Odor-evoked Calcium Research Articles

Dopamine-Dependent Plasticity Is Heterogeneously Expressed by Presynaptic Calcium Activity across Individual Boutons of the Drosophila Mushroom Body.

The Drosophila mushroom body (MB) is an important model system for studying the synaptic mechanisms of associative learning. In this system, coincidence of odor-evoked calcium influx and dopaminergic input in the presynaptic terminals of Kenyon cells (KCs), the principal neurons of the MB, triggers long-term depression (LTD), which plays a critical role in olfactory learning. However, it is controversial whether such synaptic plasticity is accompanied by a corresponding decrease in odor-evoked calcium activity in the KC presynaptic terminals. Here, we address this question by inducing LTD by pairing odor presentation with optogenetic activation of dopaminergic neurons (DANs). This allows us to rigorously compare the changes at the presynaptic and postsynaptic sites in the same conditions. By imaging presynaptic acetylcholine release in the condition where LTD is reliably observed in the postsynaptic calcium signals, we show that neurotransmitter release from KCs is depressed selectively in the MB compartments innervated by activated DANs, demonstrating the presynaptic nature of LTD. However, total odor-evoked calcium activity of the KC axon bundles does not show concurrent depression. We further conduct calcium imaging in individual presynaptic boutons and uncover the highly heterogeneous nature of calcium plasticity. Namely, only a subset of boutons, which are strongly activated by associated odors, undergo calcium activity depression, while weakly responding boutons show potentiation. Thus, our results suggest an unexpected nonlinear relationship between presynaptic calcium influx and the results of plasticity, challenging the simple view of cooperative actions of presynaptic calcium and dopaminergic input.

Towards detection of cancer biomarkers in human exhaled air by transfer-learning-powered analysis of odor-evoked calcium activity in rat olfactory bulb

Detection of volatile organic compounds in exhaled air is a promising approach to non-invasive and scalable gastric cancer screening. This work proposes a new approach for the detection of volatile organic compounds by analyzing odor-evoked calcium responses in the rat olfactory bulb. We estimate the feasibility of gastric cancer biomarker detection added to the exhaled air of healthy participants. Our detector consists of a convolutional encoder and a similarity-based classifier over encoder outputs. To minimize overfitting on a small available training set, we involve a pre-training where the encoder is trained on synthetic data representing spatiotemporal patterns similar to real calcium responses in the olfactory bulb. We estimate the classification accuracy of exhaled air samples by matching their encodings with encodings of calibration samples of two classes: 1) exhaled air and 2) a mixture of exhaled air with the cancer biomarker. On our data, the accuracy increased from 0.68 on real data up to 0.74 if pre-training on synthetic data is involved. Our work is focused on proving the feasibility of proposed new approach rather than on comparing its efficiency with existing methods. Such detection is often performed with an electronic nose, but its output becomes unstable over time due to a sensor drift. In contrast to the electronic nose, rats can robustly detect low concentrations of biomarkers over lifetime. The feasibility of gastric cancer biomarker detection in exhaled air by bio-hybrid system is shown. Pre-training of neural models for images analysis increases the accuracy of detection.

Lateral axonal modulation is required for stimulus-specific olfactory conditioning in Drosophila

Effective and stimulus-specific learning is essential for animals' survival. Two major mechanisms are known to aid stimulus specificity of associative learning. One is accurate stimulus-specific representations in neurons. The second is a limited effective temporal window for the reinforcing signals to induce neuromodulation after sensory stimuli. However, these mechanisms are often imperfect in preventing unspecific associations; different sensory stimuli can be represented by overlapping populations of neurons, and more importantly, the reinforcing signals alone can induce neuromodulation even without coincident sensory-evoked neuronal activity. Here, we report a crucial neuromodulatory mechanism that counteracts both limitations and is thereby essential for stimulus specificity of learning. In Drosophila, olfactory signals are sparsely represented by cholinergic Kenyon cells (KCs), which receive dopaminergic reinforcing input. We find that KCs have numerous axo-axonic connections mediated by the muscarinic type-B receptor (mAChR-B). By using functional imaging and optogenetic approaches, we show that these axo-axonic connections suppress both odor-evoked calcium responses and dopamine-evoked cAMP signals in neighboring KCs. Strikingly, behavior experiments demonstrate that mAChR-B knockdown in KCs impairs olfactory learning by inducing undesired changes to the valence of an odor that was not associated with the reinforcer. Thus, this local neuromodulation acts in concert with sparse sensory representations and global dopaminergic modulation to achieve effective and accurate memory formation.

Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle.

We developed a multiphoton imaging method to capture neural structure and activity in behaving flies through the intact cuticle. Our measurements showed that the fly head cuticle has surprisingly high transmission at wavelengths >900nm, and the difficulty of through-cuticle imaging is due to the air sacs and/or fat tissue underneath the head cuticle. By compressing or removing the air sacs, we performed multiphoton imaging of the fly brain through the intact cuticle. Our anatomical and functional imaging results show that 2- and 3-photon imaging are comparable in superficial regions such as the mushroom body, but 3-photon imaging is superior in deeper regions such as the central complex and beyond. We further demonstrated 2-photon through-cuticle functional imaging of odor-evoked calcium responses from the mushroom body γ-lobes in behaving flies short term and long term. The through-cuticle imaging method developed here extends the time limits of in vivo imaging in flies and opens new ways to capture neural structure and activity from the fly brain.

Distinct Characteristics of Odor-evoked Calcium and Electrophysiological Signals in Mitral/Tufted Cells in the Mouse Olfactory Bulb

Fiber photometry is a recently-developed method that indirectly measures neural activity by monitoring Ca2+ signals in genetically-identified neuronal populations. Although fiber photometry is widely used in neuroscience research, the relationship between the recorded Ca2+ signals and direct electrophysiological measurements of neural activity remains elusive. Here, we simultaneously recorded odor-evoked Ca2+ and electrophysiological signals [single-unit spikes and local field potentials (LFPs)] from mitral/tufted cells in the olfactory bulb of awake, head-fixed mice. Odors evoked responses in all types of signal but the response characteristics (e.g., type of response and time course) differed. The Ca2+ signal was correlated most closely with power in the β-band of the LFP. The Ca2+ signal performed slightly better at odor classification than high-γ oscillations, worse than single-unit spikes, and similarly to β oscillations. These results provide new information to help researchers select an appropriate method for monitoring neural activity under specific conditions.

Serotonergic afferents from the dorsal raphe decrease the excitability of pyramidal neurons in the anterior piriform cortex

The olfactory system receives extensive serotonergic inputs from the dorsal raphe, a nucleus involved in control of behavior, regulation of mood, and modulation of sensory processing. Although many studies have investigated how serotonin modulates the olfactory bulb, few have focused on the anterior piriform cortex (aPC), a region important for olfactory learning and encoding of odor identity and intensity. Specifically, the mechanism and functional significance of serotonergic modulation of the aPC remain largely unknown. Here we used pharmacologic, optogenetic, and fiber photometry techniques to examine the serotonergic modulation of neural activity in the aPC in vitro and in vivo. We found that serotonin (5-HT) reduces the excitability of pyramidal neurons directly via 5-HT2C receptors, phospholipase C, and calcium-activated potassium (BK) channels. Furthermore, endogenous serotonin attenuates odor-evoked calcium responses in aPC pyramidal neurons. These findings identify the mechanism underlying serotonergic modulation of the aPC and shed light on its potential role.

Independent Contributions of Discrete Dopaminergic Circuits to Cellular Plasticity, Memory Strength, and Valence in Drosophila

SUMMARYDopaminergic neurons play a key role in encoding associative memories, but little is known about how these circuits modulate memory strength. Here we report that different sets of dopaminergic neurons projecting to the Drosophila mushroom body (MB) differentially regulate valence and memory strength. PPL2 neurons increase odor-evoked calcium re- sponses to a paired odor in the MB and enhance behavioral memory strength when activated during olfactory classical conditioning. When paired with odor alone, they increase MB responses to the paired odor but do not drive behavioral approach or avoidance, suggesting that they increase the salience of the odor without encoding strong valence. This contrasts with the role of dopaminergic PPL1 neurons, which drive behavioral reinforcement but do not alter odor-evoked calcium responses in the MB when stimulated. These data suggest that different sets of dopaminergic neurons modulate olfactory valence and memory strength via independent actions on a memory-encoding brain region.

