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

Furthermore, I have found that the Drosophila clock neuron network features diverse modes of coupling between the various clock neuron classes. Lastly, I revealed that the Drosophila clock neuron network consists of multiple independent oscillators and requires network-wide coherence for robust circadian rhythms in activity and sleep. My thesis research greatly advances our understanding of how the circadian clock neuron network is wired, organized, and coordinated. Given that disruption of circadian rhythms is associated with increased risks of a large spectrum of diseases, including obesity, heart diseases, cancer, and mood disorders (Albrecht, 2012), my thesis research may help address the widespread adverse effects of circadian rhythm disorders. Here, I summarize the key findings and implications of my thesis research as follows.

6.1 A new approach to address functional neuronal connectivity in the Drosophila brain Chapter 2 describes a new experimental approach that my colleagues and I developed for analyzing functional neuronal connections in the Drosophila brain. In this approach, the mammalian ATP-gated cation channel P2X2 is genetically expressed in neurons of interest to render them excitable by ATP on demand, while genetically encoded fluorescent sensors are simultaneously expressed in putative postsynaptic neurons to monitor their response upon the excitation of P2X2-expressing neurons. This approach has proved powerful, versatile, yet technically facile, and is now being widely used in the field of Drosophila neurobiology (e.g., Haynes et al., 2015; Kallman et al., 2015).

6.2 Physiological connectivity within the Drosophila clock neuron network Using the experimental approach my colleagues and I developed for neuronal connectivity analysis, I have confirmed a long predicted peptidergic connection from the LNvs to the LNds mediated by PDF, and showed that it is a modulatory connection that results in cAMP increases without causing acute excitation in the LNds (Chapter 2). Furthermore, I show that within the LNv group, PDF secreted from the l-LNvs acts to specifically increase cAMP levels but not calcium levels in the s-LNvs (Fig. 6.1 and Chapter 2). Lastly, using ATP/P2X2-mediated excitation of the glutamatergic DN1ps in conjunction with whole-cell patch-clamp recording of the LNds and l-LNvs, I uncover inhibitory connections from the DN1ps to the LNds and to the lLNvs (Chapter 3). Together, I have begun to delineate the neuronal connections between important groups of clock neurons. An extension of this experimental approach will allow the characterization of functional connectivity between other groups of clock neurons, and between clock neurons and potential input and output pathways.

6.3 Electrophysiological characterization of the critical LNd clock neurons In Chapter 3, I have provided the first electrophysiological analysis of the critical LNd clock neurons, which are considered collectively as the evening oscillator of the clock neuron network (Grima et al., 2004; Stoleru et al., 2004). I found that the LNds fire spontaneous tonic and bursting patterns of action potentials, and that the LNd neuronal activity is modulated by multiple fast neurotransmitters. Specifically, the LNds are excited by acetylcholine via nicotinic acetylcholine receptors, and inhibited by GABA and glutamate via GABAA receptors and the glutamate-gated chloride channel GluClα, respectively. The LNds’ receptivity to multiple fast neurotransmitters is in striking similarity to that of the LNvs (McCarthy et al., 2011; Lelito and Shafer, 2012). Using genetic and behavioral approaches, I found that while GABAergic inhibition of the lateral clock neurons functions to promote sleep at night, glutamatergic inhibition of the same neurons functions to promote wakefulness during specific times of the day. These results advance our understanding of the neurophysiological properties of central clock neurons and reveal how the various clock neuron classes integrate distinct synaptic inputs to orchestrate circadian rhythms in sleep and activity.

6.4 Diverse modes of coupling between the various clock neuron groups In Chapter 4 and Chapter 5, using a genetic strategy to specifically speed-up or slowdown the LNv molecular clocks, I experimentally investigated if and how molecular clocks of the various clock neuron classes are coupled to the critical LNv clocks. I found that the molecular clocks of the 5th s-LNv and most of the LNds are not coupled to the LNvs, while the two pairs of sNPF-expressing LNds can only be delayed but not advanced by the LNvs. In contrast, the CRYexpressing DN1ps can be both delayed and advanced by the LNvs. These results reveal that the various classes of clock neurons do not display a uniform mode of coupling. Rather, they display unique and complex coupling relationships that vary from group to group. This may have important implications in the clock network plasticity in the face of changing environments.

