Chi-Hon Lee

A Presynaptic Gain Control Mechanism Fine-Tunes Olfactory Behavior

Early sensory processing can play a critical role in sensing environmental cues. We have investigated the physiological and behavioral function of gain control at the first synapse of olfactory processing in Drosophila. Olfactory receptor neurons (ORNs) express the GABA(B) receptor (GABA(B)R), and its expression expands the dynamic range of ORN synaptic transmission that is preserved in projection neuron responses. Strikingly, each ORN channel has a unique baseline level of GABA(B)R expression. ORNs that sense the aversive odorant CO(2) do not express GABA(B)Rs and do not have significant presynaptic inhibition. In contrast, pheromone-sensing ORNs express a high level of GABA(B)Rs and exhibit strong presynaptic inhibition. Furthermore, pheromone-dependent mate localization is impaired in flies that lack GABA(B)Rs in specific ORNs. These findings indicate that different olfactory receptor channels employ heterogeneous presynaptic gain control as a mechanism to allow an animals innate behavioral responses to match its ecological needs.

The Neural Substrate of Spectral Preference in Drosophila

Drosophila vision is mediated by inputs from three types of photoreceptor neurons; R1-R6 mediate achromatic motion detection, while R7 and R8 constitute two chromatic channels. Neural circuits for processing chromatic information are not known. Here, we identified the first-order interneurons downstream of the chromatic channels. Serial EM revealed that small-field projection neurons Tm5 and Tm9 receive direct synaptic input from R7 and R8, respectively, and indirect input from R1-R6, qualifying them to function as color-opponent neurons. Wide-field Dm8 amacrine neurons receive input from 13-16 UV-sensing R7s and provide output to projection neurons. Using a combinatorial expression system to manipulate activity in different neuron subtypes, we determined that Dm8 neurons are necessary and sufficient for flies to exhibit phototaxis toward ultraviolet instead of green light. We propose that Dm8 sacrifices spatial resolution for sensitivity by relaying signals from multiple R7s to projection neurons, which then provide output to higher visual centers.

An in vitro organ culturing system for intervertebral disc explants with vertebral endplates : a feasibility study with ovine caudal discs

Study Design. Whole ovine caudal intervertebral discs with vertebral endplates were cultured under uniaxial diurnal loading for 7 days. Objectives. To establish and characterize an organ culture system for intervertebral discs, in which disc cells may be “maintained” in their native three-dimensional environment under load. Summary of Background Data. In vitro culturing of entire discs with preserved biologic and structural integrity would be a useful model to study the effects of nutrition and mechanical loading. Methods. To maintain endplate permeability, sheep were systemically anticoagulated before death and their caudal vasculature was evacuated with saline postmortem. The first 4 caudal discs were explanted with their adjacent endplates and cultured in bioreactors under uniaxial diurnal loading (0.2 MPa for 8 hours and 0.8 MPa for 16 hours) for 4 or 7 days. Solute transport into the center of the disc was measured after 4 days of culture using a low molecular weight fluorescent marker. Cell viability, glycosaminoglycan synthesis rate, and gene expression profile were measured after 7 days of culture and compared with fresh tissue. Results. Fluorescent images showed that solutes could diffuse into the disc under both static and diurnal loading, but penetration through the endplate increased with diurnal loading. Cell viability and glycosaminoglycan synthesis rates remained unchanged after 7 days of culture. Expression of catabolic genes was significantly up-regulated, whereas anabolic genes tended to be down-regulated after 7 days. Conclusions. With this novel preparation and culturing technique, endplate permeability could be maintained, which allowed culturing of intact disc explants with endplates for up to 7 days.

Candidate neural substrates for off-edge motion detection in Drosophila.

BACKGROUND In the flys visual motion pathways, two cell types-T4 and T5-are the first known relay neurons to signal small-field direction-selective motion responses [1]. These cells then feed into large tangential cells that signal wide-field motion. Recent studies have identified two types of columnar neurons in the second neuropil, or medulla, that relay input to T4 from L1, the ON-channel neuron in the first neuropil, or lamina, thus providing a candidate substrate for the elementary motion detector (EMD) [2]. Interneurons relaying the OFF channel from L1s partner, L2, to T5 are so far not known, however. RESULTS Here we report that multiple types of transmedulla (Tm) neurons provide unexpectedly complex inputs to T5 at their terminals in the third neuropil, or lobula. From the L2 pathway, single-column input comes from Tm1 and Tm2 and multiple-column input from Tm4 cells. Additional input to T5 comes from Tm9, the medulla target of a third lamina interneuron, L3, providing a candidate substrate for L3s combinatorial action with L2 [3]. Most numerous, Tm2 and Tm9s input synapses are spatially segregated on T5s dendritic arbor, providing candidate anatomical substrates for the two arms of a T5 EMD circuit; Tm1 and Tm2 provide a second. Transcript profiling indicates that T5 expresses both nicotinic and muscarinic cholinoceptors, qualifying T5 to receive cholinergic inputs from Tm9 and Tm2, which both express choline acetyltransferase (ChAT). CONCLUSIONS We hypothesize that T5 computes small-field motion signals by integrating multiple cholinergic Tm inputs using nicotinic and muscarinic cholinoceptors.

