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Neuronal ensembles sufficient for recovery sleep and the sedative actions of α2 adrenergic agonists.

Zhang Z, Ferretti V, Güntan İ, Moro A, Steinberg EA, Ye Z, Zecharia AY, Yu X, Vyssotski AL, Brickley SG, Yustos R, Pillidge ZE, Harding EC, Wisden W, Franks NP - Nat. Neurosci. (2015)

Bottom Line: For the α2 adrenergic receptor agonist dexmedetomidine, we found that sedation and LORR were in fact distinct states, requiring different brain areas: the preoptic hypothalamic area and locus coeruleus (LC), respectively.Instead, we found that dexmedetomidine-induced sedation resembled the deep recovery sleep that follows sleep deprivation.Thus, α2 adrenergic receptor-induced sedation and recovery sleep share hypothalamic circuitry sufficient for producing these behavioral states.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, South Kensington, UK.

ABSTRACT
Do sedatives engage natural sleep pathways? It is usually assumed that anesthetic-induced sedation and loss of righting reflex (LORR) arise by influencing the same circuitry to lesser or greater extents. For the α2 adrenergic receptor agonist dexmedetomidine, we found that sedation and LORR were in fact distinct states, requiring different brain areas: the preoptic hypothalamic area and locus coeruleus (LC), respectively. Selective knockdown of α2A adrenergic receptors from the LC abolished dexmedetomidine-induced LORR, but not sedation. Instead, we found that dexmedetomidine-induced sedation resembled the deep recovery sleep that follows sleep deprivation. We used TetTag pharmacogenetics in mice to functionally mark neurons activated in the preoptic hypothalamus during dexmedetomidine-induced sedation or recovery sleep. The neuronal ensembles could then be selectively reactivated. In both cases, non-rapid eye movement sleep, with the accompanying drop in body temperature, was recapitulated. Thus, α2 adrenergic receptor-induced sedation and recovery sleep share hypothalamic circuitry sufficient for producing these behavioral states.

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Knock down of adrenergic α2A receptors in the locus coeruleus blocks dexmedetomidine-induced LORR, but not sedation. (a) Testing shRNAs for knockdown efficacy of adra2a-IRES-gfp transgene expression in HEK-293 cells (n=8). The photographs show transfected HEK-293 cells; GFP fluorescence (green) was strongly reduced with the shadra2a1 construct but not with the scramble version. The dsRED expression reveals similar transfection efficiencies. CMV, cytomegalovirus promoter/enhancer region; IRES, internal ribosome entry site; pA, polyadenylation sequence; WPRE, woodchuck post-transcriptional regulatory element. (b) AAVs expressing either dsRED-mir30-shadra2a or dsRED-mir30-shscramble transgenes were bilaterally injected into the LC of adult mice. Photographs illustrate AAV transgene expression (dsRED) in the LC as confirmed by co-staining with tyrosine hydroxylase antisera (white). ITR, inverted terminal repeats; CBA, chicken-β-actin enhancer/promoter. (c) Whole-cell recordings of action potentials of LC neurons, in acute slices from LC-scramble, and LC-adra2a-KD mice. Applying dexmedetomidine to scramble-expressing neurons hyperpolarized the membrane potential and the neurons stopped firing; by contrast dexmedetomidine had no effect on the neurons from the LC-adra2a-KD mice (P=0.7; n=7 cells). (d) Fourier transform power spectra for LC-scramble (black), and LC-adra2a-KD (red) mice in the waking state (left; n=5) and in response to 50 μg kg−1 dexmedetomidine (middle; n=4) and 400 μg kg−1 dexmedetomidine (right; n=4). Lighter shaded envelopes indicate the s.e.m. (e) Movement of LC-scramble (black; n=4-6), and LC-adra2a-KD (red; n=4–7) mice in response to sedative doses of dexmedetomidine were not significantly different (two-way ANOVA, P=0.91).
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Figure 1: Knock down of adrenergic α2A receptors in the locus coeruleus blocks dexmedetomidine-induced LORR, but not sedation. (a) Testing shRNAs for knockdown efficacy of adra2a-IRES-gfp transgene expression in HEK-293 cells (n=8). The photographs show transfected HEK-293 cells; GFP fluorescence (green) was strongly reduced with the shadra2a1 construct but not with the scramble version. The dsRED expression reveals similar transfection efficiencies. CMV, cytomegalovirus promoter/enhancer region; IRES, internal ribosome entry site; pA, polyadenylation sequence; WPRE, woodchuck post-transcriptional regulatory element. (b) AAVs expressing either dsRED-mir30-shadra2a or dsRED-mir30-shscramble transgenes were bilaterally injected into the LC of adult mice. Photographs illustrate AAV transgene expression (dsRED) in the LC as confirmed by co-staining with tyrosine hydroxylase antisera (white). ITR, inverted terminal repeats; CBA, chicken-β-actin enhancer/promoter. (c) Whole-cell recordings of action potentials of LC neurons, in acute slices from LC-scramble, and LC-adra2a-KD mice. Applying dexmedetomidine to scramble-expressing neurons hyperpolarized the membrane potential and the neurons stopped firing; by contrast dexmedetomidine had no effect on the neurons from the LC-adra2a-KD mice (P=0.7; n=7 cells). (d) Fourier transform power spectra for LC-scramble (black), and LC-adra2a-KD (red) mice in the waking state (left; n=5) and in response to 50 μg kg−1 dexmedetomidine (middle; n=4) and 400 μg kg−1 dexmedetomidine (right; n=4). Lighter shaded envelopes indicate the s.e.m. (e) Movement of LC-scramble (black; n=4-6), and LC-adra2a-KD (red; n=4–7) mice in response to sedative doses of dexmedetomidine were not significantly different (two-way ANOVA, P=0.91).

