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. Author manuscript; available in PMC: 2023 Apr 15.
Published in final edited form as: Brain Res. 2022 Feb 4;1781:147816. doi: 10.1016/j.brainres.2022.147816

Regulation of Dark Period Sleep by the Amygdala: A microinjection and optogenetics study

Laurie L Wellman 1, Gyorgy Lonart 1, Austin M Adkins 1, Larry D Sanford 1
PMCID: PMC8901558  NIHMSID: NIHMS1779285  PMID: 35131286

Abstract

The central nucleus of the amygdala (CNA) projects to brainstem regions that generate and regulate rapid eye movement sleep (REM). We used optogenetics to assess the influence of CNA inputs into reticularis pontis oralis (RPO), pedunculopontine tegmentum (PPT) and nucleus subcoeruleus (SubC) on dark period sleep. We compared these results to effects of microinjections into CNA of the GABAA agonist, muscimol (inhibition of cell bodies) and tetrodotoxin (TTX, inhibition of cell bodies and fibers of passage). For optogenetics, male Wistar rats received excitatory (AAV5-EF1a-DIO -hChR2(H134R)-EYFP) or inhibitory (AAV-EF1a-DIO-eNpHR3.0-EYFP; DIO-eNpHR3.0) opsins into CNA and AAV5-EF1a-mCherry-IRES-WGA-Cre into RPO, PPT, or SubC. This enabled only CNA neurons synaptically connected to each region to express opsin. Optic cannulae for light delivery into CNA and electrodes for determining sleep were implanted. Sleep was recorded with and without blue or amber light stimulation of CNA. Separate rats received MUS or TTX into CNA prior to recording sleep. Optogenetic activation of CNA neurons projecting to RPO enhanced REM and did not alter non-REM (NREM) whereas activation of CNA neurons projecting to PPT or SubC did not significantly affect sleep. Inhibition of CNA neurons projecting to any region did not significantly alter sleep. TTX inactivation of CNA decreased REM and increased NREM whereas muscimol inactivation did not significantly alter sleep. Thus, the amygdala can regulate decreases and increases in REM, and RPO is important for CNA promotion of REM. Fibers passing through CNA, likely from the basolateral nucleus of the amygdala, also play a role in regulating sleep.

Keywords: amygdala, central nucleus; reticularis pontis oralis; rapid eye movement sleep; optogenetics; tetrodotoxin

Graphical Abstract

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1. Introduction

A large body of work using a variety of techniques has shown that the central nucleus of the amygdala (CNA) regulates rapid eye movement sleep (REM) and REM-related neural activity. This includes both spontaneous (Sanford et al., 2002; Tang et al., 2005) and fear-conditioned changes in REM in rats (Liu et al., 2009; Liu et al., 2011), neural activity underlying ponto-geniculo-occipital (PGO) waves in cats (Calvo et al., 1996; Simon-Arceo et al., 2003) and rabbits (Cain et al., 2002; Silvestri and Kapp, 1998), and P-Waves (PGO wave equivalent) in rats (Deboer et al., 1998; Deboer et al., 1999). Studies have also demonstrated that the basolateral nucleus of the amygdala (BLA) is also involved in regulating REM (Liu et al., 2011; Wellman et al., 2016; Wellman et al., 2017a), potentially through its projections to CNA and/or the bed nucleus of the stria terminalis (BNST) which have similar direct projections to brainstem REM regulatory regions (Reviewed in (Davis and Whalen, 2001a)). The amygdala also appears to influence non-REM (NREM). Both NREM and total sleep time have been reported to be increased by electrolytic and chemical lesions of BLA (Zhu et al., 1998), and decreased by kindling stimuli in CNA (Yi et al., 2004). In addition, microinjections of prolactin into CNA produced a decrease in NREM (Sanford et al., 1998).

