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.2017 Mar 31:11:17.
doi: 10.3389/fnsys.2017.00017. eCollection 2017.

Homeostatic Changes in GABA and Glutamate Receptors on Excitatory Cortical Neurons during Sleep Deprivation and Recovery

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Homeostatic Changes in GABA and Glutamate Receptors on Excitatory Cortical Neurons during Sleep Deprivation and Recovery

Esther Del Cid-Pellitero et al. Front Syst Neurosci..

Abstract

Neuronal activity is regulated in a homeostatic manner through changes in inhibitory GABA and excitatory glutamate (Glu) AMPA (A) receptors (GluARs). Using immunofluorescent staining, we examined whether calcium/calmodulin-dependent protein kinase IIα (CaMKIIα)-labeled (+) excitatory neurons in the barrel cortex undergo such homeostatic regulation following enforced waking with associated cortical activation during the day when mice normally sleep the majority of the time. Sleep deprived mice were prevented from falling asleep by unilateral whisker stimulation and sleep recovery (SR) mice allowed to sleep freely following deprivation. In parallel with changes in c-Fos reflecting changes in activity, (β2-3 subunits of) GABAA Rs were increased on the membrane of CaMKIIα+ neurons with enforced waking and returned to baseline levels with SR in barrel cortex on sides both contra- and ipsilateral to the whisker stimulation. The GABAAR increase was correlated with increased gamma electroencephalographic (EEG) activity across conditions. On the other hand, (GluA1 subunits of) AMPA Rs were progressively removed from the membrane of CaMKIIα+ neurons by (Rab5+) early endosomes during enforced waking and returned to the membrane by (Rab11+) recycling endosomes during SR. The internalization of the GluA1Rs paralleled the expression of Arc, which mediates homeostatic regulation of AMPA receptors through an endocytic pathway. The reciprocal changes in GluA1Rs relative to GABAARs suggest homeostatic down-scaling during enforced waking and sensory stimulation and restorative up-scaling during recovery sleep. Such homeostatic changes with sleep-wake states and their associated cortical activities could stabilize excitability and activity in excitatory cortical neurons.

Keywords: AMPA; Arc; CaMKIIα; GABAA; GluA1; gamma activity.

