Principal component analysis of odor coding at the level of third‐order olfactory neurons in Drosophila

Olfactory information in Drosophila is conveyed by projection neurons from olfactory sensory neurons to Kenyon cells (KCs) in the mushroom body (MB). A subset of KCs responds to a given odor molecule, and the combination of these KCs represents a part of the neuronal olfactory code. KCs are also thought to function as coincidence detectors for memory formation, associating odor information with a coincident punishment or reward stimulus. Associative conditioning has been shown to modify KC output. This plasticity occurs in the vertical lobes of MBs containing α/α' branches of KCs, which is shown by measuring the average Ca²⁺ levels in the branch of each lobe. We devised a method to quantitatively describe the population activity patterns recorded from axons of >1000 KCs at the α/α' branches using two‐photon Ca²⁺ imaging. Principal component analysis of the population activity patterns clearly differentiated the responses to distinct odors.     

Mushroom body efferent neurons responsible for aversive olfactory memory retrieval in Drosophila

Aversive olfactory memory is formed in the mushroom bodies in Drosophila melanogaster. Memory retrieval requires mushroom body output, but the manner in which a memory trace in the mushroom body drives conditioned avoidance of a learned odor remains unknown. To identify neurons that are involved in olfactory memory retrieval, we performed an anatomical and functional screen of defined sets of mushroom body output neurons. We found that MB-V2 neurons were essential for retrieval of both short- and long-lasting memory, but not for memory formation or memory consolidation. MB-V2 neurons are cholinergic efferent neurons that project from the mushroom body vertical lobes to the middle superiormedial protocerebrum and the lateral horn. Notably, the odor response of MB-V2 neurons was modified after conditioning. As the lateral horn has been implicated in innate responses to repellent odorants, we propose that MB-V2 neurons recruit the olfactory pathway involved in innate odor avoidance during memory retrieval.     

Sensory processing in the Drosophila antennal lobe increases reliability and separability of ensemble odor representations

Here we describe several fundamental principles of olfactory processing in the Drosophila melanogaster antennal lobe (the analog of the vertebrate olfactory bulb), through the systematic analysis of input and output spike trains of seven identified glomeruli. Repeated presentations of the same odor elicit more reproducible responses in second-order projection neurons (PNs) than in their presynaptic olfactory receptor neurons (ORNs). PN responses rise and accommodate rapidly, emphasizing odor onset. Furthermore, weak ORN inputs are amplified in the PN layer but strong inputs are not. This nonlinear transformation broadens PN tuning and produces more uniform distances between odor representations in PN coding space. In addition, portions of the odor response profile of a PN are not systematically related to their direct ORN inputs, which probably indicates the presence of lateral connections between glomeruli. Finally, we show that a linear discriminator classifies odors more accurately using PN spike trains than using an equivalent number of ORN spike trains.     

Mutations in the Drosophila insulin receptor substrate, CHICO, impair olfactory associative learning

CHICO, the Drosophila homolog of vertebrate insulin receptor substrate (IRS), mediates insulin/insulin-like growth factor signaling (IIS), and reductions in chico severely disrupt cell growth and proliferation. We found extensive expression of chico in various Drosophila brain regions including the mushroom bodies (MBs), critical neural structures for olfactory learning. chico null mutants have significantly reduced brain sizes and perform poorly in an olfactory associative learning task, although their sensitivity to the odors and electric shocks used in this learning paradigm are normal. When initial memory is normalized by training for different amounts of time (short-duration training protocols), memory retention and retrieval in chico flies are indistinguishable from that of wild-type flies, demonstrating that chico mutants are defective specifically for memory formation. Inducing expression of a chico(+) transgene in neurons throughout development restores normal learning in a chico background, while inducing chico(+) specifically at the adult stage does not, suggesting that chico is required for development of a brain region required for forming olfactory associations. Significantly, expressing chico(+) in the MBs restores the number of MB neurons to wild-type amounts and also rescues chico learning defects. Our results suggest that chico-dependent growth of the MBs is essential for development of learning ability.     