6.5 The Drosophila clock neuron network consists of multiple oscillators and requires network-wide coherence for robust free-running rhythms Despite the fact that the clock neuron network features a diversity of cell types that are anatomically and neurochemically distinct, it has been long modeled as a hierarchical twooscillator network, in which the morning oscillator (the LNvs) functions as a master pacemaker in the absence of environmental cues. In Chapter 4, through the genetic speeding-up and slowing-down the LNv clocks to different extents, I find that the LNvs can set the pace of the clock network only when their intrinsic period differs less than ~2.5 hours from that of the rest of the network. In contrast to the widely accepted “Master Pacemaker” model, my results demonstrate that the clock network consists of multiple oscillatory units, each of which drives rhythms in activity. Furthermore, I find that each oscillatory unit is unified by its neuropeptide output, which might be a general organizing principle that might apply to the circadian clock neuron networks of other animals. Lastly in Chapter 5, by genetically altering the clock speed in different subsets of clock neurons, I show that coherent free-running activity rhythms require molecular clock synchrony at least in all of the lateral clock neurons as well as the DN1ps, which constitute a much larger proportion of the clock neuron network than previously thought. These findings provide insights into the organization and network coordination of the Drosophila circadian clock.

Figure 6.1.

The l-LNvs modulate cAMP levels in the s-LNvs.

(A) Averaged Epac1-camps inverse FRET plot (±SEM) of l-LNvs imaged in c929-Gal4/PdfLexA,LexAop-Epac1-camps;UAS-P2X2/+ brains, before, during and after 30s perfusion of 1mM ATP (indicated on the bottom plot in each column). ATP/P2X2 mediated excitation caused clear inverse FRET increases. (B) Averaged Epac1-camps inverse FRET plot (±SEM) of s-LNvs from the same brains as (A). Excitation of the c929 network produced inverse FRET increases in the s-LNvs. (C) Averaged Epac1-camps inverse FRET plot (±SEM) of l-LNvs imaged in a Pdfr mutant background using han5304;c929-GAL4/Pdf-LexA,LexAop-Epac1-camps;UAS-P2X2/+ brains. c929 network excitation caused clear inverse FRET increases in these neurons. (D) Averaged Epac1-camps plot (±SEM) of s-LNvs from the same brains as (C). Excitation of the c929 network failed to produce inverse FRET increases in the s-LNvs in the absence of PdfR function. (E) Averaged Epac1-camps inverse FRET plot (±SEM) of l-LNvs imaged in PdfLexA,LexAop-Epac1-camps/+;UAS-P2X2/+ brains. ATP failed to produce inverse FRET increases in the absence of the GAL4 driver. The scale bars in (E) also apply to (A) and (C). (F) Averaged Epac1-camps inverse FRET plot (±SEM) of s-LNvs from the same brains as (E). ATP caused no obvious inverse FRET increases. The scale bars in (F) also apply to (B) and (D). (G) Comparison of maximum Epac1-camps responses for the l-LNv data shown in (A), (C), and (E).

ATP (1mM) perfusion caused significant inverse FRET increases in both the experimental (“Exp” c929-GAL4/Pdf-LexA,LexAop-Epac1-camps;UAS-P2X2/+) and PdfR mutant (“-PDFR” han5304 ;c929-GAL4/Pdf- LexA,LexAop-Epac1-camps;UAS-P2X2/+) conditions, relative to the negative control lacking the GAL4 driver for P2X2 expression (“-P2X2” Pdf-LexA,LexAopEpac1-camps/+;UAS-P2X2/+). (H) Comparison of maximum Epac1-camps responses for the sLNv data shown in (B), (D), and (F). ATP (1mM) perfusion caused significant inverse FRET increases in experimental (Exp) flies relative to both PdfR mutant (-PDFR) and -P2X2 controls.

Genotypes were identical to those in (G). For (G) and (H), *** indicates P 0.001 and n.s.

indicates no significant difference (P ≥ 0.05), by Kruskal—Wallis one-way ANOVA and Dunn's multiple comparison test. The methods and materials used in this figure are the same as those described in Chapter 2.

6.6 References

Albrecht, U. (2012). Timing to Perfection: The Biology of Central and Peripheral Circadian Clocks. Neuron 74, 246–260.

Grima, B., Chélot, E., Xia, R., and Rouyer, F. (2004). Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain. Nature 431, 869–873.

Haynes, P.R., Christmann, B.L., and Griffith, L.C. (2015). A single pair of neurons links sleep to memory consolidation in drosophila melanogaster. Elife 2015, 1–24.

Kallman, B.R., Kim, H., and Scott, K. (2015). Excitation and inhibition onto central courtship neurons biases Drosophila mate choice. Elife 4, e11188.

Lelito, K.R., and Shafer, O.T. (2012). Reciprocal cholinergic and GABAergic modulation of the small ventrolateral pacemaker neurons of Drosophila’s circadian clock neuron network. J.

Neurophysiol. 107, 2096–2108.

McCarthy, E. V, Wu, Y., Decarvalho, T., Brandt, C., Cao, G., and Nitabach, M.N. (2011).

Synchronized bilateral synaptic inputs to Drosophila melanogaster neuropeptidergic rest/arousal neurons. J. Neurosci. 31, 8181–8193.

Stoleru, D., Peng, Y., Agosto, J., and Rosbash, M. (2004). Coupled oscillators control morning