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Regulation of sleep by cholinergic neurons located outside the central brain in Drosophila [1]

['Joseph D. Jones', 'Division Of Biological', 'Biomedical Systems', 'School Of Science', 'Engineering', 'University Of Missouri-Kansas City', 'Kansas City', 'Missouri', 'United States Of America', 'Brandon L. Holder']

Date: 2023-03

Sleep is a complex and plastic behavior regulated by multiple brain regions and influenced by numerous internal and external stimuli. Thus, to fully uncover the function(s) of sleep, cellular resolution of sleep-regulating neurons needs to be achieved. Doing so will help to unequivocally assign a role or function to a given neuron or group of neurons in sleep behavior. In the Drosophila brain, neurons projecting to the dorsal fan-shaped body (dFB) have emerged as a key sleep-regulating area. To dissect the contribution of individual dFB neurons to sleep, we undertook an intersectional Split-GAL4 genetic screen focusing on cells contained within the 23E10-GAL4 driver, the most widely used tool to manipulate dFB neurons. In this study, we demonstrate that 23E10-GAL4 expresses in neurons outside the dFB and in the fly equivalent of the spinal cord, the ventral nerve cord (VNC). Furthermore, we show that 2 VNC cholinergic neurons strongly contribute to the sleep-promoting capacity of the 23E10-GAL4 driver under baseline conditions. However, in contrast to other 23E10-GAL4 neurons, silencing these VNC cells does not block sleep homeostasis. Thus, our data demonstrate that the 23E10-GAL4 driver contains at least 2 different types of sleep-regulating neurons controlling distinct aspects of sleep behavior.

In this study, we sought to identify the 23E10-GAL4 dFB neurons that are important for sleep regulation by conducting a targeted, intersectional Split-GAL4 [ 33 ] screen focused on 23E10-GAL4 dFB neurons. We report here that the 23E10-GAL4 driver expresses in many non-dFB neurons in the brain and in the ventral nerve cord (VNC), the fly equivalent to the spinal cord. Furthermore, we have identified 2 non-dFB 23E10-GAL4 expressing, sleep-regulating cholinergic neurons located in the VNC. Our analyses reveal that thermogenetic and optogenetic activation of these neurons promote sleep while silencing increases wakefulness. In addition, we provide evidence that these VNC sleep-promoting neurons are not involved in sleep homeostasis while other 23E10-GAL4 expressing cells are. Thus, we conclude that 23E10-GAL4 contains at least 2 different types of sleep-regulating populations, the 2 VNC neurons we are describing in this study and another group, likely made of dFB neurons, that regulate sleep homeostasis.

Recent studies have used 23E10-GAL4 as a driver to manipulate and monitor dFB neurons [ 17 , 19 , 21 – 24 , 26 – 31 ]. Because of its somewhat restricted expression pattern and strong capacity to modulate sleep, 23E10-GAL4 has become the prominent dFB driver. Analysis of an electron microscopy-based connectome of the central complex indicated that the 23E10-GAL4 driver expresses in 31 dFB neurons in the brain [ 32 ]. However, the extent to which these 31 dFB neurons function as a homogeneous group remains unclear.

The fan-shaped body (FB) is part of the central complex in the Drosophila brain, a region organized into multiple layers that plays a role in locomotion control [ 5 ], courtship behavior [ 6 ] and memory [ 7 , 8 ], nociception [ 9 ], visual feature recognition [ 10 ] and processing [ 11 ], social behaviors [ 12 ], and feeding decision-making [ 13 ]. In addition, dFB neurons have emerged as a key sleep-regulating area as their activation promotes sleep while their neuronal silencing reduces sleep [ 14 – 22 ]. Furthermore, sleep deprivation increases the excitability of dFB neurons, suggesting a role for the dFB in sleep homeostasis [ 17 ]. Finally, it was proposed that increasing sleep pressure switches dFB neurons from an electrically silent to an electrically active state. This process is regulated by dopaminergic signaling to the dFB [ 23 ] and the accumulation of mitochondrial reactive oxygen species in dFB neurons [ 24 ]. The physiological properties of dFB neurons have led to the suggestion that these cells are functionally analogous to the ventrolateral preoptic (VLPO) nucleus, a key sleep-regulating center in the mammalian brain [ 17 , 25 ].