Specific requirement of NMDA receptors for long-term memory consolidation in Drosophila ellipsoid body

In humans and many other animals, memory consolidation occurs through multiple temporal phases and usually involves more than one neuroanatomical brain system. Genetic dissection of Pavlovian olfactory learning in Drosophila melanogaster has revealed multiple memory phases, but the predominant view holds that all memory phases occur in mushroom body neurons. Here, we demonstrate an acute requirement for NMDA receptors (NMDARs) outside of the mushroom body during long-term memory (LTM) consolidation. Targeted dsRNA-mediated silencing of Nmdar1 and Nmdar2 (also known as dNR1 or dNR2, respectively) in cholinergic R4m-subtype large-field neurons of the ellipsoid body specifically disrupted LTM consolidation, but not retrieval. Similar silencing of functional NMDARs in the mushroom body disrupted an earlier memory phase, leaving LTM intact. Our results clearly establish an anatomical site outside of the mushroom body involved with LTM consolidation, thus revealing both a distributed brain system subserving olfactory memory formation and the existence of a system-level memory consolidation in Drosophila.     

Adaptive regulation of sparseness by feedforward inhibition

In the mushroom body of insects, odors are represented by very few spikes in a small number of neurons, a highly efficient strategy known as sparse coding. Physiological studies of these neurons have shown that sparseness is maintained across thousand-fold changes in odor concentration. Using a realistic computational model, we propose that sparseness in the olfactory system is regulated by adaptive feedforward inhibition. When odor concentration changes, feedforward inhibition modulates the duration of the temporal window over which the mushroom body neurons may integrate excitatory presynaptic input. This simple adaptive mechanism could maintain the sparseness of sensory representations across wide ranges of stimulus conditions.     

Importin-α2 mediates brain development,learning and memory consolidation in Drosophila

Abstract

Neuronal development and memory consolidation are conserved processes that rely on nuclear-cytoplasmic transport of signaling molecules to regulate gene activity and initiate cascades of downstream cellular events. Surprisingly, few reports address and validate this widely accepted perspective. Here we show that Importin-α2 (Imp-α2), a soluble nuclear transporter that shuttles cargoes between the cytoplasm and nucleus, is vital for brain development, learning and persistent memory in Drosophila melanogaster. Mutations in importin-α2 (imp-α2, known as Pendulin or Pen and homologous with human KPNA2) are alleles of mushroom body miniature B (mbmB), a gene known to regulate aspects of brain development and influence adult behavior in flies. Mushroom bodies (MBs), paired associative centers in the brain, are smaller than normal due to defective proliferation of specific intrinsic Kenyon cell (KC) neurons in mbmB mutants. Extant KCs projecting to the MB β-lobe terminate abnormally on the contralateral side of the brain. mbmB adults have impaired olfactory learning but normal memory decay in most respects, except that protein synthesis-dependent long-term memory (LTM) is abolished. This observation supports an alternative mechanism of persistent memory in which mutually exclusive protein-synthesis-dependent and -independent forms rely on opposing cellular mechanisms or circuits. We propose a testable model of Imp-α2 and nuclear transport roles in brain development and conditioned behavior. Based on our molecular characterization, we suggest that mbmB is hereafter referred to as imp-α2^mbmB.     

Whole-cell recording from Kenyon cells in silkmoths

Kenyon cells (KCs), which are present in the mushroom bodies (MBs) of the insect brain, play an important role in olfactory information processing and associative learning. However, the intrinsic electrophysiological properties of KCs in silkmoth (Bombyx mori) MBs remain unknown. Here, we use whole-cell patch-clamp recordings to elucidate the functional parameters of membrane voltage and voltage-activated ionic currents of KCs in silkmoth MBs. KCs generated action potentials in response to stepping pulses of depolarizing current, and application of GABA-receptor blocker abolished inhibitory synaptic inputs and depolarized resting membrane potential. Pharmacological isolation of KC voltage-gated ionic currents revealed that KCs express a range of voltage-activated channels, including transient and non-inactivating potassium, sodium, and calcium channels. Our results provide the first electrophysiological characterization of KCs in silkmoth MBs and represent an important step toward understanding neuronal computation that underlies olfactory information processing in silkmoths.     

