Title: Activity Dependent Trans-synaptic Tracing Of Neural Circuits In Drosophila
Description: Drosophila exhibits a rich repertoire of simple and complex behaviors. In addition, the ability to allow genetic manipulations of specific neuronal populations makes the numerically simple fly brain an attractive model system to study the mechanisms that translate neural circuits to meaningful behavioral responses. Delineation of neural circuits requires development of approaches that trace functional synaptic connections. We have developed HA-Tango-trace, an activity-dependent trans-synaptic tracer to define neural circuits that convey information from the inner photoreceptors in the retina to the lobula complex in the Drosophila visual system. Elucidation of neural circuits and the mechanisms involved in translating the circuitry into a meaningful behavioral response with Tango-trace involves labeling of neurons in an activity-dependent manner based on the release of an endogenous neurotransmitter at a synapse. This strategy can be extended to any neural circuit in the brain with a known neurotransmitter in both flies and mice.

In the visual system, specific features of the visual image like motion, color, form and shape are extracted and processed in neural pathways. This information is transmitted to the brain where it must be processed to translate stimulus features into appropriate behavioral output. Here we investigate how this information is represented in higher visual centers in flies. The stochastically distributed p/yR7s and p/y R8s in the retina project to the medulla and make precise connections with four unique connectors that relay information to the lobula complex. Thus, the p/yR7s and p/y R8s process spectral information in separate pathways and relay information to the lobula and lobula plate. The projections to the lobula plate afford the opportunity for inputs to the motion pathway. Moreover, our behavioral data show that R8s influence motion-evoked behavioral responses under bright light conditions. Gap junctions between the inner and outer photoreceptors could afford an explanation for the convergence of the two pathways.

This by itself is sufficient for visual discrimination of objects during navigation or, alternatively, the postsynaptic partners of R7 and R8 may additionally provide inputs to the motion pathway. Thus, spectral and motion pathways may converge repetitively at each stage of the circuit and reorganize into pathways of behavioral significance. Furthermore, histaminergic neurons have been implicated in temperature preference and circadian rhythms. These behaviors are likely to result from neuromodulation of central brain circuits mediated by histamine. Tango assay can be used to study this other important aspect of neural circuits by measuring the intensity of signal before and after neuromodulation. This approach was successfully used to map neuromodulation of dopamine mediated sugar sensitivity in flies using dopamine tango-map. Hunger enhances behavioral sensitivity to sugar and this is mediated by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx in a DopEcR-dependent manner.

Tango-map permits the detection of increases in endogenous neuromodulator release in vivo. In addition, histamine has been detected in mechanosensory neurons in Drosophila. Auditory systems are critical to the behavior of many insects. In Drosophila melanogaster, acoustic communication is essential for making decisions related to mate selection. The projections of the HA-Tango labeled neurons overlap with the proposed higher order auditory neurons in the protocerebral areas. Further characterization of these circuits with HA-Tango-trace will provide insights into the representation of mechanosensory and auditory information that drive diverse behaviors in Drosophila. Acetylcholine is a major neurotransmitter of the olfactory and gustatory systems in Drosophila. We have designed Ach-Tango to trace connections in the olfactory and gustatory systems in an activity-dependent manner. Characterization of circuits in higher brain areas may help us understand how odor and taste percepts are formed and how these sensory modalities are processed in the higher brain centers to generate diverse olfactory and gustatory behaviors.

The studies described in this thesis provide approaches to analyze circuits and understand their functional implications. Tango-Trace is a genetically encoded trans-synaptic tracer designed to identify synaptic connections in an activity-dependent manner by chronic activation of the presynaptic neuron with a genetically targeted neuronal activator, dTrpA1 and the identification of postsynaptic partners by GFP or any other reporter of choice. Tango-Map is designed to detect volume transmission of a neuromodulator by measuring the signal intensity of the reporter before and after a neuromodulatory effect. Furthermore, deciphering the circuit mechanisms that translate into complex behaviors will provide an understanding of more complex processes in the brain like emotion, cognition and consciousness. Our understanding of the nervous system can benefit greatly from these tools.