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«Connectivity, Organization, and Network Coordination of the Drosophila Central Circadian Clock by Zepeng Yao A dissertation submitted in partial ...»

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2.1 Abstract Drosophila melanogaster is a valuable model system for the neural basis of complex behavior, but an inability to routinely interrogate physiologic connections within central neural networks of the fly brain remains a fundamental barrier to progress in the field. To address this problem, we have introduced a simple method of measuring functional connectivity based on the independent expression of the mammalian P2X2 purinoreceptor and genetically encoded Ca2+ and cAMP sensors within separate genetically defined subsets of neurons in the adult brain.

We show that such independent expression is capable of specifically rendering defined sets of neurons excitable by pulses of bath-applied ATP in a manner compatible with high-resolution Ca2+ and cAMP imaging in putative follower neurons. Furthermore, we establish that this approach is sufficiently sensitive for the detection of excitatory and modulatory connections deep within larval and adult brains. This technically facile approach can now be used in wild-type and mutant genetic backgrounds to address functional connectivity within neuronal networks governing a wide range of complex behaviors in the fly. Furthermore, the effectiveness of this approach in the fly brain suggests that similar methods using appropriate heterologous receptors might be adopted for other widely used model systems.

Originally published in J Neurophysiol 2012 Jul 15;108(2):684-96 doi: 10.1152/jn.00110.2012 with authors listed as Zepeng Yao*, Ann Marie Macara*, Katherine R. Lelito*, Tamara Y. Minosyan, and Orie T. Shafer (* denotes equal contribution).

2.2 Introduction Despite its relative simplicity the nervous system of Drosophila melanogaster is capable of producing a remarkable repertoire of complex behaviors (Weiner 1999). Work on Drosophila has identified discrete networks of neurons that govern circadian timekeeping (Nitabach and Taghert 2008), courtship (Villella et al. 2008), memory (McGuire et al. 2005), sleep (Crocker and Sehgal 2010), feeding (Melcher et al. 2007), and decision-making (e.g., Dickson 2008; Peabody et al. 2009). The study of these and other neural networks in the fly continues to enrich and inform our understanding of the neural control of animal behavior. For many of these central brain networks the pattern and physiologic basis of their constituent connections have been proposed; however, due to the electrophysiologic inaccessibility of much of the fly CNS, many aspects of these network models remain unchallenged experimentally. The development of technically feasible methods to test for the presence and physiologic nature of connections between defined neuronal classes of the fly CNS will therefore be critical for progress in the field.

The ability to address the nature of connections between pairs of identified neurons has been one of the great strengths of large invertebrate model systems (Kandel 1976). The stereotyped and large neurons of these organisms are accessible to multiple recording and stimulating electrodes, making it possible to stimulate activity in a neuron of interest while measuring electrophysiologic responses in putative follower neurons (e.g., Kandel et al.

1967; Willows and Hoyle 1969; Fig. 2.1). Unfortunately, such multielectrode experiments are not feasible for most central neural networks of the Drosophilabrain. The electrophysiologic inaccessibility of many central fly neurons has been surmounted somewhat by the use of genetically encoded sensors for neuronal excitation and second-messenger signaling (e.g., Lissandron et al. 2007; Ruta et al. 2010; Shafer et al. 2008; Tian et al. 2009; Tomchik and Davis 2009; Wang et al. 2003; Yu et al. 2003) and the physiologic responses of single deeply situated neurons can now be routinely observed in the fly brain using live imaging techniques.

Combining these techniques with an ability to acutely activate subsets of neurons would allow for existing models of neural connectivity to be tested and the downstream targets of neurons of interest to be identified physiologically.

Several genetically encoded triggers of neural excitation have been successfully used in Drosophila in conjunction with various chemical or physical triggering methods (reviewed in Venken et al. 2011). The first instance of such triggering in the fly used the photochemical excitation of neurons expressing transgenic P2X2 receptor, a mammalian ATP receptor that is not encoded by the Drosophila genome (Lima and Miesenböck 2005; Littleton and Ganetzky 2000). The mammalian thermosensitive TRPV1 channel has been used to excite fly sensory neurons using its ligand capsaicin (Marella et al. 2006) and ectopic expression of the Drosophila thermosensitive TRPA1 channel has also been used to activate multiple neuron types with pulses of high temperature (e.g., Parisky et al. 2008). Furthermore, the mammalian cold-sensitive TRPM8 channel has been used with both low-temperature pulses and menthol vapor as exogenous excitation triggers in the fly (Peabody et al. 2009). Finally, several groups have used the bacterial opsin Channelrhodopsin-2 (ChR2) to trigger neuronal excitation in Drosophila with blue light (e.g., Pulver et al. 2009; Schroll et al. 2006; Zimmermann et al.