( A ) Cartoon depicting known sleep-regulating centers in the fly brain. ( B ) Diagram of the experimental assay. Sleep was measured at 22°C for 2 days to establish baseline sleep profile. Flies were then shifted to 31°C for 24 h at the start of day 3 to increase activity of the targeted cells by activating the TrpA1 channel and then returned to 22°C. White bars (L) represent the 12 h of light and black bars (D) represent the 12 h of dark that oscillate daily. ( C, D ) Sleep profile in minutes of sleep per hour for day 2 (22°C, blue line) and day 3 (31°C, red line) for parental control female flies: 23E10-GAL4/+ (C) and UAS-TrpA1/+ (D). ( E ) Sleep profile in minutes of sleep per hour for day 2 (22°C, blue line) and day 3 (31°C, red line) for 23E10-GAL4>UAS-TrpA1 female flies. ( F ) Box plots of total sleep change in % ((total sleep on day 3-total sleep on day 2/total sleep on day 2) × 100) for data presented in (C–E). Flies expressing UAS-TrpA1 in 23E10-GAL4 significantly increase sleep when switched to 31°C compared with parental controls, Kruskal–Wallis ANOVA followed by Dunn’s multiple comparisons. ****P < 0.0001, n = 25–31 flies per genotype. ( G ) Box plots of locomotor activity counts per minute awake for flies presented in (C–E). Two-way repeated measures ANOVA followed by Sidak’s multiple comparisons test found no differences in locomotor activity between 22°C and 31°C, n.s. = not significant, n = 25–31 flies per genotype. ( H, I ) Representative confocal stacks of a female 23E10-GAL4>UAS-mCD8GFP brain (H) and VNC, (I). Green, anti-GFP; magenta, anti-nc82 (neuropile marker). ( J ) Cartoon depiction of the original goal of our Split-Gal4 screen, which was to identify which dFB neurons modulate sleep. ( K ) Sleep profile in minutes of sleep per hour for day 2 (22°C, blue line) and day 3 (31°C, red line) for empty control (Empty-AD; 23E10-DBD>TrpA1) flies. ( L ) Sleep profile in minutes of sleep per hour for day 2 (22°C, blue line) and day 3 (31°C, red line) for 23E12-AD; 23E10-DBD>TrpA1 flies. ( M ) Box plots of total sleep change in % for female control (Empty-AD; 23E10-DBD) and 23E12-AD; 23E10-DBD flies expressing UAS-TrpA1. A two-tailed unpaired t test revealed that 23E12-AD; 23E10-DBD>TrpA1 flies increase sleep significantly more than control flies when transferred to 31°C. ****P < 0.0001, n = 44–51 flies per genotype. ( N ) Box plots of locomotor activity counts per minute awake for flies presented in (M). Two-way repeated measures ANOVA followed by Sidak’s multiple comparisons test found that locomotor activity per awake time is increased in 23E12-AD; 23E10-DBD>TrpA1 flies transferred to 31°C. ****P < 0.0001, n = 44–51 flies per genotype. ( O ) Representative confocal stacks of an Empty-AD; 23E10-DBD>UAS-mCD8GFP female brain (left panel) and VNC (right panel). Green, anti-GFP; magenta, anti-nc82 (neuropile marker). ( P ) Representative confocal stacks of an 23E12-AD; 23E10-DBD>UAS-mCD8GFP female brain (left panel), VNC (middle panel left), a side view of the VNC (middle panel right) as well as a magnified view of VNC “bowtie” processes in the brain as indicated by the gray rectangle. Yellow arrows indicate TPN1-like processes in the VNC. Yellow asterisks indicate TPN1-like cell bodies. Gray arrows indicate “bowtie” neurons processes in the VNC. Gray asterisks indicate “bowtie” neurons cell bodies. Green, anti-GFP; magenta, anti-nc82 (neuropile marker). ( Q ) Diagram of the experimental assay. Sleep was measured in retinal-fed and vehicle-fed flies for 2 days without 627 nm LED stimulation to establish baseline sleep profile. LEDs were then turned on for 24 h at the start of day 3 to increase activity of the targeted cells by activating the CsChrimson channel and then turned off on day 4. White bars (L) represent the 12 h of light and black bars (D) represent the 12 h of dark that are oscillating daily. ( R ) Box plots of total sleep change in % ((total sleep on day 3-total sleep on day 2/total sleep on day 2) × 100) for control (Empty-AD; 23E10-DBD) and 23E12-AD; 23E10-DBD female flies expressing CsChrimson upon 627 nm LED stimulation. Two-way ANOVA followed by Sidak’s multiple comparisons revealed that total sleep is significantly increased in 23E12-AD; 23E10-DBD>UAS-CsChrimson female flies stimulated with 627 nm LEDs when compared with vehicle-fed flies. ****P < 0.0001, n.s. = not significant, n = 24–32 flies per genotype and condition. The raw data underlying parts F, G, M, N, and R can be found in S1 Data . The 23E10-GAL4 driver is often chosen to manipulate dFB neurons and is widely considered as a “dFB-specific” tool [ 17 , 19 , 21 – 24 , 26 – 31 ]. However, GAL4 lines commonly express in neurons outside the region of interest. To clarify the expression pattern of 23E10-GAL4, we expressed GFP under its control and identified more than 50 GFP-positive neurons in the brain, only half of which are dFB neurons (Fig 1H and 1I and S1 Table ). In addition, 23E10-GAL4 expresses in about 18 neurons in the VNC (see also S1 and S2 Movies). These results indicate that it is impossible to unequivocally assign a role in sleep promotion to 23E10-GAL4 dFB neurons (Fig 1J). Finally, since a recent study has implicated neurons located in the legs in sleep homeostasis [ 38 ], we also examined expression in the legs, gut, and ovaries in 23E10-GAL4>UAS-GFP flies. Our analysis revealed no GFP staining in these structures ( S1A–S1D Fig ). While it is possible that 23E10-GAL4 expresses in other non-CNS cells that we have not assessed in this study, we feel that this is extremely unlikely. It is even more unlikely that these non-CNS neurons would regulate sleep. Thus, we conclude that the sleep-promoting properties of the 23E10-GAL4 driver likely originate in the CNS. DANs, dopaminergic neurons; dFB, dorsal fan-shaped body; DN1, dorsal neurons 1; DPM, dorsal paired medial neurons; EB, ellipsoid body; FSB, fan-shaped body; l-LNv, large lateral neurons ventral; LPN, lateral posterior neurons; MB, mushroom body; MBONs, mushroom body output neurons; PI, pars intercerebralis; s-LNv, small lateral neurons ventral; vFB, ventral fan-shaped body; VNC, ventral nerve cord.