Partial depletion of dopaminergic neurons in the substantia nigra impairs olfaction and alters neural activity in the olfactory bulb

Olfactory dysfunction is a major non-motor symptom that appears during the early stages of Parkinson’s Disease (PD), a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra (SN). Depletion of SN dopaminergic neurons by 6-hydroxydopamine (6-OHDA) is widely used as a model for PD and ultimately results in motor deficits. However, it is largely unknown whether olfactory behavior and, more importantly, neural activity in the olfactory bulb (OB) are impaired prior to the appearance of motor deficits. We partially depleted the SN dopaminergic population in mice by injection of 6-OHDA. Seven days after injection of 6-OHDA, motor ability was unchanged but olfactory-driven behaviors were significantly impaired. Injection of 6-OHDA into the SN significantly increased the power of the ongoing local field potential in the OB for all frequency bands, and decreased odor-evoked excitatory beta responses and inhibitory high-gamma responses. Moreover, 6-OHDA treatment led to increased odor-evoked calcium responses in the mitral cells in the OB of awake mice. These data suggest that the olfactory deficits caused by depletion of the SN dopaminergic population are likely due to abnormal hyperactivity of the mitral cells in the OB.

Narrowly Confined and Glomerulus-Specific Onset Latencies of Odor-Evoked Calcium Transients in the Juxtaglomerular Cells of the Mouse Main Olfactory Bulb.

Odor information is transmitted from olfactory sensory neurons to principal neurons at the glomeruli of the olfactory bulb. The intraglomerular neuronal circuit also includes hundreds of interneurons referred to as juxtaglomerular (JG) cells. Stimulus selectivity is well correlated among many JG cells that are associated with the same glomerulus, consistent with their highly homogeneous sensory inputs. However, much less is known about the temporal aspects of their activity, including the temporal coordination of their odor-evoked responses. As many JG cells within a glomerular module respond to the same stimulus, the extent to which their activity is temporally aligned will affect the temporal profile of their population inhibitory inputs. Using random-access high-speed two-photon microscopy, we recorded the odor-evoked calcium transients of mouse JG cells and compared the onset latency and rise time among neurons putatively associated with the same and different glomeruli. Whereas the overall onset latencies of odor-evoked transients were distributed across a ∼150 ms time window, those from cells putatively associated with the same glomerulus were confined to a much narrower window of several tens of milliseconds. This result suggests that onset latency primarily depends on the associated glomerulus. We also observed glomerular specificity in the rise time. The glomerulus-specific temporal pattern of odor-evoked activity implies that the temporal patterns of inputs from the intraglomerular circuit are unique to individual glomerulus–odor pairs, which may contribute to efficient shaping of the temporal pattern of activity in the principal neurons.

Long-term optical brain imaging in live adult fruit flies

Time-lapse in vivo microscopy studies of cellular morphology and physiology are crucial toward understanding brain function but have been infeasible in the fruit fly, a key model species. Here we use laser microsurgery to create a chronic fly preparation for repeated imaging of neural architecture and dynamics for up to 50 days. In fly mushroom body neurons, we track axonal boutons for 10 days and record odor-evoked calcium transients over 7 weeks. Further, by using voltage imaging to resolve individual action potentials, we monitor spiking plasticity in dopamine neurons of flies undergoing mechanical stress. After 24 h of stress, PPL1-α’3 but not PPL1-α’2α2 dopamine neurons have elevated spike rates. Overall, our chronic preparation is compatible with a broad range of optical techniques and enables longitudinal studies of many biological questions that could not be addressed before in live flies.

