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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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Our results suggest that 30-s perfusions of 1—5 mM ATP result in significant neuronal excitation for all three neuron types we tested. To gauge the reliability of such ATP/P2X2 excitation we analyzed how often each of these 30-s ATP treatments failed to excite the P2X2expressing s-LNvs, DN1ps, and PNs. We defined a failure conservatively as any ATP-treated neuron displaying less than a 25% maximal increase in GCaMP3.0 fluorescence. For all three neuron types, failure rates were 50% for 1 mM ATP perfusions and approached zero at higher concentrations (Fig. 2.4B). Our choice of 30-s perfusions was based on previous experiments involving the bath application of neurotransmitters and receptor agonists (Lelito and Shafer 2012). We wondered if shorter applications of ATP might still yield sufficient excitation of the sLNvs, the most deeply situated of the neurons tested, using both the LexA and Gal4 expression systems. We therefore determined the failure rates for various durations of 2.5 mM ATP for sLNvs coexpressing GCaMP3.0 and P2X2 with either LexA or Gal4 drivers. For perfusion durations of 10 to 20 s, failure rates for both genotypes were all near 30%. Failure rates reached zero at perfusion durations of 25 s for LexA s-LNvs and at 30 s for GAL4 s-LNvs (Fig. 2.4F).

The ability to excite the same set of P2X2-expressing neurons repeatedly would allow for multiple sets of putative follower neurons residing in different focal planes to be investigated in the same brain preparation. We therefore asked if P2X2-mediated excitation by bath-applied ATP could be used to repeatedly stimulate deep brain neurons. Indeed, repeated 30-s perfusions of 2.5 mM ATP resulted in reliable repeated excitation of s-LNvs expressing either GAL4- or LexA-driven P2X2 (Fig. 2.4, G and H). Although the baseline fluorescence of these neurons displayed a slow and steady drop in intensity, there was no significant difference in the mean maximum GCaMP3.0 fluorescence increases displayed in response to the first and last (fifth) 30s perfusion of ATP, when compared with the baseline fluorescence preceding each ATP pulse.

For repeated excitation using the GAL4 system to coexpress GCaMP3.0 and P2X2 expression (Fig. 2.4G), the first ATP perfusion caused a mean maximum GCaMP3.0 increase of 126.6 ± 32.9% and the fifth and final pulse caused a mean increase of 114.5 ± 21.9% (P = 0.8438 by Mann—Whitney U test). For repeated excitation using the LexA system (Fig. 2.4H) the first ATP perfusion caused a mean maximum GCaMP3.0 increase of 145.3 ± 19.1% and final pulse caused a mean increase of 94.1 ± 18.8% (P = 0.0524 by Mann—Whitney U test). Thus, P2X2expressing neurons can be repeatedly activated in the same dissected brain without a significant rundown in excitation.

Based on these results, we conclude that 30-s perfusions of 1—5 mM ATP result in robust, reliable, and repeatable excitation of deep brain P2X2-expressing neurons, using either the GAL4 or LexA expression system to drive the expression of P2X2. However, we note that different neuronal types display differing profiles of excitation, indicating that specific excitation parameters should be determined empirically for every neuron class and genotype to be excited.

2.4.4 Dual binary expression of P2X2 and genetically encoded sensors allow for the specific excitation of neuronal subsets during live imaging experiments.

Having confirmed the efficacy of our LexAop-driven sensor and P2X2 elements, we next sought to confirm that the simultaneous use of the GAL4 and LexA systems could render specific neuron classes excitable by ATP during high-resolution imaging experiments. We therefore used the Pdf-LexA element to drive LexAop-GCaMP3.0 expression in both the s-LNvs and the large ventrolateral neurons (l-LNvs) of the circadian clock network, while simultaneously using the c929-GAL4 element, which is expressed by the l-LNvs but not the s-LNvs, to drive P2X2 in the l-LNvs and in the many other peptidergic neurons expressing this GAL4 driver (Fig

2.5A; Hewes et al. 2000). Thus, the l-LNvs of ;Pdf-LexA,LexAop-GCaMP3.0/c929-GAL4;UASP2X2/+ brains will express P2X2, whereas the s-LNvs will not. If the specific dual binary expression of P2X2 and GCaMP3.0 were successful, the l-LNvs would be expected to display acute GCaMP3.0 responses to bath-applied ATP, whereas the s-LNvs would not. As predicted, 30-s perfusions of 1 mM ATP resulted in the excitation of the l-LNvs, but did not excite the sLNvs imaged within the same focal planes (Fig. 2.5, B–D). This result, along with the experiments presented in the following text, indicate that the simultaneous use of the GAL4 and LexA systems for the independent expression of P2X2 and genetically encoded sensors, makes possible the specific excitation of neuronal subsets in a manner compatible with high-resolution live imaging experiments. This result also suggests that the excitation of the l-LNvs, neurons important for the control of arousal, sleep, and the integration of circadian light input (Parisky et al. 2008;Shang et al. 2008; Sheeba et al. 2008), does not result in large acute Ca2+ increases in the critical s-LNv pacemaker neurons.

