between the different classes of LNs. Additionally, we found that LNs labeled by a specific enhancer trap line (GAL4-Krasavietz), which had previously been reported as cholinergic LNs, were mostly GABAergic. The current study provides a systematic characterization of olfactory LNs in Drosophila and demonstrates that a variety of inhibitory LNs, characterized by class-specific electrophysiological and morphological properties, construct the neural circuit of the AL.

Detecting danger is one of the foremost tasks for a neural system. Larval parasitoids constitute clear danger to Drosophila, as up to 80% of fly larvae become parasitized in nature. We show that Drosophila melanogaster larvae and adults avoid sites smelling of the main parasitoid enemies, Leptopilina wasps. This avoidance is mediated via a highly specific olfactory sensory neuron (OSN) type. While the larval OSN expresses the olfactory receptor Or49a and is tuned to the Leptopilina odor iridomyrmecin, the adult expresses both Or49a and Or85f and in addition detects the wasp odors actinidine and nepetalactol. The information is transferred via projection neurons to a specific part of the lateral horn known to be involved in mediating avoidance. Drosophila has thus developed a dedicated circuit to detect a life-threatening enemy based on the smell of its semiochemicals. Such an enemy-detecting olfactory circuit has earlier only been characterized in mice and nematodes.

Extracellular recording were performed from mushroom body-extrinsic neurons while the animal was exposed to differential conditioning to two odors, the forward-paired conditioned stimulus (CSϩ; the odor that will be or has been paired with sucrose reward) and the unpaired CSϪ (the odor that will be or has been specifically unpaired with sucrose reward). A single neuron, the pedunculus-extrinsic neuron number 1 (PE1), was identified on the basis of its firing pattern, and other neurons were grouped together as non-PE1 neurons. PE1 reduces its response to CSϩ and does not change its response to CSϪafter learning. Most non-PE1 neurons do not change their responses during learning, but some decrease, and one neuron increases its response to CSϩ. PE1 receives inhibitory synaptic inputs, and neuroanatomical studies indicate closely attached GABA-immune reactive profiles originating at least partially from neurons of the protocerebral-calycal tract (PCT). Thus, either the associative reduction of odor responses originates within the PE1 via a long-term depression (LTD)-like mechanism, or PE1 receives stronger inhibition for the learned odor from the PCT neurons or from Kenyon cells. In any event, as the decreased firing of PE1 correlates with the increased probability of behavioral responses, our data suggest that the mushroom bodies exert general inhibition over sensory-motor connections, which relaxes selectively for learned stimuli.