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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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The TetTag-hM3Dq system to record and reactivate neuronal groups in the preoptic hypothalamus activated by a sedative dose of dexmedetomidine or during recovery sleep. (a) The AAV transgenes: the first contains the cfos promoter, which drives expression of tTA protein. In the presence of doxycycline (DOX), tTA cannot bind and activate its target promoter, PTRE-tight, located in the second AAV genome; when doxycycline is removed, tTA can activate hM3Dq-mCHERRY expression but only in neurons where tTA expression had been driven by the cfos promotor, reflecting neural activity. (b). The extended protocol and time-line for the experiments. (c) LPO-TetTag-hM3Dq mice. Time course of PTRE-tight-hM3Dq-mCHERRY transgene induction and decay. The photographs show coronal sections from one side of the brain stained for hM3Dq-mCHERRY expression (red), detected with mCHERRY antisera. The images were taken from animals killed at six time points: with doxycycline removed from the diet two days previously, just before dexmedetomidine-induced sedation; 2 hours after a sedative dose of dexmedetomidine; 4 days later on and back on doxycycline, 4 weeks after dexmedetomidine-induced sedation on a doxycycline diet; 4 hours after sleep deprivation; and 2 hours into recovery sleep following sleep deprivation. Induced hM3Dq-mCHERRY transgene expression was seen throughout the LPO area. Scale bar, 200 μm.
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Figure 3: The TetTag-hM3Dq system to record and reactivate neuronal groups in the preoptic hypothalamus activated by a sedative dose of dexmedetomidine or during recovery sleep. (a) The AAV transgenes: the first contains the cfos promoter, which drives expression of tTA protein. In the presence of doxycycline (DOX), tTA cannot bind and activate its target promoter, PTRE-tight, located in the second AAV genome; when doxycycline is removed, tTA can activate hM3Dq-mCHERRY expression but only in neurons where tTA expression had been driven by the cfos promotor, reflecting neural activity. (b). The extended protocol and time-line for the experiments. (c) LPO-TetTag-hM3Dq mice. Time course of PTRE-tight-hM3Dq-mCHERRY transgene induction and decay. The photographs show coronal sections from one side of the brain stained for hM3Dq-mCHERRY expression (red), detected with mCHERRY antisera. The images were taken from animals killed at six time points: with doxycycline removed from the diet two days previously, just before dexmedetomidine-induced sedation; 2 hours after a sedative dose of dexmedetomidine; 4 days later on and back on doxycycline, 4 weeks after dexmedetomidine-induced sedation on a doxycycline diet; 4 hours after sleep deprivation; and 2 hours into recovery sleep following sleep deprivation. Induced hM3Dq-mCHERRY transgene expression was seen throughout the LPO area. Scale bar, 200 μm.

Mentions: TetTagging has been developed with transgenic mice24,25. We set up the system using AAV genomes so that we could target the PO hypothalamus. Because of size constraints of the AAV genome, we generated two AAV viruses, one which contained the Pcfos-tTA transgene, and the other which contained the tet-operator promoter (PTRE-tight) linked to an hM3Dq-mCHERRY receptor reading frame (Fig. 3a). With this method, before the behavioral experiments are undertaken, the TetTag system is repressed with doxycycline in the diet. Doxycycline prevents tTA activating its target promoter, PTRE-tight, located in the second AAV genome; when doxycycline is removed, neural activity, e.g. taking place in recovery sleep or dexmedetomidine-induced sedation, can drive the cfos promoter-linked tTA expression, which in turn can activate hM3Dq-mCHERRY expression (Fig. 3a).


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)

The TetTag-hM3Dq system to record and reactivate neuronal groups in the preoptic hypothalamus activated by a sedative dose of dexmedetomidine or during recovery sleep. (a) The AAV transgenes: the first contains the cfos promoter, which drives expression of tTA protein. In the presence of doxycycline (DOX), tTA cannot bind and activate its target promoter, PTRE-tight, located in the second AAV genome; when doxycycline is removed, tTA can activate hM3Dq-mCHERRY expression but only in neurons where tTA expression had been driven by the cfos promotor, reflecting neural activity. (b). The extended protocol and time-line for the experiments. (c) LPO-TetTag-hM3Dq mice. Time course of PTRE-tight-hM3Dq-mCHERRY transgene induction and decay. The photographs show coronal sections from one side of the brain stained for hM3Dq-mCHERRY expression (red), detected with mCHERRY antisera. The images were taken from animals killed at six time points: with doxycycline removed from the diet two days previously, just before dexmedetomidine-induced sedation; 2 hours after a sedative dose of dexmedetomidine; 4 days later on and back on doxycycline, 4 weeks after dexmedetomidine-induced sedation on a doxycycline diet; 4 hours after sleep deprivation; and 2 hours into recovery sleep following sleep deprivation. Induced hM3Dq-mCHERRY transgene expression was seen throughout the LPO area. Scale bar, 200 μm.
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Related In: Results  -  Collection

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Figure 3: The TetTag-hM3Dq system to record and reactivate neuronal groups in the preoptic hypothalamus activated by a sedative dose of dexmedetomidine or during recovery sleep. (a) The AAV transgenes: the first contains the cfos promoter, which drives expression of tTA protein. In the presence of doxycycline (DOX), tTA cannot bind and activate its target promoter, PTRE-tight, located in the second AAV genome; when doxycycline is removed, tTA can activate hM3Dq-mCHERRY expression but only in neurons where tTA expression had been driven by the cfos promotor, reflecting neural activity. (b). The extended protocol and time-line for the experiments. (c) LPO-TetTag-hM3Dq mice. Time course of PTRE-tight-hM3Dq-mCHERRY transgene induction and decay. The photographs show coronal sections from one side of the brain stained for hM3Dq-mCHERRY expression (red), detected with mCHERRY antisera. The images were taken from animals killed at six time points: with doxycycline removed from the diet two days previously, just before dexmedetomidine-induced sedation; 2 hours after a sedative dose of dexmedetomidine; 4 days later on and back on doxycycline, 4 weeks after dexmedetomidine-induced sedation on a doxycycline diet; 4 hours after sleep deprivation; and 2 hours into recovery sleep following sleep deprivation. Induced hM3Dq-mCHERRY transgene expression was seen throughout the LPO area. Scale bar, 200 μm.
Mentions: TetTagging has been developed with transgenic mice24,25. We set up the system using AAV genomes so that we could target the PO hypothalamus. Because of size constraints of the AAV genome, we generated two AAV viruses, one which contained the Pcfos-tTA transgene, and the other which contained the tet-operator promoter (PTRE-tight) linked to an hM3Dq-mCHERRY receptor reading frame (Fig. 3a). With this method, before the behavioral experiments are undertaken, the TetTag system is repressed with doxycycline in the diet. Doxycycline prevents tTA activating its target promoter, PTRE-tight, located in the second AAV genome; when doxycycline is removed, neural activity, e.g. taking place in recovery sleep or dexmedetomidine-induced sedation, can drive the cfos promoter-linked tTA expression, which in turn can activate hM3Dq-mCHERRY expression (Fig. 3a).

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
Related in: MedlinePlus