Mentions: We examined dexmedetomidine’s action at α2A receptors selectively in the LC, using acute knockdown of adra2a transcripts, and determined how this affected sedation and LORR. We first selected two adra2a shRNA sequences to use in vivo. For this, four putative adra2a shRNA sequences (shadra2a) were placed into a microRNA gene, mir3028 (Fig. 1a). Two of the adra2a hairpin sequences (shadra2a1 & shadra2a2) substantially reduced GFP expression from a reporter gene, adra2a-IRES-gfp co-expressed in HEK293 cells; results using one of these, dsRED-mir30-shadra2a1, are shown (Fig. 1a). The dsRED-mir30-shadra2a1, dsRED-mir30-shadra2a2 anddsRED-mir30-shscramble cassettes were then placed into adeno-associated virus (AAV) genomes, packaged, and injected bilaterally into the LC (Fig. 1b). In the following section, similar results were obtained with both shadra2a sequences; all in vivo results are illustrated for shadra2a1. The treated animals are termed: LC-adra2a-KD and LC-scramble respectively. We obtained, on average, a knockdown to 46.3 ± 9.2 % (mean ± SE) of control adra2a transcript levels (t-test, P<0.004, compared with mRNA levels in the LC area of LC-scramble mice). We prepared acute slices from brainstem of LC-adra2a-KD and LC-scramble mice and examined the electrophysiological responses of LC noradrenergic neurons to the α2 agonist dexmedetomidine (Fig. 1c left). Dexmedetomidine (1 μM), when applied to LC-scramble neurons inhibited action potential firing, hyperpolarizing the membrane potential by 9.8 ± 2 mV (mean ± s.e.m.; n=6 cells), as shown previously for other noradrenergic α2 agonists9,10,29. However, in LC-adra2a-KD neurons, dexmedetomidine failed to block action potential firing (Fig. 1c right) and the membrane potential did not change significantly (P=0.7; n=7 cells). Thus the knockdown of adra2a gene expression by approximately 50% removed the ability of dexmedetomidine to silence LC neurons. This is in agreement with studies on heterozygote adra2a global knockout mice, which found that the adra2a allele shows strong haplo-insufficiency, whereby even at a high dose of dexmedetomidine (433 μg/kg), dexmedetomidine-induced LORR in adra2a knockout mice was abolished30.