2.4.5 Gal4-based excitation and LexA-based live imaging for an established excitatory connection in the larval brain.

We next sought to determine if our proposed method of addressing functional connectivity was sufficiently sensitive to detect an established neuronal connection in Drosophila. We were motivated to propose the present approach to circuit analysis because there are few well-established synaptic connections in our circuitry of interest, the circadian clock neuron network. One of the only fully confirmed synaptic connections in the Drosophila clock network is the excitatory connection between Bolwig's organ (BO), the maggot eye, and the LNv clock neurons, which persist through metamorphosis to become the adult s-LNvs (Fig. 2.6A; Helfrich-Förster et al. 2007). BO projects directly to the larval optic neuropil via Bolwig's nerve (BN), where its terminals reside in close apposition to LNv arbors (Helfrich-Förster et al. 2002; Malpel et al. 2002). BN expresses ChAT, an enzyme required for acetylcholine (ACh) synthesis (Yasuyama and Salvaterra 1999) and ChAT is required in BN for photic resetting of larval clock neurons (Keene et al. 2011). Dissociated and cultured larval LNvs are directly excited by bath-applied ACh and nicotine (Wegener et al. 2004). Finally, Yuan and colleagues (2011) have recently shown that blue-light stimulation of BO causes acute Ca2+ increases in the larval LNvs clock neurons. Thus, the BN to LNv connection in the larval brain offers a well-established excitatory connection in the clock network on which to test our method for addressing connectivity.

Under our experimental conditions, we found it necessary to remove the larval mouth hooks to prevent brain movement during imaging. Mouth hook removal was associated with the loss of BO, leaving only the afferent BNs associated with the eye disks and central brain (Fig.

2.6, A and B). We therefore first confirmed that excitation of BN was possible in the absence of BO by coexpressing P2X2 and GCaMP3.0 in BN using the Rh6-Gal4 driver, which is expressed in a subset of BN axons (Fig. 2.6A; Keene et al. 2011). We found that 30-s perfusions of 5 mM ATP caused reliable Ca2+ responses in BNs of ;Rh6-GAL4/UAS-GCaMP3.0;UAS-P2X2/+ brains, indicating the successful excitation of BNs (Fig. 2.6, B, D, and G).

Having confirmed successful ATP/P2X2 excitation of BN in our preparation, we asked if the predicted excitatory responses could be detected in larval LNvs in response to BN excitation.

We therefore created ;Rh6-Gal4/Pdf-lexA, LexAop-GCaMP3.0; UAS-P2X2/+ larvae to independently express P2X2 in BN and GCaMP3.0 in the LNvs (Fig. 2.6A). Consistent with previous reports, we observed no Rh6-GAL4 driver expression in the LNvs or in any other central neurons of the larval brain (e.g., Keene et al. 2011 and data not shown). All 30-s perfusions of 5 mM ATP caused significant GCaMP3.0 fluorescence increases in the LNvs of Rh6-Gal4/PdflexA, LexAop-GCaMP3.0; UAS-P2X2/+ brains (Fig. 2.6, C, E, and H). To confirm that the LNv responses to ATP perfusion were due to the specific excitation of the BN and not to the leaky expression of UAS-P2X2 in non-BN cell types or native responses of larval LNvs to ATP, we repeated the experiment on brains dissected from ;Pdf-lexA, LexAop-GCaMP3.0/+; UASP2X2/+ larvae, which lacked the R6-GAL4 element and therefore would not have driven P2X2 expression specifically in BN. The LNvs of these flies did not display significant changes in GCaMP3.0 fluorescence following 30-s perfusions of 5 mM ATP (Fig. 2.6, F and I), indicating that nonspecific UAS-P2X2expression or native ATP responses had not caused the Ca2+ responses displayed by the LNvs following the ATP/P2X2 excitation of BN. We conclude that our method of addressing connectivity was sufficiently sensitive to detect an established excitatory connection deep within the larval brain.

2.4.6 LexA-based excitation and GAL4-based live imaging to test a predicted peptidergic connection in the adult central brain.

The circadian clock neuron network of the adult fly consists of approximately 150 neurons that express conserved molecular clockwork (Nitabach and Taghert 2008).

Understanding the connective properties of this network was our motivation for developing a means for interrogating the physiologic connections between neuronal classes deep within the fly brain. The s-LNvs are critical neuronal pacemakers required for the maintenance of robust rhythms in sleep and activity in the fly under constant darkness and temperature (Grima et al.