Neural correlates of social odor recognition and the representation of individual distinctive social odors within entorhinal cortex and ventral subiculum

Recognition of individual conspecifics is important for social behavior and requires the formation of memories for individually distinctive social signals. Individual recognition is often mediated by olfactory cues in mammals, especially nocturnal rodents such as golden hamsters. In hamsters, this form of recognition requires main olfactory system input to the lateral entorhinal cortex (LEnt). Here, we tested whether neurons in LEnt and the nearby ventral subiculum (VS) would show cellular correlates of this natural form of recognition memory. Two hundred ninety single neurons were recorded from both superficial (SE) and deep layers of LEnt (DE) and VS while male hamsters investigated volatile odorants from female vaginal secretions. Many neurons encoded differences between female's odors with many discriminating between odors from different individual females but not between different odor samples from the same female. Other neurons discriminated between odor samples from one female and generalized across collections from other females. LEnt and VS neurons showed enhanced or suppressed cellular activity during investigation of previously presented odors and in response to novel odors. A majority of SE neurons decreased firing to odor repetition and increased activity to novel odors. In contrast, DE neurons often showed suppressed activity in response to novel odors. Thus, neurons in LEnt and VS of male hamsters encode information that is critical for the identification and recognition of individual females by odor cues. This study reveals cellular mechanisms in LEnt and VS that may mediate a natural form of recognition memory in hamsters. These neuronal responses were similar to those observed in rats and monkeys during performance in standard recognition memory tasks. Consequently, the present data extend our understanding of the cellular basis for recognition memory and suggest that individual recognition requires similar neural mechanisms as those employed in laboratory tests of recognition memory.     

Slow oscillations in two pairs of dopaminergic neurons gate long-term memory formation in Drosophila

A fundamental duty of any efficient memory system is to prevent long-lasting storage of poorly relevant information. However, little is known about dedicated mechanisms that appropriately trigger production of long-term memory (LTM). We examined the role of Drosophila dopaminergic neurons in the control of LTM formation and found that they act as a switch between two exclusive consolidation pathways leading to LTM or anesthesia-resistant memory (ARM). Blockade, after aversive olfactory conditioning, of three pairs of dopaminergic neurons projecting on mushroom bodies, the olfactory memory center, enhanced ARM, whereas their overactivation conversely impaired ARM. Notably, blockade of these neurons during the intertrial intervals of a spaced training precluded LTM formation. Two pairs of these dopaminergic neurons displayed sustained calcium oscillations in naive flies. Oscillations were weakened by ARM-inducing massed training and were enhanced during LTM formation. Our results indicate that oscillations of two pairs of dopaminergic neurons control ARM levels and gate LTM.     

Inhibitory interactions among olfactory glomeruli do not necessarily reflect spatial proximity

Inhibitory interactions shape the activity of output neurons in primary olfactory centers and promote contrast enhancement of odor representations. Patterns of interglomerular connectivity, however, are largely unknown. To test whether the proximity of glomeruli to one another is correlated with interglomerular inhibitory interactions, we used intracellular recording and staining methods to record the responses of projection (output) neurons (PNs) associated with glomeruli of known olfactory tuning in the primary olfactory center of the moth Manduca sexta. We focused on Toroid I, a glomerulus in the male-specific macroglomerular complex (MGC) specialized to one of the two key components of the conspecific females' sex pheromone, and the adjacent, sexually isomorphic glomerulus 35, which is highly sensitive to Z-3-hexenyl acetate (Z3-6:OAc). We used the two odorants to activate these reference glomeruli and tested the effects of olfactory activation in other glomeruli. We found that Toroid-I PNs were not inhibited by input to G35, whereas G35 PNs were inhibited by input to Toroid-I PNs. We also recorded the responses of PNs arborizing in other sexually isomorphic glomeruli to stimulation with the sex pheromone and Z3-6:OAc. We found that inhibitory responses were not related to proximity to the MGC and G35: both distant and adjacent PNs were inhibited by stimulation with the sex pheromone, some others were affected by only one odorant, and yet others by neither. Similar results were obtained in female PNs recorded in proximity to female-specific glomeruli. Our findings indicate that inhibitory interactions among glomeruli are widespread and independent of their spatial proximity.     

Biophysical mechanisms underlying olfactory receptor neuron dynamics

The responses of olfactory receptor neurons (ORNs) to odors have complex dynamics. Using genetics and pharmacology, we found that these dynamics in Drosophila ORNs could be separated into sequential steps, corresponding to transduction and spike generation. Each of these steps contributed distinct dynamics. Transduction dynamics could be largely explained by a simple kinetic model of ligand-receptor interactions, together with an adaptive feedback mechanism that slows transduction onset. Spiking dynamics were well described by a differentiating linear filter that was stereotyped across odors and cells. Genetic knock-down of sodium channels reshaped this filter, implying that it arises from the regulated balance of intrinsic conductances in ORNs. Complex responses can be understood as a consequence of how the stereotyped spike filter interacts with odor- and receptor-specific transduction dynamics. However, in the presence of rapidly fluctuating natural stimuli, spiking simply increases the speed and sensitivity of encoding.     

