Article Figures & Data
Figures
- Figure 1.
SWA decay time constant correlates with sleep need in wild-type mice. The time constant of SWA decay (τ) during an average SWS episode following differing amounts of SD is determined by a single phase exponential, regression fit in wild-type mice. The conditions for C57BL/6 mice include baseline, 4 and 6 h acute SD (A–C, respectively). D, E, The pooled group of genetic control strains (BL6_Tam, fAdK_Tam, fAdK;GFAP:CreER_Veh) experienced chronic SD (4 h SD with 2 h recovery for 8 consecutive cycles). Plots represent time of the SWS episode (plotted for each 10 s epoch; x-axis) versus normalized SWA (y-axis). F, Histogram of SWA decay during an average SWS episode determined for each SD condition shows a graded slowing in proportion to previous enforced W duration. “BL+/−St. Dev.” is a pooled control strain used for chronic SD. The τ determined under baseline conditions during either the phase of minimal sleep need (6 h, early part of the active phase, ZT12–18_BL) compared with the phase of high sleep need (6 h, early part of the inactive period, ZT0–6_BL) shows a similar increase in duration with sleep need.
- Figure 2.
SWS-SWA for an averaged SWS episode. A, SWA decay is shown during an averaged SWS episode from a wild-type strain (C57BL/6_Tam, n = 5). B, C, On transition from W to SWS, SWA increases at a similar rate (τ = ∼0.57 min) as gamma power decreases (τ = 0.58 min). B, Inset, Full averaged SWS episode shown in A with the SWA rising phase marked in red.
- Figure 3.
Neuronal AdoRA1s are necessary for SWA decay across light phase, during a SWS episode and for SWS consolidation. A, Integrated SWA/10 s epoch during SWS declines over the course of the 12 h light phase (ZT0–12 on x-axis) for a typical control wild-type mouse (fAdoRA1). B, An example of SWS-SWA/epoch across the 12 h light phase from an AdoRA1 conditional knock-out mutant mouse (fAdoRA1;CamkII:Cre) shows a lack of SWS-SWA decay. C, Pooled data for percentage decrease in SWS-SWA from the first to last hour of the light phase. Filled bar represents fAdoRA1. Open bar represents fAdoRA1;CaMKII. D, Loss of AdoRA1 attenuates both expression of SWA during SWS and abolishes the decay of SWA during an average SWS episode. The SWS-SWA amplitude is expressed as fold increase over the 24 h average waking SWA. E, Loss of AdoRA1 fragments SWS episodes, as demonstrated by cumulative distribution of SWS episode duration for fAdoRA1;CaMKII:Cre (open circles) and fAdoRA1 (filled circles) animals.
- Figure 4.
Characterization of AdK knockdown. A, Top, Representative examples of Western blots using AdK (top row) and GFAP (bottom row) antibodies from cortical and hippocampal tissue in one Veh- and one Tam-treated fAdK;GFAP:CreER mouse. Negative images and resized plots are shown. Light upper band represents a nonspecific cross reactive band that is typical for antibodies that detect protein in the 50 kDa range. Dark lower band represents both the short and long AdK isoforms that do not easily resolve (43.5 and 45 kDa, respectively). Bottom, Coomassie stain of total protein from the membrane used for the Western blots shown in top blot. This shows equivalent relative densities of Coomassie stain between genotypes and between regions and demonstrates the loading of equal amounts of protein. Thus, the similarity in GFAP expression between genotypes within the same region and the difference in GFAP expression over different regions is not attributable to differential protein loading (top and bottom blots). B, Tamoxifen decreases AdK protein as measured by quantitative immunoblotting using a monoclonal antibody against AdK (open symbols indicate Tam; closed symbols indicate Veh). AdK level is presented relative to glia-specific GFAP level because AdK is primarily expressed in glia in adults. C, Tamoxifen treatment decreases AdK mRNA as measured by quantitative PCR of fAdK;GFAP:CreER mice (open bars indicate Tam; closed bars indicate Veh). AdK values are presented relative to cyclophilin. D, Knockdown of AdK increases extracellular Ado concentration. Biosensors sensitive to the presence of Ado were used to measure Ado concentration from acute hippocampal slices taken from Veh-treated (filled bar) and Tam-treated (open bar) fAdK;GFAP:CreER animals. Tam-treated mice showed significantly higher Ado levels. E, Paired pulse facilitation, evoked by stimulation of the Schaffer collaterals and recorded as fEPSPs in the stratum radiatum of CA1, was measured in acute hippocampal slices from Veh-treated (filled bar) or Tam-treated (open bar) fAdK;GFAP:CreER animals. The ratios of the evoked response (measured by the slope of the fEPSP) of the second stimulus (S2) to the first stimulus (S1) were compared under baseline conditions (E, left axis; F, G, black traces). PPF indicating presynaptic inhibition is increased in slices obtained from Tam-treated mice. CPT, an AdoRA1 antagonist, blocks PPF (1 μm; F, G, gray traces). The Tam-treated group showed a greater sensitivity of the PPF to CPT, indicating a greater AdoRA1-mediated, tonic, presynaptic inhibition (E, right axis).
