Serotonin acts through multiple cellular targets during an olfactory critical period.

Serotonin (5-HT) is known to modulate early development during critical periods when experience drives heightened levels of plasticity in neurons. Here, we take advantage of the genetically tractable olfactory system of Drosophila to investigate how 5-HT modulates critical period plasticity in the CO2 sensing circuit of fruit flies. Our study reveals that 5HT modulation of multiple neuronal targets is necessary for experience-dependent structural changes in an odor processing circuit. The olfactory CPP is known to involve local inhibitory networks and consistent with this we found that knocking down 5-HT7 receptors in a subset of GABAergic local interneurons was sufficient to block CPP, as was knocking down GABA receptors expressed by olfactory sensory neurons (OSNs). Additionally, direct modulation of OSNs via 5-HT2B expression in the cognate OSNs sensing CO2 is also essential for CPP. Furthermore, 5-HT1B expression by serotonergic neurons in the olfactory system is also required during the critical period. Our study reveals that 5-HT modulation of multiple neuronal targets is necessary for experience-dependent structural changes in an odor processing circuit.

Olfactory Critical Periods: How Odor Exposure Shapes the Developing Brain in Mice and Flies.

Neural networks have an extensive ability to change in response to environmental stimuli. This flexibility peaks during restricted windows of time early in life called critical periods. The ubiquitous occurrence of this form of plasticity across sensory modalities and phyla speaks to the importance of critical periods for proper neural development and function. Extensive investigation into visual critical periods has advanced our knowledge of the molecular events and key processes that underlie the impact of early-life experience on neuronal plasticity. However, despite the importance of olfaction for the overall survival of an organism, the cellular and molecular basis of olfactory critical periods have not garnered extensive study compared to visual critical periods. Recent work providing a comprehensive mapping of the highly organized olfactory neuropil and its development has in turn attracted a growing interest in how these circuits undergo plasticity during critical periods. Here, we perform a comparative review of olfactory critical periods in fruit flies and mice to provide novel insight into the importance of early odor exposure in shaping neural circuits and highlighting mechanisms found across sensory modalities.

Nonspiking Interneurons in the Drosophila Antennal Lobe Exhibit Spatially Restricted Activity.

Inhibitory interneurons are important for neuronal circuit function. They regulate sensory inputs and enhance output discriminability (Olsen and Wilson, 2008; Root et al., 2008; Olsen et al., 2010). Often, the identities of interneurons can be determined by location and morphology, which can have implications for their functions (Wachowiak and Shipley, 2006). While most interneurons fire traditional action potentials, many are nonspiking. These can be seen in insect olfaction (Laurent and Davidowitz, 1994; Husch et al., 2009; Tabuchi et al., 2015) and the vertebrate retina (Gleason et al., 1993). Here, we present the novel observation of nonspiking inhibitory interneurons in the antennal lobe (AL) of the adult fruit fly, Drosophila melanogaster These neurons have a morphology where they innervate a patchwork of glomeruli. We used electrophysiology to determine whether their nonspiking characteristic is because of a lack of sodium current. We then used immunohistochemsitry and in situ hybridization to show this is likely achieved through translational regulation of the voltage-gated sodium channel gene, para Using in vivo calcium imaging, we explored how these cells respond to odors, finding regional isolation in their responses' spatial patterns. Further, their response patterns were dependent on both odor identity and concentration. Thus, we surmise these neurons are electrotonically compartmentalized such that activation of the neurites in one region does not propagate across the whole antennal lobe. We propose these neurons may be the source of intraglomerular inhibition in the AL and may contribute to regulation of spontaneous activity within glomeruli.

A Population of Interneurons Signals Changes in the Basal Concentration of Serotonin and Mediates Gain Control in the Drosophila Antennal Lobe.