Sleep is a complex behavior that has been described in a variety of species ranging from jellyfish to humans [ 1 ]. Although the precise function of sleep remains unknown, current evidence indicates that sleep is required for maintaining optimal physiological and behavioral performance [ 2 ]. Over the last 20 years, multiple studies have demonstrated that the mechanisms and regulation of sleep are largely conserved from flies to mammals [ 3 ]. As in mammals, sleep-regulating centers are found in multiple areas of the Drosophila brain ( Fig 1A ) [ 3 , 4 ]. This compartmentalized organization of sleep-regulating regions in the brain probably underlies different functions and aspects of sleep behavior. Therefore, unequivocally assigning a role for a specific neuron or group of neurons in sleep regulation is a fundamental endeavor that will help uncover the function or functions of sleep.

Results

The 23E10-GAL4 driver promotes sleep and expresses in many non-dFB neurons in the central nervous system Previous studies have reported that increasing the activity of dFB neurons using the 23E10-GAL4 driver increases sleep [19–22]. To confirm these observations, we expressed the thermogenetic TrpA1 cation channel, which is activated by transferring flies to 31°C [34], in 23E10-GAL4 expressing neurons. Individual flies were loaded in Drosophila Activity Monitors (DAM2, Trikinetics) and assessed for 2 baseline days at 22°C, before raising the temperature to 31°C for 24 h on day 3 (Fig 1B). Sleep was defined as any period of inactivity lasting for at least 5 min, as previously described [35]. Consistent with previous reports, raising the temperature changes the sleep profile of both parental controls and results in a loss of sleep [36,37] (Fig 1C and 1D), whereas activating 23E10-GAL4 neurons strongly increases sleep (Fig 1E and 1F). Importantly, activation of 23E10-GAL4 neurons does not affect the amount of locomotor activity when the flies are awake (Fig 1G). Taken together, these data confirm that activating 23E10-GAL4 neurons promotes sleep and that the increase in sleep is not caused by a general motor defect.