Although many studies have not distinguished functional roles for nuclei of the amygdala, the available evidence suggests that activation of CNA could promote REM and related activity. The bulk of pharmacological and behavioral work conducted in rats, thus far, has produced alterations in arousal that suggest that inhibiting CNA produces relatively specific reductions in REM. However, recent studies in mice using chemogenetic manipulations of GABAergic neurons in CNA failed to find that it has a role in regulating REM, but did implicate these neurons in cataplexy (Mahoney et al., 2017; Snow et al., 2017). The reason for these discrepancies is not entirely clear; though, these studies manipulated GABAergic activity in CNA during the first 3 h of darkness (Mahoney et al., 2017; Snow et al., 2017), the normal active period of mice, whereas most studies of CNA influence on REM in rodents have taken place in the light period when they exhibit the most sleep. One study that we previously conducted found that microinjections of tetrodotoxin (TTX) into CNA at the start of the dark period significantly reduced REM amounts and increased non-REM (NREM) amounts (Sanford et al., 2006). TTX inactivates both cell bodies and fibers of passage indicating that the effect may have involved altered regulation of BLA targets.

Evolving work linking REM sleep to adaptive responses to stress (Sanford et al., 2010; Suchecki et al., 2012), adaptive emotional processing (Gujar et al., 2011; van der Helm et al., 2011; Walker and van der Helm, 2009), and to the genesis of posttraumatic stress disorder (PTSD) (Mellman and Hipolito, 2006; Mellman et al., 2007) indicates that understanding how the amygdala regulates REM sleep will be important for insight into the neurobiological and neurocircuit mechanisms by which stress can produce lasting effects on behavior and sleep. While our work, and that of others, has demonstrated that the amygdala has a strong regulatory influence on REM sleep, the actual circuitry and mechanisms by which the influence is exerted is mostly unknown, though it likely involves projections to brainstem REM generator and regulatory regions. The circuitry regulating physiological signs of fear and anxiety has significant overlap with, and impact on, brain regions that regulate arousal and sleep (e.g., REM sleep promoting/generating regions (laterodorsal and pedunculopontine tegmental nuclei (LDT/PPT), the nucleus reticularis pontis oralis (RPO) and subcoeruleus (SubC)) (Morrison et al., 2000; Xi et al., 2011; Zhang et al., 2012) as well REM sleep inhibitory regions (e.g., locus coeruleus (LC); dorsal raphe nucleus (DRN)) (Steriade and McCarley, 1990b).

In this study, we used microinjection and optogenetic methods to assess the potential role of CNA in modulating dark period REM sleep. We compared microinjections of muscimol which inactivates cell bodies to microinjections of TTX which inactivated both cell bodies and fibers of passage (data reanalyzed from (Sanford et al., 2006)) on amounts and distribution of dark period sleep. We then used optogenetics to specifically target inputs from CNA into PPT, RPO and SubC and conducted studies determining the effects of activation and inhibition on dark period sleep. The data demonstrate that CNA can influence dark period REM and suggest that understanding how the amygdala regulates sleep requires considering the interactions of multiple amygdaloid nuclei and their descending projections.

2. Results

2.1. CNA influences on dark period sleep depend on fibers of passage.

We found that inactivation of CNA with muscimol (1.0 μM; n= 6) had no impact on dark period REM (vehicle: 32.0 ± 5.3; muscimol: 29.4 ± 3.1, ns). By comparison, inactivation of CNA with TTX (n = 8, 0.1 μM) prior to the dark period significantly reduced REM (vehicle: 33.8 ± 2.3; TTX: 20.1 ± 2.6, p < 0.02). Muscimol also did not alter the distribution of REM (see Fig. 1A) whereas TTX did (Fig. 1B) (Sanford et al., 2006). Muscimol did not significantly alter NREM amounts (vehicle: 174.1 ± 13.7; muscimol: 185.4 ± 17.8, ns) nor its distribution (D = 0.0972, p = 0.869). By comparison, TTX significantly increased NREM amounts (vehicle: 189.4 ± 13.9; TTX: 222.6 ± 16.6, p < .01), but its effect on the underlying distribution did not reach significance as indicated by the K-S statistic (D = 0.1771, p = 0.087).

Fig. 1.

Fig. 1.