Serotonin (5-HT) represents a quintessential neuromodulator, having been identified in nearly all animal species [1] where it functions in cognition [2], motor control [3], and sensory processing [4]. In the olfactory circuits of flies and mice, serotonin indirectly inhibits odor responses in olfactory receptor neurons (ORNs) via GABAergic local interneurons (LNs) [5, 6]. However, the effects of 5-HT in olfaction are likely complicated, because multiple receptor subtypes are distributed throughout the olfactory bulb (OB) and antennal lobe (AL), the first layers of olfactory neuropil in mammals and insects, respectively [7]. For example, serotonin has a non-monotonic effect on odor responses in Drosophila projection neurons (PNs), where low concentrations suppress odor-evoked activity and higher concentrations boost PN responses [8]. Serotonin reaches the AL via the diffusion of paracrine 5-HT through the fly hemolymph [8] and by activation of the contralaterally projecting serotonin-immunoreactive deuterocerebral interneurons (CSDns): the only serotonergic cells that innervate the AL [9, 10]. Concentration-dependent effects could arise by either the expression of multiple 5-HT receptors (5-HTRs) on the same cells or by populations of neurons dedicated to detecting serotonin at different concentrations. Here, we identify a population of LNs that express 5-HT7Rs exclusively to detect basal concentrations of 5-HT. These LNs inhibit PNs via GABAB receptors and mediate subtractive gain control. LNs expressing 5-HT7Rs are broadly tuned to odors and target every glomerulus in the antennal lobe. Our results demonstrate that serotonergic modulation at low concentrations targets a specific population of LNs to globally downregulate PN odor responses in the AL.

Local synaptic inputs support opposing, network-specific odor representations in a widely projecting modulatory neuron.

Serotonin plays different roles across networks within the same sensory modality. Previously, we used whole-cell electrophysiology in Drosophila to show that serotonergic neurons innervating the first olfactory relay are inhibited by odorants (Zhang and Gaudry, 2016). Here we show that network-spanning serotonergic neurons segregate information about stimulus features, odor intensity and identity, by using opposing coding schemes in different olfactory neuropil. A pair of serotonergic neurons (the CSDns) innervate the antennal lobe and lateral horn, which are first and second order neuropils. CSDn processes in the antennal lobe are inhibited by odors in an identity independent manner. In the lateral horn, CSDn processes are excited in an odor identity dependent manner. Using functional imaging, modeling, and EM reconstruction, we demonstrate that antennal lobe derived inhibition arises from local GABAergic inputs and acts as a means of gain control on branch-specific inputs that the CSDns receive within the lateral horn.

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels.

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between target neurons. The method relies on co-expression of a transgenic activator with the tetrodotoxin-resistant sodium channel, NaChBac, in a specific presynaptic neuron. Connections with putative post-synaptic partners are determined by whole-cell recordings in the presence of TTX, which blocks electrical activity in neurons that do not express NaChBac. This approach can be modified to work with any activator or calcium imaging as a reporter of connections. TERPS adds to the growing set of tools available for determining connectivity within networks. However, TERPS is unique in that it also reliably reports bulk or volume transmission and spillover transmission.

Serotonergic Modulation of Olfaction in Rodents and Insects.

Recent advances in genetic tools and optical imaging technology have allowed rodent and Drosophila researchers to explore the relationship between serotonergic modulation and olfactory processing at a mechanistic level previously unfeasible. Here, I review the basic organization of olfactory and serotonergic systems in both rodents and Drosophila and draw comparisons where similarities exist. I discuss circuit level models that explain many of serotonin's effects on olfactory responses in the olfactory system's inputs and outputs. Finally, I discuss models of integration within wide-field centrifugal neurons to emphasize the importance of studying serotonergic neurons directly to build more realistic models of olfactory and modulatory interactions.

The stomatogastric nervous system of the medicinal leech: its anatomy, physiology and associated aminergic neurons.

Blood feeding is an essential and signature activity of the medicinal leech species Hirudo verbana. Despite keen interest in understanding the neuronal substrates of this behavior, a major component of the nervous system associated with feeding has remained overlooked. In this study, for the first time, we report on the presence and characteristics of five stomatogastric ganglia (STGs) comprising the visceral stomatogastric nervous system (STN) of the leech. Although a brief report was published by Ruth Hanke in 1948 indicating that a ring of three ganglia (not five) was associated with the cephalic ganglia, this information was never integrated into subsequent neurobiological studies of feeding. Here, the anatomical features of the STGs are described, as are the morphological and electrophysiological characteristics of neurons originating in them. We also determined that two of the five STGs (STG-1 and STG-3) each contained two relatively large (ca. 40 µm diameter) serotonergic neurons. The STN was also enriched with dopaminergic and serotonergic arborizations; however, no intrinsic dopaminergic somata were observed. The trajectory of the serotonergic large lateral (LL) neuron, a command-like cell for feeding, was documented to project directly to the STN and not to the jaw and pharyngeal musculature as previously reported, thus reopening the important question of how the LL cell activates and coordinates biting activity with pharyngeal swallowing. Additional studies revealed that the LL cell is excited by blood serum applied to the lip and is strongly inhibited by dopamine. These findings provide a new foundation for understanding the regulation and modulation of neural networks involved in feeding.