G(o) signaling is required for Drosophila associative learning

Heterotrimeric G(o) is one of the most abundant proteins in the brain, yet relatively little is known of its neural functions in vivo. Here we demonstrate that G(o) signaling is required for the formation of associative memory. In Drosophila melanogaster, pertussis toxin (PTX) is a selective inhibitor of G(o) signaling. The postdevelopmental expression of PTX within mushroom body neurons robustly and reversibly inhibits associative learning. The effect of G(o) inhibition is distributed in both gamma- and alpha/beta-lobe mushroom body neurons. However, the expression of PTX in neurons adjacent to the mushroom bodies does not affect memory. PTX expression also does not interact genetically with a rutabaga adenylyl cyclase loss-of-function mutation. Thus, G(o) defines a new signaling pathway required in mushroom body neurons for the formation of associative memory.     

Sparsening and temporal sharpening of olfactory representations in the honeybee mushroom bodies

We explored the transformations accompanying the transmission of odor information from the first-order processing area, the antennal lobe, to the mushroom body, a higher-order integration center in the insect brain. Using Ca2+ imaging, we recorded activity in the dendrites of the projection neurons that connect the antennal lobe with the mushroom body. Next, we recorded the presynaptic terminals of these projection neurons. Finally, we characterized their postsynaptic partners, the intrinsic neurons of the mushroom body, the clawed Kenyon cells. We found fundamental differences in odor coding between the antennal lobe and the mushroom body. Odors evoked combinatorial activity patterns at all three processing stages, but the spatial patterns became progressively sparser along this path. Projection neuron dendrites and boutons showed similar response profiles, but the boutons were more narrowly tuned to odors. The transmission from projection neuron boutons to Kenyon cells was accompanied by a further sparsening of the population code. Activated Kenyon cells were highly odor specific. Furthermore, the onset of Kenyon cell responses to projection neurons occurred within the first 200 ms and complex temporal patterns were transformed into brief phasic responses. Thus two types of transformations occurred within the MB: sparsening of a combinatorial code, mediated by pre- and postsynaptic processing within the mushroom body microcircuits, and temporal sharpening of postsynaptic Kenyon cell responses, probably involving a broader loop of inhibitory recurrent neurons.     

Non-redundant odor coding by sister mitral cells revealed by light addressable glomeruli in the mouse

Sensory inputs frequently converge on the brain in a spatially organized manner, often with overlapping inputs to multiple target neurons. Whether the responses of target neurons with common inputs become decorrelated depends on the contribution of local circuit interactions. We addressed this issue in the olfactory system using newly generated transgenic mice that express channelrhodopsin-2 in all of the olfactory sensory neurons. By selectively stimulating individual glomeruli with light, we identified mitral/tufted cells that receive common input (sister cells). Sister cells had highly correlated responses to odors, as measured by average spike rates, but their spike timing in relation to respiration was differentially altered. In contrast, non-sister cells correlated poorly on both of these measures. We suggest that sister mitral/tufted cells carry two different channels of information: average activity representing shared glomerular input and phase-specific information that refines odor representations and is substantially independent for sister cells.     

The Drosophila DCO mutation suppresses age-related memory impairment without affecting lifespan

The study of age-related memory impairment (AMI) has been hindered by a lack of AMI-specific mutants. In a screen for such mutants in Drosophila melanogaster, we found that heterozygous mutations of DCO (DCO/+), which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), delay AMI more than twofold without affecting lifespan or memory at early ages. AMI is restored when a DCO transgene is expressed in mushroom bodies, structures important for olfactory memory formation. Furthermore, increasing cAMP and PKA activity in mushroom bodies causes premature AMI, whereas reducing activity suppresses AMI. In Drosophila AMI consists of a specific reduction in memory dependent on the amnesiac (amn) gene. amn encodes putative neuropeptides that have been proposed to regulate cAMP levels in mushroom bodies. Notably, both the memory and AMI defects of amn mutants are restored in amn;DCO/+ double mutants, suggesting that AMI is caused by an age-related disruption of amn-dependent memory via PKA activity in mushroom bodies.     

