Mushroom body output neurons MBON-a1/a2 define an odor intensity channel that regulates behavioral odor discrimination learning in larval Drosophila.

The sensitivity of animals to sensory input must be regulated to ensure that signals are detected and also discriminable. However, how circuits regulate the dynamic range of sensitivity to sensory stimuli is not well understood. A given odor is represented in the insect mushroom bodies (MBs) by sparse combinatorial coding by Kenyon cells (KCs), forming an odor quality representation. To address how intensity of sensory stimuli is processed at the level of the MB input region, the calyx, we characterized a set of novel mushroom body output neurons that respond preferentially to high odor concentrations. We show that a pair of MB calyx output neurons, MBON-a1/2, are postsynaptic in the MB calyx, where they receive extensive synaptic inputs from KC dendrites, the inhibitory feedback neuron APL, and octopaminergic sVUM1 neurons, but relatively few inputs from projection neurons. This pattern is broadly consistent in the third-instar larva as well as in the first instar connectome. MBON-a1/a2 presynaptic terminals innervate a region immediately surrounding the MB medial lobe output region in the ipsilateral and contralateral brain hemispheres. By monitoring calcium activity using jRCamP1b, we find that MBON-a1/a2 responses are odor-concentration dependent, responding only to ethyl acetate (EA) concentrations higher than a 200-fold dilution, in contrast to MB neurons which are more concentration-invariant and respond to EA dilutions as low as 10-4. Optogenetic activation of the calyx-innervating sVUM1 modulatory neurons originating in the SEZ (Subesophageal zone), did not show a detectable effect on MBON-a1/a2 odor responses. Optogenetic activation of MBON-a1/a2 using CsChrimson impaired odor discrimination learning compared to controls. We propose that MBON-a1/a2 form an output channel of the calyx, summing convergent sensory and modulatory input, firing preferentially to high odor concentration, and might affect the activity of downstream MB targets.

Octopaminergic neurons have multiple targets in Drosophila larval mushroom body calyx and can modulate behavioral odor discrimination.

Discrimination of sensory signals is essential for an organism to form and retrieve memories of relevance in a given behavioral context. Sensory representations are modified dynamically by changes in behavioral state, facilitating context-dependent selection of behavior, through signals carried by noradrenergic input in mammals, or octopamine (OA) in insects. To understand the circuit mechanisms of this signaling, we characterized the function of two OA neurons, sVUM1 neurons, that originate in the subesophageal zone (SEZ) and target the input region of the memory center, the mushroom body (MB) calyx, in larval Drosophila We found that sVUM1 neurons target multiple neurons, including olfactory projection neurons (PNs), the inhibitory neuron APL, and a pair of extrinsic output neurons, but relatively few mushroom body intrinsic neurons, Kenyon cells. PN terminals carried the OA receptor Oamb, a Drosophila α1-adrenergic receptor ortholog. Using an odor discrimination learning paradigm, we showed that optogenetic activation of OA neurons compromised discrimination of similar odors but not learning ability. Our results suggest that sVUM1 neurons modify odor representations via multiple extrinsic inputs at the sensory input area to the MB olfactory learning circuit.

A single GABAergic neuron mediates feedback of odor-evoked signals in the mushroom body of larval Drosophila.

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 manner 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.

Targeting expression to projection neurons that innervate specific mushroom body calyx and antennal lobe glomeruli in larval Drosophila.

The first and secondary olfactory centers in the olfactory pathway in Drosophila are organized into neuropil structures called glomeruli. The antennal lobe (AL), the first olfactory center in larval Drosophila, is organized in 21 glomeruli. Each AL glomerulus receives innervation from a specific olfactory sensory neuron (OSN), and is therefore identifiable anatomically by the position of the OSN terminal. Olfactory projection neurons (PNs) send a dendrite to a single AL glomerulus and an axon that usually terminates in a single glomerulus in the mushroom body (MB) calyx, a secondary olfactory center, and in the lateral horn. By random labeling of single PNs that express GH146-GAL4, it was previously shown that PNs stereotypically innervate specific AL and calyx glomeruli, and most of these connections have been mapped. Here we report the pattern of innervation of GAL4 lines that drive expression of reporter genes in single or a few PNs, including PNs not identified by the widely used GH146-GAL4 driver. We have mapped the AL and calyx glomeruli innervated by these labeled PNs. This study provides a collection of GAL4 lines to molecularly mark the connections between specific AL and calyx glomeruli. It thus confirms and extends the previous map of AL-calyx connectivity that was based only on randomly labeled single PNs, and provides tools for targeted manipulation of specific PNs for developmental and functional studies.

Localized olfactory representation in mushroom bodies of Drosophila larvae.

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.

Stereotypic and random patterns of connectivity in the larval mushroom body calyx of Drosophila.

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.

Embryonic and larval development of the Drosophila mushroom bodies: concentric layer subdivisions and the role of fasciclin II.

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the arthropod brain. In order to understand the cellular and genetic processes that control the early development of MBs, we have performed high-resolution neuroanatomical studies of the embryonic and post-embryonic development of the Drosophila MBs. In the mid to late embryonic stages, the pioneer MB tracts extend along Fasciclin II (FAS II)-expressing cells to form the primordia for the peduncle and the medial lobe. As development proceeds, the axonal projections of the larval MBs are organized in layers surrounding a characteristic core, which harbors bundles of actin filaments. Mosaic analyses reveal sequential generation of the MB layers, in which newly produced Kenyon cells project into the core to shift to more distal layers as they undergo further differentiation. Whereas the initial extension of the embryonic MB tracts is intact, loss-of-function mutations of fas II causes abnormal formation of the larval lobes. Mosaic studies demonstrate that FAS II is intrinsically required for the formation of the coherent organization of the internal MB fascicles. Furthermore, we show that ectopic expression of FAS II in the developing MBs results in severe lobe defects, in which internal layers also are disrupted. These results uncover unexpected internal complexity of the larval MBs and demonstrate unique aspects of neural generation and axonal sorting processes during the development of the complex brain centers in the fruit fly brain.