Rapid adaptive remote focusing microscope for sensing of volumetric neural activity.

The ability to record neural activity in the brain of a living organism at cellular resolution is of great importance for defining the neural circuit mechanisms that direct behavior. Here we present an adaptive two-photon microscope optimized for extraction of neural signals over volumes in intact Drosophila brains, even in the presence of specimen motion. High speed volume imaging was made possible through reduction of spatial resolution while maintaining the light collection efficiency of a high resolution, high numerical aperture microscope. This enabled simultaneous recording of odor-evoked calcium transients in a defined volume of mushroom body Kenyon cell bodies in a live fruit fly.

The role of connexin 43 in mediating odor response

Connexin proteins are the hemichannels that form gap junctions to regulate the intercellular communication. Connexin 43 (Cx43) is the most common gap junction protein that expresses in many cell types, including the olfactory sensory neurons. Phosphorylation is a crucial step to regulate the function of Cx43. Gap junction was found to modulate the odor response, but the specific role is still elusive. Here, we report that gap junctions play a role in odor-evoked calcium response in both heterologous cell system and primary olfactory sensory neurons. This regulation is mediated through gap junction protein Cx43. Overexpression of full length Cx43 can counteract the inhibitory effect of gap junction or connexin blockers on odor-evoked [Ca2+]i increase in hana3A cells. Carboxy-terminal of Cx43 (Cx43CT) has the similar function as the full length of Cx43. Furthermore, we found that expression level of phosphorylation of Cx43 at S368 is dynamic with the stimulation of odor in hana3A cells. Expression level of phosphorylated Cx43 at S368 was decreased when gap junction or connexin inhibitors were applied. Phosphorylation of Cx43 during odor or inhibitor stimulation may be mediated by ERK and JNK signaling pathway. Altogether our data suggest that expression of Cx43 can regulate the odor response. This study provides a clue to indicate the possible protective mechanism of gap junction in odor response.

Olfactory Neuromodulation of Motion Vision Circuitry in Drosophila

SummaryIt is well established that perception is largely multisensory [1]; often served by modalities such as touch, vision, and hearing that detect stimuli emanating from a common point in space [2, 3]; and processed by brain tissue maps that are spatially aligned [4]. However, the neural interactions among modalities that share no spatial stimulus domain yet are essential for robust perception within noisy environments remain uncharacterized. Drosophila melanogaster makes its living navigating food odor plumes. Odor acts to increase the strength of gaze-stabilizing optomotor reflexes [5] to keep the animal aligned within an invisible plume, facilitating odor localization in free flight [6–8]. Here, we investigate the cellular mechanism for cross-modal behavioral interactions. We characterize a wide-field motion-selective interneuron of the lobula plate that shares anatomical and physiological similarities with the “Hx” neuron identified in larger flies [9, 10]. Drosophila Hx exhibits cross-modal enhancement of visual responses by paired odor, and presynaptic inputs to the lobula plate are required for behavioral odor tracking but are not themselves the target of odor modulation, nor is the neighboring wide-field “HSE” neuron [11]. Octopaminergic neurons mediating increased visual responses upon flight initiation [12] also show odor-evoked calcium modulations and form connections with Hx dendrites. Finally, restoring synaptic vesicle trafficking within the octopaminergic neurons of animals carrying a null mutation for all aminergic signaling [13] is sufficient to restore odor-tracking behavior. These results are the first to demonstrate cellular mechanisms underlying visual-olfactory integration required for odor localization in fruit flies, which may be representative of adaptive multisensory interactions across taxa.

Calcium Imaging in the Drosophila Olfactory System with a Genetic Indicator

Insects show sophisticated odor-mediated behaviors controlled by an olfactory system that is genetically and anatomically simpler than that of vertebrates, providing an attractive system to investigate the mechanistic link between behavior and odor perception. Advances in neuroscience have been facilitated by modern optical imaging technologies--both in instrumentation and in probe design--that permit the visualization of functional neural circuits. Imaging calcium activity in genetically defined populations of neurons provides an important tool for investigating the function of neural circuits. This article describes a two-photon imaging system for monitoring neural activity in the Drosophila antennal lobe. Odor-evoked calcium activity is followed by measuring the specific expression of the calcium-sensitive green fluorescent protein G-CaMP in Drosophila antennae-brain preparations.