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REM amounts plotted cumulatively across the 12 h dark period after inactivation of CNA with muscimol (MUS, A) or tetrodotoxin (TTX, B). Inactivating CNA with MUS (n=6) prior to the dark period had no effect on REM whereas microinjections of TTX into CNA (n=8) prior to dark period significantly reduced REM amounts (adapted from (Sanford et al., 2006)) and altered the underlying distribution of REM compared to control. The K-S (Kolmogorov-Smirnoff) D. statistic were used for comparing distributions of dark period REM. CNA: central nucleus of the amygdala.

2.2. Optogenetic activation of CNA inputs into RPO, but not PPT or SubC, enhanced dark period REM.

Dark period sleep was recorded under non-stimulated and under blue light stimulation (excitation) of opsin expressing CNA neurons. Stimulation with blue light into CNA of animals with paired constructs in CNA and RPO resulted in significantly higher REM amounts compared to baseline sleep, but had no significant effects on REM in animals with paired constructs in PPT or SubC (Fig. 2A). The increase in REM produced by stimulation of CNA inputs into RPO was accompanied by a significant alteration in the distribution of REM compared to baseline, whereas there were no effects on the distribution of REM in the PPT and SubC groups (Fig. 3A–C). Analyses of the data for the RPO group also found significantly increased total REM in the first two 4 h blocks of the 12 h dark period (Fig 4A), but without significant increases in either REM episode number or duration (Fig. 4C, E).

Fig. 2.

Fig. 2.

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Effects of blue light stimulation (excitation) of CNA on select regions of REM generator and its effects on total dark period REM (A) and NREM (B) sleep. Stimulation of projections into RPO enhanced REM. **, p < .01. No other significant effects were observed. CNA: central nucleus of the amygdala; PPT: pedunculopontine tegmentum (n = 6); RPO: reticularis pontis oralis (n = 6); SubC: nucleus subcoeruleus (n = 5).

Fig. 3.

Fig. 3.

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REM amounts plotted cumulatively across the 12 h dark period under different optogenetic stimulation regiments. A. Effects of intermittent blue light stimulation of CNA projections into RPO (n=5) on REM amounts. B. Effects of intermittent blue light stimulation of CNA projections into PPT (n=6) on REM amounts. C. Effects of intermittent blue light stimulation of CNA projections into SubC (n=5) on REM amounts. Only stimulation of projections into RPO enhanced REM. D. Effects of amber light stimulation (NpHR; inhibition) of CNA projections into brainstem REM generator/regulatory regions. Inhibition was provided only on alternate hours. Plots are collapsed (total n=10) across subsets of rats that were prepared for optogenetic inhibition of CNA inputs into either one or two brainstem regions (CNA/RPO (n=2), CNA/PPT (n=2), CNA/SubC (n=1) CNA/PPT/RPO (n=3), CNA/SubC/RPO (n=3)). The K-S (Kolmogorov-Smirnoff) D statistic were used for comparing distributions of dark period REM. CNA: central nucleus of the amygdala; PPT: pedunculopontine tegmentum; RPO: reticularis pontis oralis; SubC: nucleus subcoeruleus.

Fig. 4.

Fig. 4.

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Effects of blue light stimulation (excitation) of central nucleus of the amygdala on projections to the reticularis pontis oralis on select REM and NREM parameters plotted in 4 h blocks (BLK) across the dark period (total REM (A) and NREM (B) sleep, number of REM (C) and NREM episodes (D), and REM (E) and NREM (F) duration. Stimulation of projections into RPO enhanced REM during BLKs 1 and 2, *, p < .05 (n=6). No other significant effects were observed.

Neither overall amount (Fig. 2B), episode number, episode duration (Fig. 4B, D, F), nor distribution of NREM (not shown) were altered in any group with activation of CNA.