Visual circuit development in Drosophila.

Fly visual circuits are organized into lattice-like arrays and layers. Recent genetic studies have provided insights into how these reiterated structures are assembled through stepwise processes and how precise connections are established during development. Afferent-derived morphogens, such as Hedgehog, play a key role in organizing the overall structure by inducing and recruiting target neurons and glia. In turn, the target-derived ligand DWnt4 guides Frizzled2-expressing photoreceptor afferents to their proper destination. Photoreceptor afferents select specific synaptic targets by forming adhesive interactions and regulating actin cytoskeleton in growth cones. Target specificity is probably achieved by restricting the expression of adhesive molecules, such as Capricious, to appropriate presynaptic and postsynaptic partners, and by differentially regulating the function of broadly expressed adhesive molecules such as N-cadherin.

Photoreceptor-derived activin promotes dendritic termination and restricts the receptive fields of first-order interneurons in Drosophila

How neurons form appropriately sized dendritic fields to encounter their presynaptic partners is poorly understood. The Drosophila medulla is organized in layers and columns and innervated by medulla neuron dendrites and photoreceptor axons. Here, we show that three types of medulla projection (Tm) neurons extend their dendrites in stereotyped directions and to distinct layers within a single column for processing retinotopic information. In contrast, the Dm8 amacrine neurons form a wide dendritic field to receive ∼16 R7 photoreceptor inputs. R7- and R8-derived Activin selectively restricts the dendritic fields of their respective postsynaptic partners, Dm8 and Tm20, to the size appropriate for their functions. Canonical Activin signaling promotes dendritic termination without affecting dendritic routing direction or layer. Tm20 neurons lacking Activin signaling expanded their dendritic fields and aberrantly synapsed with neighboring photoreceptors. We suggest that afferent-derived Activin regulates the dendritic field size of their postsynaptic partners to ensure appropriate synaptic partnership.

Focusing Transgene Expression in Drosophila by Coupling Gal4 With a Novel Split-LexA Expression System

Here we report the development of a ternary version of the LexA::VP16/LexAop system in which the DNA-binding and trans-activating moieties are independently targeted using distinct promoters to achieve highly restricted, intersectional expression patterns. This Split LexA system can be concatenated with the Gal4/upstream activating sequence system to refine the expression patterns of existing Gal4 lines with minimal genetic manipulations.

Multiple Redundant Medulla Projection Neurons Mediate Color Vision in Drosophila

Abstract The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons, we have uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. We developed a novel aversive operant conditioning assay for intensity-independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs (light-emitting diodes). The flies’ optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow-spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad-spectrum photoreceptors R1–R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. We hypothesize that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate ultraviolet (UV) spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons.

The Genetic Analysis of Functional Connectomics in Drosophila

Fly and vertebrate nervous systems share many organizational features, such as layers, columns and glomeruli, and utilize similar synaptic components, such as ion channels and receptors. Both also exhibit similar network features. Recent technological advances, especially in electron microscopy, now allow us to determine synaptic circuits and identify pathways cell-by-cell, as part of the flys connectome. Genetic tools provide the means to identify synaptic components, as well as to record and manipulate neuronal activity, adding function to the connectome. This review discusses technical advances in these emerging areas of functional connectomics, offering prognoses in each and identifying the challenges in bridging structural connectomics to molecular biology and synaptic physiology, thereby determining fundamental mechanisms of neural computation that underlie behavior.

Mapping chromatic pathways in the Drosophila visual system

In Drosophila, color vision and wavelength‐selective behaviors are mediated by the compound eyes narrow‐spectrum photoreceptors R7 and R8 and their downstream medulla projection (Tm) neurons Tm5a, Tm5b, Tm5c, and Tm20 in the second optic neuropil or medulla. These chromatic Tm neurons project axons to a deeper optic neuropil, the lobula, which in insects has been implicated in processing and relaying color information to the central brain. The synaptic targets of the chromatic Tm neurons in the lobula are not known, however. Using a modified GFP reconstitution across synaptic partners (GRASP) method to probe connections between the chromatic Tm neurons and 28 known and novel types of lobula neurons, we identify anatomically the visual projection neurons LT11 and LC14 and the lobula intrinsic neurons Li3 and Li4 as synaptic targets of the chromatic Tm neurons. Single‐cell GRASP analyses reveal that Li4 receives synaptic contacts from over 90% of all four types of chromatic Tm neurons, whereas LT11 is postsynaptic to the chromatic Tm neurons, with only modest selectivity and at a lower frequency and density. To visualize synaptic contacts at the ultrastructural level, we develop and apply a “two‐tag” double‐labeling method to label LT11s dendrites and the mitochondria in Tm5cs presynaptic terminals. Serial electron microscopic reconstruction confirms that LT11 receives direct contacts from Tm5c. This method would be generally applicable to map the connections of large complex neurons in Drosophila and other animals. J. Comp. Neurol. 524:213–227, 2016.