The larval brain of Drosophila is a useful model to study olfactory processing because of its cellular simplicity. The early stages of central olfactory processing involve the detection of odor features, but the coding mechanisms that transform them into a representation in higher brain centers is not clear. Here we examine the pattern of connectivity of the main neurons that process olfactory information in the calyx (dendritic region) of the mushroom bodies, a higher brain center essential for associative olfactory learning. The larval calyx has a glomerular organization. We generated a map of calyx glomeruli, using both anatomical criteria and the pattern of innervation by subsets of its input neurons (projection neurons), molecularly identified by GAL4 markers. Thus, we show that projection neurons innervate calyx glomeruli in a stereotypic manner. By contrast, subsets of mushroom body neurons (Kenyon cells) that are labeled by GAL4 markers show no clear preference for specific glomeruli. Clonal subsets of Kenyon cells show some preference for subregions of the calyx, implying that they receive distinct input. However, at the level of individual glomeruli, dendritic terminals of larval-born Kenyon cells innervate about six glomeruli, apparently randomly. These results are consistent with a model in which Kenyon cells process olfactory information by integrating different inputs from several calyx glomeruli in a combinatorial manner.Kenyon cells ͉ MARCM ͉ olfactory learning ͉ projection neurons ͉ antennal lobe M uch is known about the early steps in detection of olfactory signals and about the circuitry that processes them in the first olfactory relay center of the brain, the antennal lobe (AL) in Drosophila or the olfactory bulb in mammals (1). From these centers, information is carried by second-order olfactory neurons to the secondary olfactory centers: by projection neurons (PNs) to the mushroom body (MB) and lateral horn in Drosophila (2-4) and by mitral͞tufted cells to a number of areas of olfactory cortex in mammals (5). Second-order neurons have stereotypic target areas within at least some of these secondary centers (2-4, 6, 7), and therefore carry a spatial representation of the olfactory map to there. However, the organization of the circuitry at secondary centers, which underlies information processing in both systems, is not well understood. Work in Drosophila and locusts suggests that, in contrast to the broad odor response tuning of PNs, the responses of MB neurons (Kenyon cells, KCs) to the same odors are usually rare and selective (8-10), and electrophysiological studies (9) suggest a model in which KCs act as coincidence detectors of odor input from PNs. In the mammalian piriform cortex, both anatomical and physiological data suggest that olfactory cortical neurons also integrate olfactory information in a combinatorial manner (reviewed in ref. 5).To understand connectivity in the olfactory circuitry, Drosophila offers a simpler system with fewer cells than in mammals. In the larva, single o...