2004;Renn et al. 1999; Shafer and Taghert 2009; Stoleru et al. 2004). A large and growing body of anatomic, genetic, and physiologic evidence suggests that the clock neuron network is coordinated through modulatory connections between the s-LNvs and the various classes of dorsal clock neurons. The s-LNvs project to the dorsal brain, where their terminals comingle with terminals from the dorsal clock neuron classes (Helfrich-Förster et al. 2007; Kaneko and Hall 2000). The s-LNvs express the neuropeptide pigment-dispersing factor (PDF), the genetic loss of which causes a weakening or loss of free-running behavioral rhythms (Helfrich-Förster 1995; Renn et al. 1999; Shafer and Taghert 2009) and a loss of synchronization among various clock neuron classes (Lin et al. 2004). PDF signals through PDFR, a G-protein—coupled receptor (GPCR) that signals through cAMP increases (Hyun et al. 2005; Lear et al.

2005; Mertens et al. 2005) and is expressed by dorsal clock neurons (Im and Taghert 2010).

Finally, the dorsal neuron classes respond to bath-applied PDF peptide with cAMP increases (Shafer et al. 2008). Taken together, these findings provide strong evidence for a neuromodulatory connection between the s-LNvs and dorsal clock neurons in the adult fly brain.

Thus, the current prevailing model predicts that the excitation of the s-LNvs will result in acute cAMP increases within dorsal clock neurons.

Nevertheless, the physiologic nature of this proposed connection has not been confirmed experimentally. Indeed, recent work has shown that the s-LNvs also expressshort neuropeptide F (sNPF) (Johard et al. 2009), which encodes four peptides whose GPCR would likely antagonize PDFR signaling (Garczynski et al. 2007; Mertens et al. 2002; Reale et al. 2004). The coexpression of potentially antagonistic peptides in the s-LNvs suggests that the excitation of these neurons might in fact cause cAMP decreases in dorsal clock neuron classes. Determining the functional nature of this proposed connection therefore requires the ability to experimentally interrogate its physiology. We therefore set out to determine the nature of the predicted connection between the s-LNv pacemakers and the LNds, which are among the predicted neuronal targets of the s-LNvs (Im and Taghert 2010; Shafer et al. 2008) and are thought to play a critical role in the control of the fly's evening bout of daily activity (Grima et al. 2004; Stoleru et al. 2004).

To investigate the proposed connection between the s-LNvs and the LNd clock neurons, we drove P2X2 expression specifically in the l-LNvs and s-LNvs using Pdf-LexA, while driving GCaMP3.0 or Epac1-camps expression with Clock(856)-GAL4, which is expressed throughout most of clock neuron network (Fig. 2.7A; Gummadova et al. 2009). Note that although PdfLexA drives LexAop-P2X2 in both the l-LNvs and s-LNvs, only the s-LNvs send projections to the dorsal brain, whereas the l-LNvs project to both optic lobes (Fig. 2.7A; Helfrich-Förster et al.

2007). For brains dissected from ;Clock(856)-GAL4,UAS-GCaMP3.0/+;Pdf-LexA,LexAopP2X2/+ flies, excitation of the l-LNvs and s-LNvs with 30-s perfusions of 1 mM ATP caused clear increases in GCaMP3.0 fluorescence in the LNvs, but had no measurable effects on the LNds residing in the same optical sections, suggesting that LNv excitation does not cause large acute Ca2+ increases or acute excitation in the LNds (Fig. 2.7B). In contrast, 30-s perfusions of 1 mM ATP across ;Clock(856)-GAL4,UAS-Epac1-camps/+;Pdf-LexA,LexAop-P2X2/+ brains resulted in significant increases in Epac1-camps inverse FRET within the LNds, consistent with cAMP increases in response to LNv excitation (Fig. 2.7, C and D). Direct ATP/P2X2 excitation of the l-LNvs and s-LNvs caused significant increases in Epac1-camps inverse FRET (Fig.

2.7, E and F, and data not shown), indicating a strong coupling of neuronal excitation and cAMP production in these neurons. The large increase in LNd inverse Epac1-camps FRET was preceded by a small and transient decrease in inverse FRET (Fig. 2.7C). However, this decrease was not caused by LNv excitation, because we observed a similar initial decrease in mean inverse FRET in control brains lacking the Pdf-LexA element for driving LexAop-P2X2 expression in the LNvs (Fig. 2.7C).

The LNd cAMP response to bath-applied ATP required P2X2 expression in the LNvs, because brains carrying the LexAop-P2X2 element but lacking the Pdf-LexA driver failed to show cAMP increases in either the LNds or LNvs (Fig. 2.7, C–F; “—P2X2”). Furthermore, the LNd cAMP response to LNv excitation required functional PDF receptor, because ATP perfusion over brains from PdfR5304;Clock(856-GAL4,UAS-Epac1-camps/+;Pdf-LexA,LexAop-P2X2/+ flies failed to produce significant changes in LNd Epac1-camps inverse FRET levels (Fig.

2.7, C and D; “—PDFR”), despite clear excitation of LNvs within the same optical sections (Fig.

2.7, E and F; “—PDFR”).

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