Antennal Lobe Processing Correlates to Moth Olfactory Behavior

Animals typically perceive their olfactory environment as a complex blend of natural odor cues. In insects, the initial processing of odors occurs in the antennal lobe (AL). Afferent peripheral input from olfactory sensory neurons (OSNs) is modified via mostly inhibitory local interneurons (LNs) and transferred by projection neurons (PNs) to higher brain centers. Here we performed optophysiological studies in the AL of the moth, Manduca sexta, and recorded odor-evoked calcium changes in response to antennal stimulation with five monomolecular host volatiles and their artificial mixture. In a double staining approach, we simultaneously measured OSN network input in concert with PN output across the glomerular array. By comparing odor-evoked activity patterns and response intensities between the two processing levels, we show that host mixtures could generally be predicted from the linear summation of their components at the input of the AL, but output neurons established a unique, nonlinear spatial pattern separate from individual component identities. We then assessed whether particularly high levels of signal modulation correspond to behavioral relevance. One of our mixture components, phenyl acetaldehyde, evoked significant levels of nonlinear input-output modulation in observed spatiotemporal activation patterns that were unique from the other individual odorants tested. This compound also accelerated behavioral activity in subsequent wind tunnel tests, whereas another compound that did not exhibit high levels of modulation also did not affect behavior. These results suggest that the high degree of input-output modulation exhibited by the AL for specific odors can correlate to behavioral output.

A Late-Phase, Long-Term Memory Trace Forms in the γ Neurons ofDrosophilaMushroom Bodies after Olfactory Classical Conditioning

Using functional optical imaging in vivo, we demonstrate that the γ mushroom body (MB) neurons of Drosophila melanogaster respond with axonal calcium influx when odors or electric shock stimuli are presented to the fly. Pairing of odor and electric shock stimuli in a single training trial or multiple, massed training trials failed to modify the odor-evoked calcium signal when flies were tested at several different times after training. In contrast, animals that received multiple but spaced odor-shock pairings exhibited a robust increase in calcium influx into the MB axons when tested between 18 and 48 h after training. This time window for the γ neuron memory trace is displaced relative to the modifications that occur between 9 and 24 h after training in the α branch of the α/β MB neurons. The α/β and the γ neuron long-term memory traces were both blocked by expressing a repressor of the transcription factor cAMP response element-binding protein or a calcium/calmodulin-dependent kinase II hairpin RNA. These results demonstrate that behavioral long-term olfactory memory is encoded as modifications of calcium influx into distinct MB neurons during overlapping but different windows of time after training.

Neuropeptide feedback modifies odor-evoked dynamics in Caenorhabditis elegans olfactory neurons.

Many neurons release classical transmitters together with neuropeptide cotransmitters whose functions are incompletely understood. Here we define the relationship between two transmitters in the olfactory system of Caenorhabditis elegans, showing that a neuropeptide-to-neuropeptide feedback loop alters sensory dynamics in primary olfactory neurons. The AWC olfactory neuron is glutamatergic and also expresses the peptide NLP-1. nlp-1 mutants have increased AWC-dependent behaviors, suggesting that NLP-1 limits the normal response. The receptor for NLP-1 is the G protein-coupled receptor NPR-11, which acts in postsynaptic AIA interneurons. Feedback from AIA interneurons modulates odor-evoked calcium dynamics in AWC olfactory neurons and requires INS-1, a neuropeptide released from AIA. The neuropeptide feedback loop dampens behavioral responses to odors on short and long timescales. Our results point to neuronal dynamics as a site of behavioral regulation and reveal the ability of neuropeptide feedback to remodel sensory networks on multiple timescales.