Identified Serotonergic Modulatory Neurons Have Heterogeneous Synaptic Connectivity within the Olfactory System of Drosophila.

Modulatory neurons project widely throughout the brain, dynamically altering network processing based on an animal's physiological state. The connectivity of individual modulatory neurons can be complex, as they often receive input from a variety of sources and are diverse in their physiology, structure, and gene expression profiles. To establish basic principles about the connectivity of individual modulatory neurons, we examined a pair of identified neurons, the "contralaterally projecting, serotonin-immunoreactive deutocerebral neurons" (CSDns), within the olfactory system of Drosophila Specifically, we determined the neuronal classes providing synaptic input to the CSDns within the antennal lobe (AL), an olfactory network targeted by the CSDns, and the degree to which CSDn active zones are uniformly distributed across the AL. Using anatomical techniques, we found that the CSDns received glomerulus-specific input from olfactory receptor neurons (ORNs) and projection neurons (PNs), and networkwide input from local interneurons (LNs). Furthermore, we quantified the number of CSDn active zones in each glomerulus and found that CSDn output is not uniform, but rather heterogeneous, across glomeruli and stereotyped from animal to animal. Finally, we demonstrate that the CSDns synapse broadly onto LNs and PNs throughout the AL but do not synapse upon ORNs. Our results demonstrate that modulatory neurons do not necessarily provide purely top-down input but rather receive neuron class-specific input from the networks that they target, and that even a two cell modulatory network has highly heterogeneous, yet stereotyped, pattern of connectivity.SIGNIFICANCE STATEMENT Modulatory neurons often project broadly throughout the brain to alter processing based on physiological state. However, the connectivity of individual modulatory neurons to their target networks is not well understood, as modulatory neuron populations are heterogeneous in their physiology, morphology, and gene expression. In this study, we use a pair of identified serotonergic neurons within the Drosophila olfactory system as a model to establish a framework for modulatory neuron connectivity. We demonstrate that individual modulatory neurons can integrate neuron class-specific input from their target network, which is often nonreciprocal. Additionally, modulatory neuron output can be stereotyped, yet nonuniform, across network regions. Our results provide new insight into the synaptic relationships that underlie network function of modulatory neurons.

Functional integration of a serotonergic neuron in the Drosophila antennal lobe.

Serotonin plays a critical role in regulating many behaviors that rely on olfaction and recently there has been great effort in determining how this molecule functions in vivo. However, it remains unknown how serotonergic neurons that innervate the first olfactory relay respond to odor stimulation and how they integrate synaptically into local circuits. We examined the sole pair of serotonergic neurons that innervates the Drosophila antennal lobe (the first olfactory relay) to characterize their physiology, connectivity, and contribution to pheromone processing. We report that nearly all odors inhibit these cells, likely through connections made reciprocally within the antennal lobe. Pharmacological and immunohistochemical analyses reveal that these neurons likely release acetylcholine in addition to serotonin and that exogenous and endogenous serotonin have opposing effects on olfactory responses. Finally, we show that activation of the entire serotonergic network, as opposed to only activation of those fibers innervating the antennal lobe, may be required for persistent serotonergic modulation of pheromone responses in the antennal lobe.

Leg-tracking and automated behavioural classification in Drosophila.

Much remains unknown about how the nervous system of an animal generates behaviour, and even less is known about the evolution of behaviour. How does evolution alter existing behaviours or invent novel ones? Progress in computational techniques and equipment will allow these broad, complex questions to be explored in great detail. Here we present a method for tracking each leg of a fruit fly behaving spontaneously upon a trackball, in real time. Legs were tracked with infrared-fluorescent dyes invisible to the fly, and compatible with two-photon microscopy and controlled visual stimuli. We developed machine-learning classifiers to identify instances of numerous behavioural features (for example, walking, turning and grooming), thus producing the highest-resolution ethological profiles for individual flies.

Distinct roles of TRP channels in auditory transduction and amplification in Drosophila.