2.3. Optogenetic inhibition of CNA inputs into brainstem regions did not alter dark period sleep.

Subsets of rats were prepared for optogenetic inhibition of CNA inputs into either one or two brainstem regions (CNA/RPO (n=2), CNA/PPT (n=2), CNA/SubC (n=1), CNA/PPT/RPO (n=3), CNA/SubC/RPO (n=3)). Because we saw no noticeable effects of optogenetic inhibition on sleep in any of these subsets, and because muscimol inactivation of CNA produced no significant effects on either REM or NREM, we collapsed the animals into one group for analysis.

Optogenetic inhibition of CNA produced no significant alteration in NREM (n=11; CTL: 166.1 ± 14.5; NpHR: 175.6 ± 13.1, ns) or REM (n=11; CTL: 50.1 ± 6.9; NpHR: 51.9 ± 6.8, ns) amounts. There also was no alteration in the distribution of dark period REM (Fig 3D) or NREM (not shown) amounts.

3. Discussion

This study explored CNA regulation of dark period sleep using both microinjection and optogenetic methods. Core microinjection findings revealed TTX inactivation decreased REM and increased NREM whereas muscimol inactivation produced no significant alterations in dark period sleep. Optogenetic stimulation of CNA in animals with paired constructs in CNA and RPO enhanced REM and did not alter NREM whereas simulation of CNA with paired constructs in PPT or SubC did not significantly affect either stage of sleep. Concordant with our findings with muscimol, optogenetic inhibition of CNA cells with projections to PPT, RPO, or SubC produced no significant alterations in dark period sleep. Together, these data demonstrate that the amygdala can regulate both decreases and increases in REM sleep, and indicate that RPO is an important projection area for mediating the ability of CNA to promote REM.

3.1. Microinjection Experiments

Our finding that microinjections of muscimol into CNA failed to alter dark period REM is consistent with recent chemogenetic studies that also failed to find an effect on spontaneous dark period REM (Mahoney et al., 2017; Snow et al., 2017). However, microinjections of low concentrations of muscimol into CNA during the light period, when sleep drive is higher, produced relatively selective decreases in total REM and number of REM episodes that lasted up to 6 h whereas microinjections of bicuculline into CNA produced significant increases in REM (Sanford et al., 2002). Light period microinjections did not produce significant alterations in NREM or wakefulness. This suggests that CNA activation may play a role in promoting REM during the normal inactive period when brain activity is more conducive to sleep. Our current optogenetic with projections into RPO also suggest that externally applied CNA activation can enhance REM when brain activity is more conducive to promoting wakefulness.

Several studies have demonstrated that CNA plays a role in regulating stress-induced alterations in REM. Whereas inescapable footshock stress often produces significant decreases in REM, avoidable (Datta, 2000; Kant et al., 1995; Smith et al., 1980; Smith and Lapp, 1986) and escapable (Sanford et al., 2010; Yang et al., 2010) footshock can be followed by significant increases in REM. Blocking inactivation of CNA during inescapable footshock with the GABAA antagonist, bicuculline, prevents footshock induced reductions in REM and can decrease stress-induced c-Fos activation in LC (Liu et al., 2009) in a manner consistent with the changes in REM (i.e., increased activation of LC was associated with decreased REM). By comparison, footshock preceded by both vehicle and muscimol microinjections selectively reduced REM and increased Fos expression in LC (Liu et al., 2009). These findings are consistent with our suggestion that CNA activation can enhance REM when the brain is in an aroused state.

The TTX microinjection data presented in this paper were partially reanalyzed from a prior contribution (Sanford et al., 2006). We only included the lower of two dosages (2.5 ng/0.1 μl and not 5.0 ng/0.2 μl) from the original study as they produced virtually identical alterations in sleep and the lower volume would have been less likely to impact regions outside CNA. The re-analysis demonstrated that TTX produced a significant alteration in distribution as well as the amount of dark period REM. Interestingly, the same dosage administered in the light period did not significantly reduce REM or NREM amounts, but did reduce NREM latency (Tang et al., 2005). The higher dose also reduced NREM latency, but only increased NREM amount in the first h of recording (Tang et al., 2005). Together, these findings also support the suggestion that the relative influence of the amygdala on sleep may depend on the general level of arousal or sleep drive.