Neuronal ensembles sufficient for recovery sleep and the sedative actions of α2 adrenergic agonists.

Zhang Z, Ferretti V, Güntan İ, Moro A, Steinberg EA, Ye Z, Zecharia AY, Yu X, Vyssotski AL, Brickley SG, Yustos R, Pillidge ZE, Harding EC, Wisden W, Franks NP - Nat. Neurosci. (2015)

Knock down of adrenergic α2A receptors in the locus coeruleus blocks dexmedetomidine-induced LORR, but not sedation. (a) Testing shRNAs for knockdown efficacy of adra2a-IRES-gfp transgene expression in HEK-293 cells (n=8). The photographs show transfected HEK-293 cells; GFP fluorescence (green) was strongly reduced with the shadra2a1 construct but not with the scramble version. The dsRED expression reveals similar transfection efficiencies. CMV, cytomegalovirus promoter/enhancer region; IRES, internal ribosome entry site; pA, polyadenylation sequence; WPRE, woodchuck post-transcriptional regulatory element. (b) AAVs expressing either dsRED-mir30-shadra2a or dsRED-mir30-shscramble transgenes were bilaterally injected into the LC of adult mice. Photographs illustrate AAV transgene expression (dsRED) in the LC as confirmed by co-staining with tyrosine hydroxylase antisera (white). ITR, inverted terminal repeats; CBA, chicken-β-actin enhancer/promoter. (c) Whole-cell recordings of action potentials of LC neurons, in acute slices from LC-scramble, and LC-adra2a-KD mice. Applying dexmedetomidine to scramble-expressing neurons hyperpolarized the membrane potential and the neurons stopped firing; by contrast dexmedetomidine had no effect on the neurons from the LC-adra2a-KD mice (P=0.7; n=7 cells). (d) Fourier transform power spectra for LC-scramble (black), and LC-adra2a-KD (red) mice in the waking state (left; n=5) and in response to 50 μg kg−1 dexmedetomidine (middle; n=4) and 400 μg kg−1 dexmedetomidine (right; n=4). Lighter shaded envelopes indicate the s.e.m. (e) Movement of LC-scramble (black; n=4-6), and LC-adra2a-KD (red; n=4–7) mice in response to sedative doses of dexmedetomidine were not significantly different (two-way ANOVA, P=0.91).
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Figure 1: Knock down of adrenergic α2A receptors in the locus coeruleus blocks dexmedetomidine-induced LORR, but not sedation. (a) Testing shRNAs for knockdown efficacy of adra2a-IRES-gfp transgene expression in HEK-293 cells (n=8). The photographs show transfected HEK-293 cells; GFP fluorescence (green) was strongly reduced with the shadra2a1 construct but not with the scramble version. The dsRED expression reveals similar transfection efficiencies. CMV, cytomegalovirus promoter/enhancer region; IRES, internal ribosome entry site; pA, polyadenylation sequence; WPRE, woodchuck post-transcriptional regulatory element. (b) AAVs expressing either dsRED-mir30-shadra2a or dsRED-mir30-shscramble transgenes were bilaterally injected into the LC of adult mice. Photographs illustrate AAV transgene expression (dsRED) in the LC as confirmed by co-staining with tyrosine hydroxylase antisera (white). ITR, inverted terminal repeats; CBA, chicken-β-actin enhancer/promoter. (c) Whole-cell recordings of action potentials of LC neurons, in acute slices from LC-scramble, and LC-adra2a-KD mice. Applying dexmedetomidine to scramble-expressing neurons hyperpolarized the membrane potential and the neurons stopped firing; by contrast dexmedetomidine had no effect on the neurons from the LC-adra2a-KD mice (P=0.7; n=7 cells). (d) Fourier transform power spectra for LC-scramble (black), and LC-adra2a-KD (red) mice in the waking state (left; n=5) and in response to 50 μg kg−1 dexmedetomidine (middle; n=4) and 400 μg kg−1 dexmedetomidine (right; n=4). Lighter shaded envelopes indicate the s.e.m. (e) Movement of LC-scramble (black; n=4-6), and LC-adra2a-KD (red; n=4–7) mice in response to sedative doses of dexmedetomidine were not significantly different (two-way ANOVA, P=0.91).
Mentions: We examined dexmedetomidine’s action at α2A receptors selectively in the LC, using acute knockdown of adra2a transcripts, and determined how this affected sedation and LORR. We first selected two adra2a shRNA sequences to use in vivo. For this, four putative adra2a shRNA sequences (shadra2a) were placed into a microRNA gene, mir3028 (Fig. 1a). Two of the adra2a hairpin sequences (shadra2a1 & shadra2a2) substantially reduced GFP expression from a reporter gene, adra2a-IRES-gfp co-expressed in HEK293 cells; results using one of these, dsRED-mir30-shadra2a1, are shown (Fig. 1a). The dsRED-mir30-shadra2a1, dsRED-mir30-shadra2a2 anddsRED-mir30-shscramble cassettes were then placed into adeno-associated virus (AAV) genomes, packaged, and injected bilaterally into the LC (Fig. 1b). In the following section, similar results were obtained with both shadra2a sequences; all in vivo results are illustrated for shadra2a1. The treated animals are termed: LC-adra2a-KD and LC-scramble respectively. We obtained, on average, a knockdown to 46.3 ± 9.2 % (mean ± SE) of control adra2a transcript levels (t-test, P<0.004, compared with mRNA levels in the LC area of LC-scramble mice). We prepared acute slices from brainstem of LC-adra2a-KD and LC-scramble mice and examined the electrophysiological responses of LC noradrenergic neurons to the α2 agonist dexmedetomidine (Fig. 1c left). Dexmedetomidine (1 μM), when applied to LC-scramble neurons inhibited action potential firing, hyperpolarizing the membrane potential by 9.8 ± 2 mV (mean ± s.e.m.; n=6 cells), as shown previously for other noradrenergic α2 agonists9,10,29. However, in LC-adra2a-KD neurons, dexmedetomidine failed to block action potential firing (Fig. 1c right) and the membrane potential did not change significantly (P=0.7; n=7 cells). Thus the knockdown of adra2a gene expression by approximately 50% removed the ability of dexmedetomidine to silence LC neurons. This is in agreement with studies on heterozygote adra2a global knockout mice, which found that the adra2a allele shows strong haplo-insufficiency, whereby even at a high dose of dexmedetomidine (433 μg/kg), dexmedetomidine-induced LORR in adra2a knockout mice was abolished30.