Auditory receptor cells rely on mechanically gated channels to transform sound stimuli into neural activity. Several TRP channels have been implicated in Drosophila auditory transduction, but mechanistic studies have been hampered by the inability to record subthreshold signals from receptor neurons. Here, we develop a non-invasive method for measuring these signals by recording from a central neuron that is electrically coupled to a genetically defined population of auditory receptor cells. We find that the TRPN family member NompC, which is necessary for the active amplification of sound-evoked motion by the auditory organ, is not required for transduction in auditory receptor cells. Instead, NompC sensitizes the transduction complex to movement and precisely regulates the static forces on the complex. In contrast, the TRPV channels Nanchung and Inactive are required for responses to sound, suggesting they are components of the transduction complex. Thus, transduction and active amplification are genetically separable processes in Drosophila hearing.

Asymmetric neurotransmitter release enables rapid odour lateralization in Drosophila.

In Drosophila, most individual olfactory receptor neurons (ORNs) project bilaterally to both sides of the brain. Having bilateral rather than unilateral projections may represent a useful redundancy. However, bilateral ORN projections to the brain should also compromise the ability to lateralize odours. Nevertheless, walking or flying Drosophila reportedly turn towards the antenna that is more strongly stimulated by odour. Here we show that each ORN spike releases approximately 40% more neurotransmitter from the axon branch ipsilateral to the soma than from the contralateral branch. As a result, when an odour activates the antennae asymmetrically, ipsilateral central neurons begin to spike a few milliseconds before contralateral neurons, and at a 30 to 50% higher rate than contralateral neurons. We show that a walking fly can detect a 5% asymmetry in total ORN input to its left and right antennal lobes, and can turn towards the odour in less time than it requires the fly to complete a stride. These results demonstrate that neurotransmitter release properties can be tuned independently at output synapses formed by a single axon onto two target cells with identical functions and morphologies. Our data also show that small differences in spike timing and spike rate can produce reliable differences in olfactory behaviour.

Decision points: the factors influencing the decision to feed in the medicinal leech.

The decision to feed is a complex task that requires making several small independent choices. Am I hungry? Where do I look for food? Is there something better I'd rather be doing? When should I stop? With all of these questions, it is no wonder that decisions about feeding depend on several sensory modalities and that the influences of these sensory systems would be evident throughout the nervous system. The leech is uniquely well suited for studying these complicated questions due to its relatively simple nervous system, its exceptionally well-characterized behaviors and neural circuits, and the ease with which one can employ semi-intact preparations to study the link between physiology and decision-making. We will begin this review by discussing the cellular substrates that govern the decision to initiate and to terminate a bout of feeding. We will then discuss how feeding temporarily blocks competing behaviors from being expressed while the animal continues to feed. Then we will review what is currently known about how feeding affects long-term behavioral choices of the leech. Finally, we conclude with a short discussion of the advantages of the leech's decision-making circuit's design and how this design might be applicable to all decision circuits.

Smelling on the fly: sensory cues and strategies for olfactory navigation in Drosophila.

Navigating toward (or away from) a remote odor source is a challenging problem that requires integrating olfactory information with visual and mechanosensory cues. Drosophila melanogaster is a useful organism for studying the neural mechanisms of these navigation behaviors. There are a wealth of genetic tools in this organism, as well as a history of inventive behavioral experiments. There is also a large and growing literature in Drosophila on the neural coding of olfactory, visual, and mechanosensory stimuli. Here we review recent progress in understanding how these stimulus modalities are encoded in the Drosophila nervous system. We also discuss what strategies a fly might use to navigate in a natural olfactory landscape while making use of all these sources of sensory information. We emphasize that Drosophila are likely to switch between multiple strategies for olfactory navigation, depending on the availability of various sensory cues. Finally, we highlight future research directions that will be important in understanding the neural circuits that underlie these behaviors.

Feeding-mediated distention inhibits swimming in the medicinal leech.

An animal's response to a stimulus depends on many factors such as age, hormonal state, experience, and its behavioral state. For example, an animal may suppress a behavior that is inappropriate or incompatible with its current state. In this study, we show that, as a medicinal leech feeds, the distention that it incurs inhibits its expression of swimming. Distention slows the swimming pattern and decreases the number of swim cycles elicited by a test electrical stimulation; large distentions inhibit swimming altogether. We have previously shown that the ingestive phase of feeding inhibits behaviors by presynaptic inhibition of mechanosensory neurons. Distention has its effects downstream (e.g., gating and central pattern generating interneurons) from these sensory neurons and thus represents a novel mechanism for choosing between conflicting behaviors during feeding. Because removing the leech's gut surgically did not eliminate the effects of body distention, we conclude that the receptors mediating the distention-induced suppression of swimming are likely to be located in the animal's body wall. Together with previous findings, these new data show that leeches rely on two different decision-making networks to ensure that a biologically important behavior is not disrupted by other behaviors.

