Neuronal excitability as a regulator of circuit remodeling.

Postnatal remodeling of neuronal connectivity shapes mature nervous systems.1,2,3 The pruning of exuberant connections involves cell-autonomous and non-cell-autonomous mechanisms, such as neuronal activity. Indeed, experience-dependent competition sculpts various excitatory neuronal circuits.4,5,6,7,8,9 Moreover, activity has been shown to regulate growth cone motility and the stability of neurites and synaptic connections.10,11,12,13,14 However, whether inhibitory activity influences the remodeling of neuronal connectivity or how activity influences remodeling in systems in which competition is not clearly apparent is not fully understood. Here, we use the Drosophila mushroom body (MB) as a model to examine the role of neuronal activity in the developmental axon pruning of γ-Kenyon cells. The MB is a neuronal structure in insects, implicated in associative learning and memory,15,16 which receives mostly olfactory input from the antennal lobe.17,18 The MB circuit includes intrinsic neurons, called Kenyon cells (KCs), which receive inhibitory input from the GABAergic anterior paired lateral (APL) neuron among other inputs. The γ-KCs undergo stereotypic, steroid-hormone-dependent remodeling19,20 that involves the pruning of larval neurites followed by regrowth to form adult connections.21 We demonstrate that silencing neuronal activity is required for γ-KC pruning. Furthermore, we show that this is mechanistically achieved by cell-autonomous expression of the inward rectifying potassium channel 1 (irk1) combined with inhibition by APL neuron activity likely via GABA-B-R1 signaling. These results support the Hebbian-like rule "use it or lose it," where inhibition can destabilize connectivity and promote pruning while excitability stabilizes existing connections.

Pruning deficits of the developing Drosophila mushroom body result in mild impairment in associative odour learning and cause hyperactivity.

The principles of how brain circuits establish themselves during development are largely conserved across animal species. Connections made during embryonic development that are appropriate for an early life stage are frequently remodelled later in ontogeny via pruning and subsequent regrowth to generate adult-specific connectivity. The mushroom body of the fruit fly Drosophila melanogaster is a well-established model circuit for examining the cellular mechanisms underlying neurite remodelling. This central brain circuit integrates sensory information with learned and innate valences to adaptively instruct behavioural decisions. Thereby, the mushroom body organizes adaptive behaviour, such as associative learning. However, little is known about the specific aspects of behaviour that require mushroom body remodelling. Here, we used genetic interventions to prevent the intrinsic neurons of the larval mushroom body (γ-type Kenyon cells) from remodelling. We asked to what degree remodelling deficits resulted in impaired behaviour. We found that deficits caused hyperactivity and mild impairment in differential aversive olfactory learning, but not appetitive learning. Maintenance of circadian rhythm and sleep were not affected. We conclude that neurite pruning and regrowth of γ-type Kenyon cells is not required for the establishment of circuits that mediate associative odour learning per se, but it does improve distinct learning tasks.

Visualization of learning-induced synaptic plasticity in output neurons of the Drosophila mushroom body γ-lobe.

By learning, through experience, which stimuli coincide with dangers, it is possible to predict outcomes and act pre-emptively to ensure survival. In insects, this process is localized to the mushroom body (MB), the circuitry of which facilitates the coincident detection of sensory stimuli and punishing or rewarding cues and, downstream, the execution of appropriate learned behaviors. Here, we focused our attention on the mushroom body output neurons (MBONs) of the γ-lobes that act as downstream synaptic partners of the MB γ-Kenyon cells (KCs) to ask how the output of the MB γ-lobe is shaped by olfactory associative conditioning, distinguishing this from non-associative stimulus exposure effects, and without the influence of downstream modulation. This was achieved by employing a subcellularly localized calcium sensor to specifically monitor activity at MBON postsynaptic sites. Therein, we identified a robust associative modulation within only one MBON postsynaptic compartment (MBON-γ1pedc > α/ß), which displayed a suppressed postsynaptic response to an aversively paired odor. While this MBON did not undergo non-associative modulation, the reverse was true across the remainder of the γ-lobe, where general odor-evoked adaptation was observed, but no conditioned odor-specific modulation. In conclusion, associative synaptic plasticity underlying aversive olfactory learning is localized to one distinct synaptic γKC-to-γMBON connection.

The anterior paired lateral neuron normalizes odour-evoked activity in the Drosophila mushroom body calyx.