The failure of muscimol to produce a significant alteration in spontaneous dark period REM, and the finding that TTX reduced REM and enhanced NREM, suggest that CNA and BLA work in concert to regulate sleep. CNA (Liu et al., 2009) and BLA (Wellman et al., 2014) also appear to play complementary roles in mediating the effects of stress and fearful memories on REM. Based on current information, CNA appears to directly regulate REM generator regions whereas BLA is critical for forming fear memories that can differentially increase or decrease REM (Wellman et al., 2013; Wellman et al., 2016). The projections of the pathway inactivated by TTX remains to be determined, though they are likely mediated through at least partially through BNST (Davis and Whalen, 2001b).

3.2. Optogenetic Experiments

The amygdala, via CNA and potentially BNST, has direct projections to regions (e.g., LDT, PPT, RPO, SubC and LC and DRN) in the brainstem (Bernard et al., 1993; Krettek and Price, 1978; Petrov et al., 1994; Peyron et al., 1998; Price et al., 1987) that play significant roles in regulating sleep and arousal, and the stress response. It is through influences on these neurons that the amygdala is thought to regulate REM and its related phenomena. In this study, we concentrated on three of these regions, PPT, RPO and SubC, which have roles in inducing and/or generating REM.

REM sleep is thought to be initiated by cholinergic projections from the PPT that activate REM-on neurons in the RPO that are components of the REM generator (Steriade and McCarley, 1990a) and glutamatergic neurons in SubC that are thought to regulate REM sleep and its defining features such as muscle paralysis and cortical activation (Reviewed in (Fraigne et al., 2015)), as well as PGO/P-wave generation (Datta et al., 1998). The SubC is also considered a REM induction site, e.g., local microinjection of bicuculline into the dorsal SubC during the light period significantly increased the amount and reduced the latency to REM (Pollock and Mistlberger, 2003) whereas its inactivation with TTX reduces REM (Sanford et al., 2005). However, we found no significant effect of optogenetic stimulation of CNA inputs into either region on REM sleep amounts. This does not preclude a role for CNA regulation of other REM phenomena. Several studies have demonstrated that CNA regulates PGO/P-wave activity (Cain et al., 2002; Calvo et al., 1996; Deboer et al., 1998; Deboer et al., 1999; Silvestri and Kapp, 1998; Simon-Arceo et al., 2003) which may be mediated via projections to SubC. Additionally, PPT is activated in both wakefulness and REM and it plays a role in modulating arousal, posture and locomotion, as well as REM (Steriade and McCarley, 1990a), thereby suggesting that CNA inputs may be involved in modulating other functions.

The CNA has direct excitatory (glutamatergic) projections to presumptive REM generator neurons in RPO (Fung et al., 2011; Zhang et al., 2012). These projections are capable of exerting powerful postsynaptic excitation that drives activation of RPO neurons and have been suggested to be capable of producing REM sleep (Xi et al., 2011). We did not see evidence that optogenetically activating CNA produced REM, but we did find a significant overall increase in REM and a significant alteration in the distribution of REM. This suggests that activation of CNA inputs into RPO is not sufficient to produce REM, but can enhance it. The increases in REM were observed only in the first 8 h of the dark period, and not in the last 4 h of recording. This also suggests that the effects of CNA activation were potentially reliant on other sleep- or REM-related neural activity that may have differed across time in the dark period. There is also the possibility that the efficacy of stimulation may have declined over time, potentially from heat damage to neural tissue which can happen with extended optogenetic stimulation (Mahmoudi et al., 2017). In either case, the results demonstrate that CNA activity can influence dark period REM sleep amounts via inputs into RPO.