2009). The fact that ChR2 is maximally activated by blue wavelengths makes it problematic for use in live imaging experiments, since GFP-based sensors must be excited with the same wavelengths that activate opsin conductance (Guo et al. 2009). The recent development of redshifted optogenetic controls (Yizhar et al. 2011) and Ca2+ sensors (Zhao et al. 2011) may ultimately circumvent this problem, but these newly developed tools have not yet been successfully introduced to Drosophila. The use of temperature pulses to trigger the opening of TRPA1 or TRPM8 channels during live imaging experiments is also problematic, because acute shifts in temperature can cause significant movement of imaging targets within the explanted brain during high-resolution imaging, which makes the analysis of single-neuron somata difficult (Q. Zhang and O. Shafer, unpublished observations). For these reasons we have opted for ligandgated triggering of transgenic receptors as a means for acute neuronal excitation. The feasibility of combining ATP excitation of P2X2-expressing fly neurons to attain biologically relevant neural excitation during behavioral and physiologic experiments has already been established for both larval and adult nervous systems (e.g., Hu et al. 2010; Lima and Miesenböck 2005). We have therefore chosen ATP/P2X2 excitation for use in our live imaging experiments.





In Drosophila the Gal4/UAS system is a powerful and versatile method of transgene expression that has been the tool of choice for directing sensor expression in specific neuronal classes within the fly brain (Brand and Perrimon 1993; Venken et al. 2011). The recent development of alternative binary expression systems, the LexA and Q systems (Lai and Lee 2006; Potter et al. 2010), now makes it possible to independently direct P2X2 and sensor expression within different neuronal classes. Here we have used the simultaneous use of the Gal4 and LexA systems for the independent dual binary expression of P2X2 and genetically encoded sensors of Ca2+ or cAMP, thereby allowing for the acute excitation of defined neuronal populations during the simultaneous live imaging of Ca2+ and cAMP dynamics within putative neuronal targets (Fig. 2.1).

Here we establish the feasibility of the simultaneous use of the GAL4 and LexA systems to render defined groups of neurons excitable by pulses of bath-applied ATP while simultaneously and independently expressing the Ca2+ sensor GCaMP3.0 or the cAMP sensor Epac1-camps in putative follower neurons. We present proof of principle experiments that establish the efficacy of this method for detecting established and/or predicted excitatory and modulatory connections within larval and adult brains, concentrating on the well-characterized circadian clock neuron network of the fly (Nitabach and Taghert 2008), the constituent physiologic connections of which have remained largely unexamined. The LexAopP2X2, LexAop-GCaMP3.0, and LexAop-Epac1-camps lines we have used for these studies, along with large and growing number of existing GAL4, UAS, and LexA lines, constitute a useful and technically facile toolkit for the interrogation of central neuronal networks in the Drosophila brain.

2.3 Methods 2.3.1 Fly stocks and rearing Flies were reared on cornmeal-yeast-sucrose media at 25°C under a 12:12 light:dark cycle or under the diurnal conditions of the lab. All Gal4 and UAS lines used in this study have been previously described: Pdf(M)-Gal4;; and ;Pdf(bmrj)-Gal4; (Renn et al. 1999), ;UASGCaMP3.0; (Tian et al. 2009), ;;UAS-P2X2(Lima and Miesenböck 2005), ;;Clock(4.1M)Gal4 (Zhang et al. 2010a,b), ;Clock(-856[8.2/2])-Gal4; (Gummadova et al. 2009), ;c929Gal4; (Hewes et al. 2000), ;Rh6-Gal4; (Pichaud and Desplan 2001), ;UAS-Epac1camps(50A); (Shafer and Taghert 2009), and ;Cha(7.4)-Gal4/CyO; (Salvaterra and Kitamoto 2001). The ;Pdf-LexA;line has also been described previously (Shang et al. 2008). The creation of the LexAop-P2X2, LexAop-GCaMP3.0, and LexAop-Epac1-camps lines is described in the following text. Stable lines carrying combinations of these elements were created using standard Drosophila genetic techniques.

2.3.2 Creation of LexAop P2X2 and sensor lines We used the LexA-response element containing pLOT vector (Lai and Lee 2006) for the creation of LexAop-GCaM3.0,LexAop-Epac1-camps, and LexAop-P2X2 plasmids. GCaMP3.0 (Tian et al. 2009) was obtained in a pEGFP-N1 vector from Addgene (Cambridge, MA; plasmid # 22692) and digested with EagI. The resulting GCaMP3.0-containing fragment was gel purified, digested with BglII, and subsequently PCR purified using a QIAquick PCR Purification Kit (Qiagen, Valencia, CA). In parallel, pLOT vector was digested with EagI and BglII, and treated with CIP alkaline phosphatase (New England Biolabs, Ipswich, MA) following manufacturer's instructions. The GCaMP3.0 fragment was ligated with the linearized pLOT vector with a Quick Ligation Kit from New England Biolabs. Epac1-camps (Nikolaev et al.