To identify and memorize discrete but similar environmental inputs, the brain needs to distinguish between subtle differences of activity patterns in defined neuronal populations. The Kenyon cells (KCs) of the Drosophila adult mushroom body (MB) respond sparsely to complex olfactory input, a property that is thought to support stimuli discrimination in the MB. To understand how this property emerges, we investigated the role of the inhibitory anterior paired lateral (APL) neuron in the input circuit of the MB, the calyx. Within the calyx, presynaptic boutons of projection neurons (PNs) form large synaptic microglomeruli (MGs) with dendrites of postsynaptic KCs. Combining electron microscopy (EM) data analysis and in vivo calcium imaging, we show that APL, via inhibitory and reciprocal synapses targeting both PN boutons and KC dendrites, normalizes odour-evoked representations in MGs of the calyx. APL response scales with the PN input strength and is regionalized around PN input distribution. Our data indicate that the formation of a sparse code by the KCs requires APL-driven normalization of their MG postsynaptic responses. This work provides experimental insights on how inhibition shapes sensory information representation in a higher brain centre, thereby supporting stimuli discrimination and allowing for efficient associative memory formation.

Unc13A and Unc13B contribute to the decoding of distinct sensory information in Drosophila.

The physical distance between presynaptic Ca2+ channels and the Ca2+ sensors triggering the release of neurotransmitter-containing vesicles regulates short-term plasticity (STP). While STP is highly diversified across synapse types, the computational and behavioral relevance of this diversity remains unclear. In the Drosophila brain, at nanoscale level, we can distinguish distinct coupling distances between Ca2+ channels and the (m)unc13 family priming factors, Unc13A and Unc13B. Importantly, coupling distance defines release components with distinct STP characteristics. Here, we show that while Unc13A and Unc13B both contribute to synaptic signalling, they play distinct roles in neural decoding of olfactory information at excitatory projection neuron (ePN) output synapses. Unc13A clusters closer to Ca2+ channels than Unc13B, specifically promoting fast phasic signal transfer. Reduction of Unc13A in ePNs attenuates responses to both aversive and appetitive stimuli, while reduction of Unc13B provokes a general shift towards appetitive values. Collectively, we provide direct genetic evidence that release components of distinct nanoscopic coupling distances differentially control STP to play distinct roles in neural decoding of sensory information.

Circuit reorganization in the Drosophila mushroom body calyx accompanies memory consolidation.

The formation and consolidation of memories are complex phenomena involving synaptic plasticity, microcircuit reorganization, and the formation of multiple representations within distinct circuits. To gain insight into the structural aspects of memory consolidation, we focus on the calyx of the Drosophila mushroom body. In this essential center, essential for olfactory learning, second- and third-order neurons connect through large synaptic microglomeruli, which we dissect at the electron microscopy level. Focusing on microglomeruli that respond to a specific odor, we reveal that appetitive long-term memory results in increased numbers of precisely those functional microglomeruli responding to the conditioned odor. Hindering memory consolidation by non-coincident presentation of odor and reward, by blocking protein synthesis, or by including memory mutants suppress these structural changes, revealing their tight correlation with the process of memory consolidation. Thus, olfactory long-term memory is associated with input-specific structural modifications in a high-order center of the fly brain.

Visualization of naive and learned odor representations using in vivo calcium imaging and immunohistochemical bouton mapping of single Drosophila mushroom body neurons.

This protocol enables the quantification of odor-evoked calcium activity in mushroom body Kenyon cells of the Drosophila melanogaster brain at the single bouton level. We also present subsequent characterization of naive and learned odor representations in the context of olfactory coding. This approach to analyzing the neuronal basis of associative learning provides a substrate for similar studies, perhaps in other animals, to probe the attributes of a neuronal memory trace at the level of synapses distributed across neurons. For complete details on the use and execution of this protocol, please refer to Bilz et al. (2020).

Visualization of a Distributed Synaptic Memory Code in the Drosophila Brain.

During associative conditioning, animals learn which sensory cues are predictive for positive or negative conditions. Because sensory cues are encoded by distributed neurons, one has to monitor plasticity across many synapses to capture how learned information is encoded. We analyzed synaptic boutons of Kenyon cells of the Drosophila mushroom body γ lobe, a brain structure that mediates olfactory learning. A fluorescent Ca2+ sensor was expressed in single Kenyon cells so that axonal boutons could be assigned to distinct cells and Ca2+ could be measured across many animals. Learning induced directed synaptic plasticity in specific compartments along the axons. Moreover, we show that odor-evoked Ca2+ dynamics across boutons decorrelate as a result of associative learning. Information theory indicates that learning renders the stimulus representation more distinct compared with naive stimuli. These data reveal that synaptic boutons rather than cells act as individually modifiable units, and coherence among them is a memory-encoding parameter.