Electrical stimulation of either the CNA or PPT evoked short-latency excitatory postsynaptic potentials (EPSPs) in the same neurons within RPO, and the amplitude of PPT-evoked EPSPs from RPO neurons was increased when stimulation of PPT was preceded by stimulation of CNA (Xi et al., 2012). This suggests that CNA and PPT may work together to produce activation within RPO that can produce REM. However, we found no evidence that CNA projections into PPT alone can promote REM. This may be due to differences in electrical and optogenetic stimulation or influences on different types of cells. Electrical stimulation would not distinguish between effects of activating CNA or fibers of passage originating in BLA whereas optogenetic stimulation in our studies would have only impacted CNA neurons projecting into CNA. Additionally, PPT contains REM-on (~13%), Wake-on (~27%) and Wake-REM-on (~60%) cells, which are the most common type (Datta and Siwek, 2002). The high discharge rate of these cells during waking is thought to contribute to the activated (desynchronized) cortical EEG typical of wakefulness via projections to the thalamus (el Mansari et al., 1989; Steriade and McCarley, 1990b). REM-on neurons also project to the thalamus, where they may work with the Wake-REM-on neuronal projections to promote the activated EEG characteristic of REM sleep (el Mansari et al., 1989; Steriade and McCarley, 1990b). Activity in the majority of this class of cells in PPT increases prior to wakefulness and remains very active until 5 to 8 sec before the end of wakefulness indicating that the increased neuronal activity of these cells may also be involved in the induction of wakefulness (Datta and Siwek, 2002). Thus, potential CNA effects on these cells might not necessarily promote REM. Interestingly, activation of CNA projections into LC inhibits neuronal firing which could assist in promoting REM (Wellman et al., 2017b) given the putative inhibitory role of LC noradrenergic neurons on REM (Steriade and McCarley, 1990b).

3.3. Conclusion

Most of the effort aimed at determining how the amygdala influences REM has focused on CNA inputs into brainstem REM generator and regulatory regions. This work has provided insight into some aspects of the role of the amygdala in REM regulation by suggesting that CNA activation can directly promote REM through projections to RPO. However, the significant reduction in REM (and increase in NREM) produced by inactivation of fibers of passage in CNA illustrates the existence of a significant sleep regulatory pathway that has not been explored. Those fibers project, at least partially, to BNST (Davis and Whalen, 2001b) which has downstream projections similar to those of CNA (Krettek and Price, 1978; Price et al., 1987). This suggests that the ability of the amygdala to enhance or reduce REM may be regulated functionally through BLA control of CNA and BNST and their downstream projections. Functional studies similar to those conducted in this study for CNA could begin to determine if this is the case.

In summary, microinjection, optogenetic, and behavioral studies demonstrate the amygdala is capable of producing both decreases and increases in REM, and its regulation by fear memory. Decreases in REM were produced only when fibers of passage from BLA were blocked in CNA, and increases in REM were produced only when CNA projections to RPO were activated. These findings suggest that BLA and CNA work in concert to regulate REM sleep through influences on brainstem REM generator and regulatory regions. They also suggest a significant role for BLA fibers passing through CNA, potentially to BNST, in mediating REM. Delineating these pathways will be crucial for understanding the neural circuitry that controls the interactions between emotion and sleep.

4. Experimental Procedure

4.1. Subjects

The subjects were ninety-day-old Wistar rats obtained from Harlan Laboratories (Frederick, MD). Upon arrival, the rats were individually housed in polycarbonate cages and given ad lib access to food and water. The rooms were kept on a 12:12 light:dark cycle with lights on from 07:00 to 19:00 h. Light intensity during the light period was 100–110 lux and less than 1 lux during the dark period. Ambient room temperature was maintained at 24.5 ± 0.5 °C.

4.2. Surgery

Beginning one week following arrival, the rats were anesthetized with isoflurane (5% induction; 2% maintenance) and implanted with skull screw electrodes for recording the electroencephalogram (EEG) and stainless steel wire electrodes sutured to the dorsal neck musculature for recording the electromyogram (EMG). Leads from the recording electrodes were routed to a 9-pin miniature plug that mated to one attached to a recording cable. The recording plug and cannulas were affixed to the skull with dental acrylic and stainless steel anchor screws. Rats serving in the microinjection studies received guide cannula (26 ga.) implanted with their tips aimed 1.0 mm above CNA [P: 2.2 (Bregma), L: 4.0, DV: 7.5 bilateral] (Kruger et al., 1995). Rats serving in the optogenetic studies received bilateral microinjections of the appropriate excitatory or inhibitory construct and were also implanted with bilateral optic cannulas for light delivery into CNA. Ibuprofen (15 mg/kg) was made available in their water supply for relief of post-operative pain. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Experimental Animals and were approved by Eastern Virginia Medical School’s Animal Care and Use Committee.