Odor discrimination in higher brain centers is essential for behavioral responses to odors. One such center is the mushroom body (MB) of insects, which is required for odor discrimination learning. The calyx of the MB receives olfactory input from projection neurons (PNs) that are targets of olfactory sensory neurons (OSNs) in the antennal lobe (AL). In the calyx, olfactory information is transformed from broadly-tuned representations in PNs to sparse representations in MB neurons (Kenyon cells). However, the extent of stereotypy in olfactory representations in the calyx is unknown. Using the anatomically-simple larval olfactory system of Drosophila in which odor ligands for the entire set of 21 OSNs are known, we asked how odor identity is represented in the MB calyx. We first mapped the projections of all larval OSNs in the glomeruli of the AL, and then followed the connections of individual PNs from the AL to different calyx glomeruli. We thus established a comprehensive olfactory map from OSNs to a higher olfactory association center, at a single-cell level. Stimulation of single OSNs evoked strong neuronal activity in 1 to 3 calyx glomeruli, showing that broadening of the strongest PN responses is limited to a few calyx glomeruli. Stereotypic representation of single OSN input in calyx glomeruli provides a mechanism for MB neurons to detect and discriminate olfactory cues.calyx ͉ genetically-encoded calcium indicator ͉ olfactory sensory neurons ͉ projection neurons W e now understand much about how odor information is detected and conveyed to the brain by sensory neurons, but less about how this information is represented in higher brain centers to influence behavioral outputs. The mushroom body (MB) of insects, which in Drosophila is essential for odor discrimination learning, provides a model to understand olfactory coding in higher association centers (1, 2). In the periphery, odor identity is detected by sets of olfactory sensory neurons (OSNs) whose specificities are determined by the olfactory receptor (OR) that they express (3, 4). OSNs that express the same OR converge on defined glomeruli in the first olfactory center of the brain, the antennal lobe (AL), analogously to the convergence of OSNs on olfactory bulb glomeruli in mammals. Projection neurons (PNs) then carry olfactory information from single AL glomeruli to the higher brain, the MB, and the lateral horn. However, excitatory interneurons that innervate multiple AL glomeruli lead to broadening of PN specificity compared with OSNs (5, 6). PNs then connect to Kenyon cells (KCs) in the calyx of the MB, where the representation of odor qualities is radically transformed; individual KCs respond much more selectively to odors than do either OSNs or PNs (7-9).The extent of stereotypy in olfactory processing in the calyx has been a matter of debate, in contrast to the clearly stereotypic connections between OSNs and PNs in the AL. In Drosophila adults, apparently stereotypic projections of PNs and KCs have been defined anatomically only at the leve...

Inhibition has a central role in defining the selectivity of the responses of higher order neurons to sensory stimuli. However, the circuit mechanisms of regulation of these responses by inhibitory neurons are still unclear. In Drosophila, the mushroom bodies (MBs) are necessary for olfactory memory, and by implication for the selectivity of learned responses to specific odors. To understand the circuitry of inhibition in the calyx (the input dendritic region) of the MBs, and its relationship with MB excitatory activity, we used the simple anatomy of the Drosophila larval olfactory system to identify any inhibitory inputs that could contribute to the selectivity of MB odor responses. We found that a single neuron accounts for all detectable GABA innervation in the calyx of the MBs, and that this neuron has pre-synaptic terminals in the calyx and post-synaptic branches in the MB lobes (output axonal area). We call this neuron the larval anterior paired lateral (APL) neuron, because of its similarity to the previously described adult APL neuron. Reconstitution of GFP partners (GRASP) suggests that the larval APL makes extensive contacts with the MB intrinsic neurons, Kenyon Cells (KCs), but few contacts with incoming projection neurons (PNs). Using calcium imaging of neuronal activity in live larvae, we show that the larval APL responds to odors, in a mannner that requires output from KCs. Our data suggest that the larval APL is the sole GABAergic neuron that innervates the MB input region and carries inhibitory feedback from the MB output region, consistent with a role in modulating the olfactory selectivity of MB neurons.

Abstract. Leech neurons in culture sprout rapidly when attached to extracts from connective tissue surrounding the nervous system. Laminin-like molecules that promote sprouting have now been isolated from this extracellular matrix. Two mAbs have been prepared that react on immunoblots with a =220-and a =340-kD polypeptide, respectively. These antibodies have been used to purify molecules with cross-shaped structures in the electron microscope. The molecules, of =103 kD on nonreducing SDS gels, have subunits of =340, 220, and 160-180 kD. Attachment to the laminin-like molecules was sufficient to initiate sprouting by single isolated leech neurons in defined medium. This demonstrates directly a function for a laminin-related invertebrate protein. The mAbs directed against the =220-kD chains of the lamininlike leech molecule labeled basement membrane extracellular matrix in leech ganglia and nerves. A polyclonal antiserum against the =220-kD polypeptide inhibited neurite outgrowth. Vertebrate laminin did not mediate the sprouting of leech neurons; similarly, the leech molecule was an inert substrate for vertebrate neurons. Although some traits of structure, function, and distribution are conserved between vertebrate laminin and the invertebrate molecule, our results suggest that the functional domains differ.