Aralar Sequesters GABA into Hyperactive Mitochondria, Causing Social Behavior Deficits.

Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. Here, we show that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. We identify the regulation of GABA availability by mitochondrial activity as a biologically relevant mechanism and demonstrate its contribution to social behavior. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. We identify Aralar as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases our understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions.

Stochastic and Arbitrarily Generated Input Patterns to the Mushroom Bodies Can Serve as Conditioned Stimuli in Drosophila.

Single neurons in the brains of insects often have individual genetic identities and can be unambiguously identified between animals. The overall neuronal connectivity is also genetically determined and hard-wired to a large degree. Experience-dependent structural and functional plasticity is believed to be superimposed onto this more-or-less fixed connectome. However, in Drosophila melanogaster, it has been shown that the connectivity between the olfactory projection neurons (OPNs) and Kenyon cells, the intrinsic neurons of the mushroom body, is highly stochastic and idiosyncratic between individuals. Ensembles of distinctly and sparsely activated Kenyon cells represent information about the identity of the olfactory input, and behavioral relevance can be assigned to this representation in the course of associative olfactory learning. Previously, we showed that in the absence of any direct sensory input, artificially and stochastically activated groups of Kenyon cells could be trained to encode aversive cues when their activation coincided with aversive stimuli. Here, we have tested the hypothesis that the mushroom body can learn any stochastic neuronal input pattern as behaviorally relevant, independent of its exact origin. We show that fruit flies can learn thermogenetically generated, stochastic activity patterns of OPNs as conditioned stimuli, irrespective of glomerular identity, the innate valence that the projection neurons carry, or inter-hemispheric symmetry.

Odor-Induced Multi-Level Inhibitory Maps in Drosophila.

Optical imaging of intracellular Ca2+ influx as a correlate of neuronal excitation represents a standard technique for visualizing spatiotemporal activity of neuronal networks. However, the information-processing properties of single neurons and neuronal circuits likewise involve inhibition of neuronal membrane potential. Here, we report spatially resolved optical imaging of odor-evoked inhibitory patterns in the olfactory circuitry of Drosophila using a genetically encoded fluorescent Cl- sensor. In combination with the excitatory component reflected by intracellular Ca2+ dynamics, we present a comprehensive functional map of both odor-evoked neuronal activation and inhibition at different levels of olfactory processing. We demonstrate that odor-evoked inhibition carried by Cl- influx is present both in sensory neurons and second-order projection neurons (PNs), and is characterized by stereotypic, odor-specific patterns. Cl--mediated inhibition features distinct dynamics in different neuronal populations. Our data support a dual role of inhibitory neurons in the olfactory system: global gain control across the neuronal circuitry and glomerulus-specific inhibition to enhance neuronal information processing.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously naïve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

Slow presynaptic mechanisms that mediate adaptation in the olfactory pathway of Drosophila.

The olfactory system encodes odor stimuli as combinatorial activity of populations of neurons whose response depends on stimulus history. How and on which timescales previous stimuli affect these combinatorial representations remains unclear. We use in vivo optical imaging in Drosophila to analyze sensory adaptation at the first synaptic step along the olfactory pathway. We show that calcium signals in the axon terminals of olfactory receptor neurons (ORNs) do not follow the same adaptive properties as the firing activity measured at the antenna. While ORNs calcium responses are sustained on long timescales, calcium signals in the postsynaptic projection neurons (PNs) adapt within tens of seconds. We propose that this slow component of the postsynaptic response is mediated by a slow presynaptic depression of vesicle release and enables the combinatorial population activity of PNs to adjust to the mean and variance of fluctuating odor stimuli.

Developmental Coordination during Olfactory Circuit Remodeling in Drosophila.