4.3. Microinjections:

4.3.1. Drugs

Muscimol (muscimol hydrobromide, 5-aminomethyl-3-hydroxyisoxazole) was obtained from Sigma–Aldrich (St. Louis, MO, USA). It was prepared in pyrogen-free distilled water (dW; 1.0 μM) and was sonicated for 20 min to ensure that the drug was dissolved completely. TTX (0.1 μM /0.1 μl) was prepared in saline. Fresh solutions were prepared for each substance for each experimental day.

4.3.2. Procedure:

For microinjections, injection cannulas (33 ga.) were secured in place within the guide cannulas, and projecting 1.0 mm beyond the tip of the guide cannulas for delivery of drug into the target region. The injection cannulas were connected to one end of a section of polyethylene tubing that had the other end connected to 5.0 μl Hamilton syringes. The injection cannulas and tubing were prefilled with the solution to be injected. Once the cannulas were in place, 0.2 (muscimol) or 0.1 (TTX) μL,or vehicle, was bilaterally infused over 3 min. The cannulas were left in place one min pre- and post-injection to allow for maximal absorption of the solution.

4.4. Optogenetics

4.4.1. Opsin Constructs

We used optogenetics to assess the influence of CNA inputs into RPO, PPT and SubC on dark period sleep. Freshly thawed excitatory (AAV5-EF1a-DIO -hChR2(H134R)-EYFP; DIO-hChR2) or inhibitory (AAV-EF1a-DIO-eNpHR3.0-EYFP; DIO-eNpHR3.0) constructs were microinected into the CNA and AAV5-EF1a-mCherry-IRES-WGA-Cre (mCherry/WGA-Cre) was microinjected into PPT [P: 7.8, L: 1.9, V: 6.9], RPO [P: 8.8, L: 1.2, V: 8.4] or SubC [P: 9.3, L: 1.2, V: 7.3]. These constructs are designed such that only those CNA neurons that were synaptically connected to the specific brainstem region would express the light sensitive opsin. Vectors in CNA code for double floxed and inverted open reading frame opsins, DIO-hChR2 or DIO-eNpHR3.0. When a neuron in RPO, PPT or SubC synapses with neurons in CNA and expressed WGA-Cre, it is transneurally transferred to CNA neurons, where Cre activity flips the opsin gene(s) into its correct orientation thereby allowing its expression (Fenno et al., 2014; Fenno et al., 2017). Subsequent expression of the opsin(s) gene enables excitation of ChR2 expressing neurons/projections by blue light or inhibition of the NpHR expressing neurons/projections by amber light.

4.4.2. Stimulation:

For optogenetic studies, each rat was attached to a recording cable and optic fibers for stimulation 2 h prior to lights off. Both were routed through commutators that allowed the rats free movement within their home cage. In the optogenetic conditions, the program controlling stimulation was automatically started at lights off, and stimulation continued throughout the dark period. ChR animals were stimulated with pulsatile (10 Hz) 470nm wavelength (blue) excitatory LED light directly into CNA for 1 hour with a 1 hour delay between sequences for the entirety of the dark period. NpHR were stimulated with continuous amber light on alternate hours throughout the dark period using the same schedule. Light output was measured by an optical power meter and adjusted to produce ~10 mW at the fiber tip for both blue and amber light. In the control conditions, the rats were identically connected with cable and optic fibers and left undisturbed throughout the dark period without stimulation. Stimulation and control conditions were conducted in a counterbalanced order.