Local inhibition modulates odor-evoked synchronization of glomerulus-specific output neurons

At the first stage of olfactory processing in the brain, synchronous firing across glomeruli may help to temporally bind multiple and spatially distributed input streams activated by a given odor. This hypothesis, however, has never been tested in an organism in which the odor-tuning properties of several spatially identifiable glomeruli are known. Using the sphinx moth, an insect that meets these specific criteria, we recorded odor-evoked responses simultaneously from pairs of projection neurons (PNs) innervating the same or different glomeruli in the macroglomerular complex (MGC), which is involved in processing pheromonal information. PNs that branched in the same glomerulus and were activated by the same pheromone component also showed the strongest coincident responses to each odor pulse. Glomerulus-specific PN pairs were also inhibited by the pheromone component that selectively activated PNs in the neighboring glomerulus, and about 70% of all intraglomerular pairs showed increased synchronization when stimulated with a mixture of the two odorants. Thus, when two adjacent glomeruli receive their inputs simultaneously, the temporal tuning of output from each glomerulus is enhanced by reciprocal and inhibitory interglomerular interactions.     

Keratinocyte intercellular adhesion molecule-1 (ICAM-1) expression precedes dermal T lymphocytic infiltration in allergic contact dermatitis (Rhus dermatitis).

The ability of small molecules such as urushiol, present as a wax on the poison ivy leaf surface, to cause allergic contact dermatitis (rhus dermatitis) has fascinated immunologists for decades. Current dogma suggests that these epicutaneously applied catechol-containing molecules serve as haptens to conjugate with larger proteins via reactive o-quinone intermediates. These complexes are then recognized as foreign antigens by the immune system and elicit a hypersensitivity reaction. Phorbol ester can directly induce cultured keratinocyte (KC) intercellular adhesion molecule-1 (ICAM-1) expression via a protein kinase C (PK-C)-dependent mechanism. As urushiol is also a known PK-C agonist, we asked if topical application of a poison ivy/oak mixture could directly induce epidermal KC ICAM-1 expression. During the pre-erythematous phase of this reaction (4 to 20 hours), epidermal KCs expressed ICAM-1; this "initiation phase" preceded the appearance of activated memory T lymphocytes in the papillary dermis, and thus appeared to be nonlymphokine mediated. A near-contiguous cellular-adhesion molecular network was identified by ICAM-1 staining of basal KCs, dermal dendrocytes, and endothelial cells. During the second 24-hour period with the onset of erythema and edema, there was an "amplification phase" of more intense KC ICAM-1 expression coupled with relatively weak KC HLA-DR expression that coincided with dermal and epidermal T-cell infiltration. This suggests the presence of lymphokines, such as gamma interferon, during the amplification phase because of KC HLA-DR expression. On cultured KCs, urushiol directly induced ICAM-1 expression but not HLA-DR. Thus, in addition to functioning as an antigenic hapten, urushiol directly induces KC ICAM-1 expression. The KC ICAM-1 expression may then alter the dynamic trafficking of memory T cells in the epidermis, so as to initiate cutaneous inflammation in a nonantigen specific manner. This initiation phase is followed by T-cell infiltration and consequent lymphokine production that significantly amplifies the original stimulus. Thus much can still be learned about the molecular pathophysiology of this common type of cutaneous inflammation.     

Olfactory input to the lateral hypothalamus of the old world monkey

Responses of lateral hypothalamic neurons to 8 odors were studied in chronic unanesthetized old world monkeys (Macaca irus). Many neurons (54.5%) responded to a single odor only, and the number of neurons responding to 2, 3 and 4 odors decreased successively. No neuron responded to as many as 5 odors. Thus, the presence of olfactory input and a highly discriminative ability for odors were found in the lateral hypothalamic area (LHA). Neuronal responses to the same odors were also studied in the septum (Spt). In anesthetized old world monkeys, evoked potentials were recorded in the LHA and in areas of the Spt and the nucleus accumbens (Acc) during stimulation of the olfactory bulb (OB). When the Spt (and probably the Acc with it) was subsequently destroyed, OB-evoked potentials in the LHA disappeared. Next, by injecting horseradish peroxidase (HRP) into the LHA, an olfactory pathway to the LHA was examined. Labeled neurons were found mainly in the Spt and the Acc, and only partly in other areas. However, labeled neurons were scarcely found in the prepyriform (PPF)-entorhinal (ER) area or in the olfactory tubercle (OT). The present study thus shows that an olfactory pathway to the LHA passes through the Spt and probably also the Acc, but not through the PPF-ER areas nor through the OT in the old world monkey.