- Figure 5.
Glial adenosine kinase knockdown increases SWA. A, Average, normalized SWA during SWS and waking (left y-axis). Under baseline conditions, fAdK;GFAP:CreER_Tam mice (open bars) show a significant increase in SWA during SWS and waking compared with Veh-treated mice (filled bars). Tam-treated mice spent the same amount of time in SWS compared with fAdK;GFAP:CreER_Veh mice (right y-axis). B, The circadian distribution of SWS-SWA is illustrated by the percentage SWA in 2 h bins of the average 48 h baseline SWS-SWA for each animal and is similar between groups. C–E, Power spectrum distribution in Tam- and Veh-treated fAdK;GFAP:CreER mice (open symbols indicate Tam; closed symbols indicate Veh) during waking (C), SWS (D), and REM (E). F, SWA across states is shown over 48 h of chronic, partial SD with 4 h/2 h cycles of SD and recovery. SWA is greater during both SD (TMon) and during the recovery period (TMoff) in the Tam-treated compared with the control mice. G, Tam-treated mice (open bars) show a greater percentage increase of SWA from enforced wake to SD recovery sleep (left y-axis, TMon to TMoff) compared with Veh-treated mice (filled bars). The same two genotypes were also compared for the percentage increase from baseline SWS time to SD recovery SWS time (right y-axis, base to TMoff). Unlike the greater increase in SWA in Tam-treated mice, both groups show similar increases in SWS time following SD.
- Figure 6.
Reduction of glial, but not neuronal, AdK slows SWA decay during an SWS episode and induces increased SWS consolidation. A, B, Tam-treated mice have a significantly slowed decay rate (τ = 22.5 min) compared with Veh-treated mice (τ = 8.6 min). C, Cumulative distribution of SWS episode durations is shifted to the right (more consolidated) in Tam-treated fAdK;GFAP:CreERVeh-treated mice. D, E, The time constant of SWA decay between control fAdK_Tam and neuronal AdK knock-out animals, fAdK;CaMKII:Cre (τ = 11.1 vs 10.1 min) was similar. F, Mice lacking neuronal AdK (fAdK;CaMKII:Cre) have a similar SWS episode duration distribution as fAdK_Tam mice.
- Figure 7.
The decay time constant for SWA during the SWS episode is influenced by the genes coding for AdoRA and glial AdK. A, Histogram of the time constants for decay (τ) of SWA during an average SWS episode under baseline conditions for different genotypes. Loss of neuronal AdoRA1s (fAdoRA1;CaMKII:Cre) results in loss of decay. Reduction of glial AdK expression (fAdK;GFAP:CreER_Tam) increases τ by >10× the SD (SD = 1.1 min). B, Diagram depicting systems-level relationships between waking, which is facilitated by arousal and increases sleep need, and sleep function, which decreases sleep need and presumably enhances arousal. Increased waking results in increased Ado tone that increases sleep need indices. C, At the local circuit level, the expression of sleep need is mediated by Ado acting on neuronal AdoRA1 to facilitate rebound SWA in response to prolonged enforced waking. During SWS, Ado flows down its concentration gradient into glia by glial equilibrative transporters. There it is metabolized by the low-capacity but high-affinity glial enzyme, AdK, thus reducing the activation of neuronal AdoRA1 and thereby controlling the rate of SWA decay.