A Behavioral Switch: cGMP and PKC Signaling in Olfactory Neurons Reverses Odor Preference in C. elegans

Innate chemosensory preferences are often encoded by sensory neurons that are specialized for attractive or avoidance behaviors. Here, we show that one olfactory neuron in Caenorhabditis elegans, AWC(ON), has the potential to direct both attraction and repulsion. Attraction, the typical AWC(ON) behavior, requires a receptor-like guanylate cyclase GCY-28 that acts in adults and localizes to AWC(ON) axons. gcy-28 mutants avoid AWC(ON)-sensed odors; they have normal odor-evoked calcium responses in AWC(ON) but reversed turning biases in odor gradients. In addition to gcy-28, a diacylglycerol/protein kinase C pathway that regulates neurotransmission switches AWC(ON) odor preferences. A behavioral switch in AWC(ON) may be part of normal olfactory plasticity, as odor conditioning can induce odor avoidance in wild-type animals. Genetic interactions, acute rescue, and calcium imaging suggest that the behavioral reversal results from presynaptic changes in AWC(ON). These results suggest that alternative modes of neurotransmission can couple one sensory neuron to opposite behavioral outputs.

Synergistic behavioral responses of female oriental fruit moths (Lepidoptera:Tortricidae) to synthetic host plant-derived mixtures are mirrored by odor-evoked calcium activity in their antennal lobes

Attraction of many gravid female herbivore insects to suitable host plants is mediated largely by olfactory cues. Behaviorally, synergism among odor mixtures constituents underlies this attraction in some systems. Yet, the representation of synergistic odor-mixture effects is unknown in the antennal lobe, the first processing center for olfactory information in insect brains. Using both behavioral and physiological data we demonstrate that in the oriental fruit moth, Cydia (Grapholita) molesta, a minor constituent of a plant-derived synthetic mixture plays a key role in behavioral discrimination and in neural representation of mixtures. Behaviorally, minute amounts of benzonitrile added to an unattractive 4-compound mixture resulted in a bioactive 5-compound mixture that was as attractive to mated female moths as the natural blend. Physiologically, the bioactive benzonitrile-containing mixture elicited strong activation of one additional, new type of glomerulus that showed specific synergisms for this mixture. The specific pattern of activated glomeruli elicited by the addition of benzonitrile demonstrates a physiological correlate to the behaviorally observed synergism, and emphasizes the key role of a minor component of a complex mixture. While minor constituents of mixtures are often overlooked, they may, as conclusively documented here, be determinant for successful recognition and behavioral discrimination of suitable host plants by herbivore insects.

The Relationship between Blood Flow and Neuronal Activity in the Rodent Olfactory Bulb

In the brain, neuronal activation triggers an increase in cerebral blood flow (CBF). Here, we use two animal models and several techniques (two-photon imaging of CBF and neuronal calcium dynamics, intracellular and extracellular recordings, local pharmacology) to analyze the relationship between neuronal activity and local CBF during odor stimulation in the rodent olfactory bulb. Application of glutamate receptor antagonists or tetrodotoxin directly into single rat olfactory glomeruli blocked postsynaptic responses but did not affect the local odor-evoked CBF increases. This suggests that in our experimental conditions, odor always activates more than one glomerulus and that silencing one of a few clustered glomeruli does not affect the vascular response. To block synaptic transmission more widely, we then superfused glutamate antagonists over the surface of the olfactory bulb in transgenic G-CaMP2 mice. This was for two reasons: (1) mice have a thin olfactory nerve layer compared to rats and this will favor drug access to the glomerular layer, and (2) transgenic G-CaMP2 mice express the fluorescent calcium sensor protein G-CaMP2 in mitral cells. In G-CaMP2 mice, odor-evoked, odor-specific, and concentration-dependent calcium increases in glomeruli. Superfusion of glutamate receptor antagonists blocked odor-evoked postsynaptic calcium signals and CBF responses. We conclude that activation of postsynaptic glutamate receptors and rises in dendritic calcium are major steps for neurovascular coupling in olfactory bulb glomeruli.