4.5. Sleep Recording:

The rats were allowed a post-surgery recovery period of 14 days prior to beginning the experiment. All rats were habituated to the recording cable and chamber over 3 consecutive days. Then, the rats were habituated to the 5 min handling procedure necessary for microinjections over 2 consecutive days and a baseline following handling (BH) was recorded.

Home cages were changed at least 3 days prior to each treatment day. The same room was used for animal housing and sleep recording. The microinjections and behavioral testing were conducted in a separate room from that used for recording.

All experimental manipulations were conducted during the 12 h of the light period such that sleep recording would begin at the start of the dark period. This resulted in 12 h of uninterrupted dark period recording on each experimental day.

For recording sleep, each animal, in its home cage, was placed on a rack outfitted for electrophysiological recording and a lightweight, shielded cable was connected to the miniature plug on the rat’s head. The cable was attached to a commutator that permitted free movement of the rat within its cage. EEG and EMG signals were processed by a Grass Model 12 polygraph equipped with model 12A5 amplifiers and routed to an A/D board (Model USB-2533, Measurement Computing) housed in a personal computer. The signals were digitized at 256 Hz and collected in 10 s epochs using the SleepWave™ (Biosoft Studio) data collection program.

4.6. Data Analyses

4.6.1. Sleep:

Computerized EEG and EMG records were visually scored by trained observers blind to drug condition in 10 s epochs to determine wakefulness, NREM and REM. Wakefulness was scored based on the presence of low-voltage, fast EEG and high amplitude, tonic EMG levels. NREM was characterized by the presence of spindles interspersed with slow waves, lower muscle tone and no gross body movements. REM was scored continuously during the presence of low voltage, fast EEG, theta rhythm, and muscle atonia.

4.6.2. Statistics:

Twelve h totals of NREM (min) and total REM (min) were examined using paired t-tests to compare experimental and control conditions. Hourly amounts of NREM and REM were examined using Kolmogorov-Smirnov two sample tests to determine whether either microinjections or optogenetic manipulations had altered the underlying distribution of either sleep state across the dark period. Subsequent to the primary analyses, select parameters (totals, number of episodes, and duration of episodes) of REM and NREM for animals with optogenetic stimulation of CNA-RPO projections were analyzed in three 4 h blocks to determine the time course of sleep changes across the dark period. Comparison between experimental and control conditions were conducted using repeated measures ANOVA followed by Holm-Sidak tests for comparisons between means where warranted.

4.6.3. Histology:

To localize the microinjection sites in, brain slices (40 μm) were made through the amygdala and the sections were mounted on slides and stained with cresyl violet. The sections were then examined in conjunction with a stereotaxic atlas (Kruger et al., 1995) to confirm cannula placements. Though there were rostral-caudal variations in the placements among animals, the histology indicated that MUS, TTX, or vehicle would have been infused primarily into CNA and all animals were used in the data analyses.

We also determined the distribution of Cre-mCherry+ cells in brainstem regions and of Cre-dependent expression of eYFP+ cells in CNA and double expression in brainstem regions. An example for RPO is shown in Fig. 5.

Fig. 5.

Fig. 5.

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Histological confirmation of placement. A. Brightfield image of RPO. B. Distribution of Cre-mCherry+ cells in RPO (image observed under mCherry filter, Nikon, C-FL Texas Red HC HISN). C. Brightfield image of CNA. D. Distribution of Cre-dependent expression of eYFP+ cells in CNA (image observed under eYFP filter, Nikon, Yellow GFP BP HYP). e-g. Image series in RPO showing Cre-mCherry+ (e), opsin-eYFP+ from CNA (f), and merged image (g). CNA: central nucleus of the amygdala; PPT: pedunculopontine tegmentum; RPO: reticularis pontis oralis; SubC: nucleus subcoeruleus; xscp: decussation superior cerebellar peduncle; ec: external capsule.

Highlights.

  • The central nucleus of the amygdala (CNA) regulates rapid eye movement sleep (REM).

  • Influence of CNA on REM requires projections to nucleus reticularis pontis oralis.

  • Influences of amygdala on non-REM requires amygdala basolateral nucleus.

Footnotes

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