The 23E10-GAL4 driver contains sleep-promoting neurons that are not dFB neurons To shed light on the role of individual 23E10-GAL4 expressing neurons in sleep regulation, we employed a Split-GAL4 strategy [33] to access a reduced number of 23E10-GAL4 expressing cells. We focused primarily on the dFB in designing this screen (Fig 1J), a choice dictated by the number of studies supporting a role for this structure in sleep regulation [14–17,21,23,24,28,39]. Central to the Split-GAL4 technology is the fact that the functional GAL4 transcription factor can be separated into 2 non-functional fragments, a GAL4 DNA-binding domain (DBD) and an activation domain (AD). Different enhancers are used to express these 2 fragments and the functional GAL4 transcription factor is reconstituted only in the cells in which both enhancers are active [33]. In this screen, we created new Split-GAL4 drivers by combining AD and DBD lines that are putatively expressing in the dFB, as assessed by the expression pattern of their corresponding GAL4 lines. The complete description and results of this ongoing Split-GAL4 dFB-based screen are beyond the scope of this manuscript and will be described elsewhere. However, when performing this screen, we identified a line (23E12-AD; 23E10-DBD) that strongly promotes sleep when thermogenetically activated with the TrpA1 channel (Fig 1L and 1M) when compared with an enhancer-less (empty) AD construct combined with 23E10-DBD (Fig 1K and 1M). Analysis of activity counts during awake time reveals that the increase in sleep observed when activating neurons contained in the 23E12-AD; 23E10-DBD line is not caused by a reduction in locomotor activity. On the contrary, these flies display an increase in waking activity upon neuronal activation (Fig 1N). These data demonstrate that activating 23E12-AD; 23E10-DBD neurons does not create a state of paralysis or motor defects as flies do not display any motor deficits when not sleeping. The increase in waking activity is likely explained by the need for a fly to perform tasks that are mutually exclusive to sleep in a reduced amount of waking time. Although increases in total sleep are indicative of increased sleep quantity, this measurement does not provide information about its quality or depth. To assess whether sleep quality is modulated by 23E12-AD; 23E10-DBD neurons, we analyzed sleep consolidation during the day and night in these flies and found that daytime and nighttime sleep bout duration is significantly increased in 23E12-AD; 23E10-DBD>UAS-TrpA1 flies upon neuronal activation (S2A and S2B Fig). Because increased sleep bout duration is believed to be an indication of increased sleep depth [40], these data suggest that activating 23E12-AD; 23E10-DBD neurons not only increases sleep quantity, but also probably increases sleep depth. In addition, since sleep in Drosophila is sexually dimorphic [41–43], we also systematically assessed male flies in our experiments. As seen in S2C–S2F Fig, we obtained almost identical behavioral data when analyzing male flies using our thermogenetic activation approach. Because the goal of our screen was to identify sleep-regulating dFB neurons in the 23E10-GAL4 pattern of expression, we expected that the 23E12-AD; 23E10-DBD Split-GAL4 line would express in dFB cells. Surprisingly, our anatomical analyses revealed that it does not express in any dFB neurons (Fig 1P and S3 and S4 Movies). Instead, this line expresses in 2 clusters of 4 to 5 neurons located in the anterior ventrolateral protocerebrum and in 4 VNC cells located in the metathoracic ganglion. A close examination of the anatomy of these 4 VNC neurons indicates that they have processes in the brain. Two of these neurons show a specific pattern of expression, with brain processes located in the superior medial protocerebrum and were named “bowtie” neurons (right panel in Fig 1P (see also S3 Movie)). An inspection of the expression pattern of 23E10-GAL4 (S1 Movie) confirms that these “bowtie” processes are present in this GAL4 driver, but they are difficult to observe because of their very close proximity to 23E10-GAL4 dFB projections. To further confirm that “bowtie” processes are unequivocally present in 23E10-GAL4, we have produced a confocal stack focused only on these structures in a 23E10-GAL4>UAS-mCD8GFP brain (S1E Fig). 23E12-AD; 23E10-DBD additionally labels 2 neurons in the VNC whose somas are located very close to the “bowtie” neurons. These neurons have characteristic processes in each leg ganglion (yellow arrows in Fig 1P-VNC image). Based on their anatomy, we hypothesize that these cells could be taste projection neurons 1 (TPN1) that receive inputs from sweet gustatory receptor leg neurons (GRNs) and convey this information to the brain in the subesophageal zone (SEZ) [44]. Since all the neurons contained in the 23E12-AD; 23E10-DBD Split-GAL4 are part of the 23E10-GAL4 expression pattern, these data indicate that this driver contains non-dFB sleep-promoting cells.

Optogenetic confirmation of sleep-promoting capacity of 23E12-AD; 23E10-DBD neurons Because identifying non-dFB 23E10-GAL4 sleep-promoting neurons was unexpected, we sought to confirm our thermogenetic findings using an optogenetic approach with CsChrimson [45] (Fig 1Q). In this setup, optogenetic activation of 23E12-AD; 23E10-DBD neurons strongly increases total sleep, as well as daytime and nighttime sleep bout duration (Figs 1R, S3B and S3D). These effects are not seen in control or vehicle-fed 23E12-AD; 23E10-DBD>UAS-CsChrimson flies (Figs 1R, S3A and S3C). Assessment of waking locomotor activity reveals that optogenetic activation does not affect waking activity in long sleeping retinal-fed females (S3E and S3F Fig), ruling out the possibility that locomotor defects are the underlying cause of the sleep phenotypes observed. Similar behavioral data were obtained with male flies (S4 Fig). Taken together, our anatomical and behavioral data clearly demonstrate that 23E10-GAL4 contains non-dFB sleep-promoting neurons.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002012

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