2004) was sequentially digested from the pUAST-Epac1-camps plasmid (Shafer et al. 2008) using XhoI and BglII, and PCR purified. This Epac1-camps fragment was cloned into pLOT as above using sequential XhoI and BglII restriction digests of pLOT. The P2X2 trimer (Lima and Miesenböck 2005) was obtained as the Gateway entry clone pENTRA1_P2X2 from G.

Miesenböck (Oxford University). We created a pLOT Gateway vector by cutting pLOT with KPN1, generating blunt ends using T4 DNA Polymerase (Invitrogen), and inserting the chloramphenicol/ccdB-resistant Gateway cassette A using T4 DNA Ligase following manufacturer's instructions (Invitrogen). We transformed OmniMAX 2T1R cells (Invitrogen) with the resulting pLOT-Gateway vector, selected ampicillin- and chloramphenicol-resistant clones for vector propagation, and purified the pLOT-Gateway vector using a Qiagen Mini Prep kit (Qiagen). The transfer of the P2X2 trimer from pENTRA1_P2X2 to the pLOT-Gateway vector was accomplished via LR recombination reaction according to manufacturer's instructions (Invitrogen) using LR II clonase (Invitrogen).

All three LexAop plasmids were extracted and purified using a Qiagen Mini Prep kit.

Purified plasmids were sent to Genetic Services, Inc. (Cambridge, MA), where they were injected into w1118 embryos. We isolated and mapped several independent transgenic lines for each LexAop element using standard fly genetic techniques. The specific lines used here were: w;LexAop-GCaMP3.0(4B);, w;LexAop-Epac1-camps(1A);, w;LexAop-P2X2(7);, and w;;LexAop-P2X2(1).

2.3.3 Dissections, solutions, and test compound delivery Flies were anesthetized on CO2 and brains were dissected into room temperature hemolymph-like saline (HL3) consisting of (in mM): 70 NaCl, 5 KCl, 1.5 CaCl2, 20 MgCl2, 10 NaHCO3, 5 trehalose, 115 sucrose, 5 HEPES; pH 7.1 (Stewart et al. 1994). For larval brain dissections, third instar (nonwandering) larvae were removed from the food and brains were dissected directly into HL3, keeping the eye disks and ventral nerve cord intact. Mouth hooks continued to move after dissections and were therefore removed to prevent brain movement during imaging experiments. All brains were allowed to adhere to the bottom of 35-mm FALCON culture dishes (Becton Dickenson Labware, Franklin Lakes, NJ) under a drop of HL3 contained within a petri dish insert (Bioscience Tools, San Diego, CA) for directing perfusion flow. Brains were imaged 5 to 10 min after dissection to allow for optimum baseline stabilization and settling of the brain to the dish. Perfusion flow was established over the brain with a gravityfed PS-8H perfusion system (Bioscience Tools). Test compounds were delivered to mounted brains by switching perfusion flow from the main HL3 line to another channel containing diluted compound for desired durations followed by a return to HL3 flow. All test compounds were dissolved in HL3. To control for the effects of switching channels, we perfused HL3 for 30 s from a second vehicle channel as a vehicle control. Adenosine 5[prime]-triphosphate disodium salt hydrate (ATP), guanosine 5[prime]-triphosphate disodium salt hydrate (GTP), and carbamoylcholine chloride (carbachol) were purchased from Sigma-Aldrich (St. Louis, MO).

2.3.4 Live imaging and analysis Live imaging was performed using an Olympus FV1000 laser-scanning microscope (Olympus, Center Valley, PA) under a ×20 (0.50 N/A W, UMPlan FL N) or ×60 (1.10 N/A W, FUMFL N) objective (Olympus, Center Valley, PA). Regions of interest (ROIs) were selected over single neuronal somata or, in the case of Bolwig's nerve, over the length of a nerve. For GCaMP3.0 imaging experiments, frames were scanned with a 488-nm laser at 1—10 Hz for 5 min and GCaMP emission was directed to a photomultiplier tube by means of a DM405/488 dichroic mirror. Scanning frequencies for GCaMP3.0 imaging were kept constant within experiments, but varied between experiments. Experiments involving multiple neuronal classes demanded larger scanning areas and therefore lower scan rates. Epac1-camps FRET imaging was performed by scanning frames with a 440-nm laser at a frequency of 1 Hz for 5 min. CFP and YFP emission was separated by means of a SDM510 dichroic mirror.



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