Developmental neuronal remodeling is crucial for proper wiring of the adult nervous system. While remodeling of individual neuronal populations has been studied, how neuronal circuits remodel-and whether remodeling of synaptic partners is coordinated-is unknown. We found that the Drosophila anterior paired lateral (APL) neuron undergoes stereotypic remodeling during metamorphosis in a similar time frame as the mushroom body (MB) ɣ-neurons, with whom it forms a functional circuit. By simultaneously manipulating both neuronal populations, we found that cell-autonomous inhibition of ɣ-neuron pruning resulted in the inhibition of APL pruning in a process that is mediated, at least in part, by Ca2+-Calmodulin and neuronal activity dependent interaction. Finally, ectopic unpruned MB ɣ axons display ectopic connections with the APL, as well as with other neurons, at the adult, suggesting that inhibiting remodeling of one neuronal type can affect the functional wiring of the entire micro-circuit.

Neural Control of Startle-Induced Locomotion by the Mushroom Bodies and Associated Neurons in Drosophila.

Startle-induced locomotion is commonly used in Drosophila research to monitor locomotor reactivity and its progressive decline with age or under various neuropathological conditions. A widely used paradigm is startle-induced negative geotaxis (SING), in which flies entrapped in a narrow column react to a gentle mechanical shock by climbing rapidly upwards. Here we combined in vivo manipulation of neuronal activity and splitGFP reconstitution across cells to search for brain neurons and putative circuits that regulate this behavior. We show that the activity of specific clusters of dopaminergic neurons (DANs) afferent to the mushroom bodies (MBs) modulates SING, and that DAN-mediated SING regulation requires expression of the DA receptor Dop1R1/Dumb, but not Dop1R2/Damb, in intrinsic MB Kenyon cells (KCs). We confirmed our previous observation that activating the MB α'ß', but not αß, KCs decreased the SING response, and we identified further MB neurons implicated in SING control, including KCs of the γ lobe and two subtypes of MB output neurons (MBONs). We also observed that co-activating the αß KCs antagonizes α'ß' and γ KC-mediated SING modulation, suggesting the existence of subtle regulation mechanisms between the different MB lobes in locomotion control. Overall, this study contributes to an emerging picture of the brain circuits modulating locomotor reactivity in Drosophila that appear both to overlap and differ from those underlying associative learning and memory, sleep/wake state and stress-induced hyperactivity.

SIFamide Translates Hunger Signals into Appetitive and Feeding Behavior in Drosophila.

Animal behavior is, on the one hand, controlled by neuronal circuits that integrate external sensory stimuli and induce appropriate motor responses. On the other hand, stimulus-evoked or internally generated behavior can be influenced by motivational conditions, e.g., the metabolic state. Motivational states are determined by physiological parameters whose homeostatic imbalances are signaled to and processed within the brain, often mediated by modulatory peptides. Here, we investigate the regulation of appetitive and feeding behavior in the fruit fly, Drosophila melanogaster. We report that four neurons in the fly brain that release SIFamide are integral elements of a complex neuropeptide network that regulates feeding. We show that SIFamidergic cells integrate feeding stimulating (orexigenic) and feeding suppressant (anorexigenic) signals to appropriately sensitize sensory circuits, promote appetitive behavior, and enhance food intake. Our study advances the cellular dissection of evolutionarily conserved signaling pathways that convert peripheral metabolic signals into feeding-related behavior.

Spermidine Suppresses Age-Associated Memory Impairment by Preventing Adverse Increase of Presynaptic Active Zone Size and Release.

Memories are assumed to be formed by sets of synapses changing their structural or functional performance. The efficacy of forming new memories declines with advancing age, but the synaptic changes underlying age-induced memory impairment remain poorly understood. Recently, we found spermidine feeding to specifically suppress age-dependent impairments in forming olfactory memories, providing a mean to search for synaptic changes involved in age-dependent memory impairment. Here, we show that a specific synaptic compartment, the presynaptic active zone (AZ), increases the size of its ultrastructural elaboration and releases significantly more synaptic vesicles with advancing age. These age-induced AZ changes, however, were fully suppressed by spermidine feeding. A genetically enforced enlargement of AZ scaffolds (four gene-copies of BRP) impaired memory formation in young animals. Thus, in the Drosophila nervous system, aging AZs seem to steer towards the upper limit of their operational range, limiting synaptic plasticity and contributing to impairment of memory formation. Spermidine feeding suppresses age-dependent memory impairment by counteracting these age-dependent changes directly at the synapse.

Optogenetics in Drosophila Neuroscience.