Bottom Line: For the α2 adrenergic receptor agonist dexmedetomidine, we found that sedation and LORR were in fact distinct states, requiring different brain areas: the preoptic hypothalamic area and locus coeruleus (LC), respectively.Instead, we found that dexmedetomidine-induced sedation resembled the deep recovery sleep that follows sleep deprivation.Thus, α2 adrenergic receptor-induced sedation and recovery sleep share hypothalamic circuitry sufficient for producing these behavioral states.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Imperial College London, South Kensington, UK.

ABSTRACT
Do sedatives engage natural sleep pathways? It is usually assumed that anesthetic-induced sedation and loss of righting reflex (LORR) arise by influencing the same circuitry to lesser or greater extents. For the α2 adrenergic receptor agonist dexmedetomidine, we found that sedation and LORR were in fact distinct states, requiring different brain areas: the preoptic hypothalamic area and locus coeruleus (LC), respectively. Selective knockdown of α2A adrenergic receptors from the LC abolished dexmedetomidine-induced LORR, but not sedation. Instead, we found that dexmedetomidine-induced sedation resembled the deep recovery sleep that follows sleep deprivation. We used TetTag pharmacogenetics in mice to functionally mark neurons activated in the preoptic hypothalamus during dexmedetomidine-induced sedation or recovery sleep. The neuronal ensembles could then be selectively reactivated. In both cases, non-rapid eye movement sleep, with the accompanying drop in body temperature, was recapitulated. Thus, α2 adrenergic receptor-induced sedation and recovery sleep share hypothalamic circuitry sufficient for producing these behavioral states.

Show MeSH