Species-specific behavioral patterns correlate with differences in synaptic connections between homologous mechanosensory neurons.

We characterized the behavioral responses of two leech species, Hirudo verbana and Erpobdella obscura, to mechanical skin stimulation and examined the interactions between the pressure mechanosensory neurons (P cells) that innervate the skin. To quantify behavioral responses, we stimulated both intact leeches and isolated body wall preparations from the two species. In response to mechanical stimulation, Hirudo showed local bending behavior, in which the body wall shortened only on the side of the stimulation. Erpobdella, in contrast, contracted both sides of the body in response to touch. To investigate the neuronal basis for this behavioral difference, we studied the interactions between P cells. Each midbody ganglion has four P cells; each cell innervates a different quadrant of the body wall. Consistent with local bending, activating any one P cell in Hirudo elicited polysynaptic inhibitory potentials in the other P cells. In contrast, the P cells in Erpobdella had excitatory polysynaptic connections, consistent with the segment-wide contraction observed in this species. In addition, activating individual P cells caused asymmetrical body wall contractions in Hirudo and symmetrical body wall contractions in Erpobdella. These results suggest that the different behavioral responses in Erpobdella and Hirudo are partly mediated by interactions among mechanosensory cells.

Behavioral choice by presynaptic inhibition of tactile sensory terminals.

When presented with multiple stimuli, animals generally choose to respond only to one input. The neuronal mechanisms determining such behavioral choices are poorly understood. We found that the medicinal leech had greatly diminished responses to moderate mechanosensory input as it fed. Feeding dominated other responses by suppressing transmitter release from mechanosensory neurons onto all of their neuronal targets. The effects of feeding on synaptic transmission could be mimicked by serotonin. Furthermore, the serotonin antagonist mianserin blocked feeding-induced decreases in synaptic transmission. These results indicate that feeding predominates behaviors by using serotonin at an early stage of sensory processing, namely on presynaptic terminals of mechanosensory neurons.

Photochemical control of endogenous ion channels and cellular excitability.

Light-activated ion channels provide a precise and noninvasive optical means for controlling action potential firing, but the genes encoding these channels must first be delivered and expressed in target cells. Here we describe a method for bestowing light sensitivity onto endogenous ion channels that does not rely on exogenous gene expression. The method uses a synthetic photoisomerizable small molecule, or photoswitchable affinity label (PAL), that specifically targets K+ channels. PALs contain a reactive electrophile, enabling covalent attachment of the photoswitch to naturally occurring nucleophiles in K+ channels. Ion flow through PAL-modified channels is turned on or off by photoisomerizing PAL with different wavelengths of light. We showed that PAL treatment confers light sensitivity onto endogenous K+ channels in isolated rat neurons and in intact neural structures from rat and leech, allowing rapid optical regulation of excitability without genetic modification.

Bursting in leech heart interneurons: cell-autonomous and network-based mechanisms.

Rhythmic activity within the heartbeat pattern generator of the medicinal leech is based on the alternating bursting of mutually inhibitory pairs of oscillator heart interneurons (half-center oscillators). Bicuculline methiodide has been shown to block mutual inhibition between these interneurons and to cause them to spike tonically while recorded intracellularly (Schmidt and Calabrese, 1992). Using extracellular recording techniques, we show here that oscillator and premotor heart interneurons continue to burst when pharmacologically isolated with bicuculline, although the bursting is not robust in some preparations. We propose that a nonspecific leak current introduced by the intracellular microelectrode suppresses endogenous bursting activity to account for the discrepancy with results using intracellular recording. A two-parameter bifurcation diagram (E(leak) vs g(leak)) of a mathematical model of a single heart interneuron shows a narrow stripe of parameter values where bursting occurs, separating large zones of tonic spiking and silence. A similar analysis performed for a half-center oscillator model outlined a much larger area of bursting. Bursting in the half-center oscillator model is also less sensitive to variation in the maximal conductances of voltage-gated currents than in the single-neuron model. Thus, in addition to ensuring appropriate bursting characteristics such as period, phase, and duty cycles, the half-center configuration enhances oscillation robustness, making them less susceptible to random or imposed changes in membrane parameters. Endogenous bursting, in turn, ensures appropriate bursting if the strength of mutual inhibition is weakened and limits the minimum period of the half-center oscillator to a period near that of the single neuron.