Optogenetic techniques enable one to target specific neurons with light-sensitive proteins, e.g., ion channels, ion pumps, or enzymes, and to manipulate their physiological state through illumination. Such artificial interference with selected elements of complex neuronal circuits can help to determine causal relationships between neuronal activity and the effect on the functioning of neuronal circuits controlling animal behavior. The advantages of optogenetics can best be exploited in genetically tractable animals whose nervous systems are, on the one hand, small enough in terms of cell numbers and to a certain degree stereotypically organized, such that distinct and identifiable neurons can be targeted reproducibly. On the other hand, the neuronal circuitry and the behavioral repertoire should be complex enough to enable one to address interesting questions. The fruit fly Drosophila melanogaster is a favorable model organism in this regard. However, the application of optogenetic tools to depolarize or hyperpolarize neurons through light-induced ionic currents has been difficult in adult flies. Only recently, several variants of Channelrhodopsin-2 (ChR2) have been introduced that provide sufficient light sensitivity, expression, and stability to depolarize central brain neurons efficiently in adult Drosophila. Here, we focus on the version currently providing highest photostimulation efficiency, ChR2-XXL. We exemplify the use of this optogenetic tool by applying it to a widely used aversive olfactory learning paradigm. Optogenetic activation of a population of dopamine-releasing neurons mimics the reinforcing properties of a punitive electric shock typically used as an unconditioned stimulus. In temporal coincidence with an odor stimulus this artificially induced neuronal activity causes learning of the odor signal, thereby creating a light-induced memory.

Identified Serotonin-Releasing Neurons Induce Behavioral Quiescence and Suppress Mating in Drosophila.

Animals show different levels of activity that are reflected in sensory responsiveness and endogenously generated behaviors. Biogenic amines have been determined to be causal factors for these states of arousal. It is well established that, in Drosophila, dopamine and octopamine promote increased arousal. However, little is known about factors that regulate arousal negatively and induce states of quiescence. Moreover, it remains unclear whether global, diffuse modulatory systems comprehensively affecting brain activity determine general states of arousal. Alternatively, individual aminergic neurons might selectively modulate the animals' activity in a distinct behavioral context. Here, we show that artificially activating large populations of serotonin-releasing neurons induces behavioral quiescence and inhibits feeding and mating. We systematically narrowed down a role of serotonin in inhibiting endogenously generated locomotor activity to neurons located in the posterior medial protocerebrum. We identified neurons of this cell cluster that suppress mating, but not feeding behavior. These results suggest that serotonin does not uniformly act as global, negative modulator of general arousal. Rather, distinct serotoninergic neurons can act as inhibitory modulators of specific behaviors. SIGNIFICANCE STATEMENT: An animal's responsiveness to external stimuli and its various types of endogenously generated, motivated behavior are highly dynamic and change between states of high activity and states of low activity. It remains unclear whether these states are mediated by unitary modulatory systems globally affecting brain activity, or whether distinct neurons modulate specific neuronal circuits underlying particular types of behavior. Using the model organism Drosophila melanogaster, we find that activating large proportions of serotonin-releasing neurons induces behavioral quiescence. Moreover, distinct serotonin-releasing neurons that we genetically isolated and identified negatively affect aspects of mating behavior, but not food uptake. This demonstrates that individual serotoninergic neurons can modulate distinct types of behavior selectively.

Optical dissection of experience-dependent pre- and postsynaptic plasticity in the Drosophila brain.

Drosophila represents a key model organism for dissecting neuronal circuits that underlie innate and adaptive behavior. However, this task is limited by a lack of tools to monitor physiological parameters of spatially distributed, central synapses in identified neurons. We generated transgenic fly strains that express functional fluorescent reporters targeted to either pre- or postsynaptic compartments. Presynaptic Ca(2+) dynamics are monitored using synaptophysin-coupled GCaMP3, synaptic transmission is monitored using red fluorescent synaptophysin-pHTomato, and postsynaptic Ca(2+) dynamics are visualized using GCaMP3 fused with the postsynaptic matrix protein, dHomer. Using two-photon in vivo imaging of olfactory projection neurons, odor-evoked activity across populations of synapses is visualized in the antennal lobe and the mushroom body calyx. Prolonged odor exposure causes odor-specific and differential experience-dependent changes in pre- and postsynaptic activity at both levels of olfactory processing. The approach advances the physiological analysis of synaptic connections across defined groups of neurons in intact Drosophila.