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Figures

Figure 1
Figure 1
Behavior and sleep-wake stage classification. Behavior was classified automatically from video frames and sleep-wake stages visually from telemetric electroencephalographic (EEG).(A) Examples of wake behaviors and stages. In channel 1 (VTM #11), the video frame is fixed at the end (marked by blue arrowhead, above the EEG) of an episode, which was behaviorally classified as “walk right” (blue highlighted row, top left and blue bar, below) and which occurred during a 2 s epoch (and within a 10 s epoch) when the EEG activity was characterized by prominent high frequency beta and gamma (20.5–58 Hz) activity riding upon high theta activity (6.5–10 Hz) and was therefore classified as active or attentive Wake (aW). In channel 2 (VTM #7), the video is fixed at the end (marked by blue arrowhead, above) of an episode behaviorally classified as “remain low” (blue highlighted row, top right and blue bar, below) which occurred over multiple 2 s epochs (and within a 10 s epoch) when the EEG activity was characterized by less prominent high frequency activity riding upon low theta activity (4.5–6 Hz) and was therefore classified as quiet Wake (qW).(B) Examples of sleep behaviors and stages. In channel 2 (VTM #7), the video frame is fixed at the end (marked by blue arrowhead, above) of an episode which was classified behaviorally as “sleep” (blue highlighted row, top right and blue line, below) and which occurred during a 10 s epoch characterized by continuous prominent irregular slow wave activity in the delta range (0.5–4 Hz) and thus classified as slow wave sleep (SWS). In channel 1 (VTM #11), the video is fixed at the end (marked by blue arrowhead, above) of an episode which was also classified behaviorally as “sleep” (blue highlighted row, top left and blue line, below), which occurred during a 10 s epoch when the EEG was characterized by prominent rhythmic high theta activity (6.5–10 Hz) and thus classified as paradoxical sleep (PS). Screen display from HomeCageScan (Clever Systems) with which two mice were recorded simultaneously in adjacent cages. Records taken from baseline recordings. Calibration bars for EEG, vertical = 0.5 mV, horizontal = 0.5 s.
Figure 2
Figure 2
Behavior and EEG changes elicited by whisker stimulation during sleep deprivation (SD). As evident in the video frame (the time of which is marked by the blue arrowhead above the EEG), the whisker stimulation was performed using a soft brush inserted into the cage. In this case, the mouse had been in “remain low” or “walk slowly” then again “remain low” posture/behavior (blue highlighted row, top and blue bar., below) appearing to prepare to sleep. The whisker stimulation was then initiated and maintained for 1 s (bar) during which the animal moved (“walk left”). The EEG changed from irregular to rhythmic theta with gamma on both right (R) and left (L) parietal Cx during the stimulation of the left whiskers. After the stimulation, the mouse appeared to remain awake, while the EEG was characteristic first of qW followed by some aW such that this10 s period would be classified as qW. Calibration bars for EEG, vertical = 0.5 mV, horizontal = 0.5 s.
Figure 3
Figure 3
Behavioral and EEG changes during SD and sleep recovery (SR).(A) Experimental procedure using unilateral whisker stimulation to maintain mice awake during the day for 2 or 4 h of SD and preceding 2 h of SR. Mice were recorded by video alone (VM,n = 16) for behavior or video plus telemetry (VTM,n = 12) for behavior and EEG (over contra- and ipsilateral barrel cortices) for 2 h prior to termination (at ~16:00). They were divided into four groups: sleep control (SC), SD2, SD4 and SR (n = 4 VM and 3 VTM per group).(B) The number of whisker stimulations that were necessary to prevent mice from going to sleep increased significantly during 4 h SD (in SD4 and SR groups,n = 14).(C) The percentage of sleep-wake stages based upon video behavioral and telemetric EEG records in the four experimental groups (of VTM,n = 12) varied significantly across stages between groups.(D) The percentage of waking and sleep behaviors based upon video behavioral scoring together with EEG scoring in the four experimental groups varied significantly and differentially across behaviors between groups (of VTM,n = 12).(E–G) Average EEG gamma (30.5–58 Hz) and delta (0.5–4 Hz) integrated amplitude (mV per s) over the right barrel cortex varied significantly across and between sleep-wake stages(E–F) and behaviors(G) in baseline (of VTM,n = 12).(H) The relative total energy of gamma amplitude (calculated as percent of baseline value for each mouse) during the 2 h prior to termination in the four groups (of VTM,n = 12) varied significantly between groups on both sides of the barrel cortex. Large *indicates a significant (p < 0.05) main effect or interaction using one, two or three-way analyses of variances (ANOVAs; see text). Small *indicates a significant difference (p < 0.05) between the value on the left (marked by an arrowhead) and every other value to the right shown by a small vertical line for pairs connected by a horizontal bar, except where † indicates a trend (p < 0.10) of that difference between the pairs, usingpost hoc paired comparisons. Error bars indicate SEM.
Figure 4
Figure 4
Changes in expression of immediate early gene (IEG) proteins and receptors following SD and SR. The percentage of cells that were considered double-labeled in dual-immunostained sections in the barrel cortex ipsilateral and/or contralateral to the whisker stimulation in the four groups of SC, SD2, SD4 and SR mice(Figures 5–10).(A) Percent calcium/calmodulin-dependent protein kinase IIα (CaMKIIα)+ cells which were c-Fos+ varied significantly and differentially on the two sides of the cortex between groups.(B) Percent CaMKIIα+ cells which were Arc+ varied significantly on both sides of the cortex between groups.(C) Percentage of CaMKIIα+ cells which were GABAA (β2-3 subunit) R+ varied significantly on both sides of the cortex between groups.(D) Percentage of CaMKIIα+ cells which were glutamate (GluA1) (subunit AMPA) R+ varied significantly between groups.(E) Percentage of GluA1 receptor (GluA1R)+ cells which were Rab5+ or Rab11+ on the contralateral side of the cortex varied significantly between groups. Large*indicates significant main effect of group using two-way ANOVAs with side(A–D) or one-way ANOVA for the contralateral side(E), and small *indicates significant difference (p < 0.05) between the value on the left (marked by an arrowhead) and those indicated by a line and connected by bars to the right, except where † indicates a trend (p < 0.10), usingpost hoc paired comparisons (see text).N = 12 VTM in(A–D) andn = 12 VM and VTM in(E). Error bars indicate SEM.
Figure 5
Figure 5
c-Fos expression in CaMKIIα+ pyramidal cells following SD and SR. Fluorescent microscopic images of sections dual-immunostained for c-Fos (red), as a marker of neuronal activity, and CaMKIIα (green), as a marker of pyramidal cells, in layer V of barrel cortex on the side contralateral to whisker stimulation.(A) In SC mouse, c-Fos immunostaining is not apparent in the CaMKIIα+ cells.(B) In SD2 mouse, c-Fos is apparent in the nucleus of CaMKIIα+ cells (arrowheads).(C) In SD4 mouse, c-Fos is still apparent in the nucleus of CaMKIIα+ cells (arrowheads).(D) In SR mouse, c-Fos is no longer apparent in the nucleus of CaMKIIα+ cells. Scale bar, 20 μm.
Figure 6
Figure 6
Arc expression in CaMKIIα+ pyramidal cells following SD and SR. Fluorescent microscopic images of dual-immunostained sections for Arc (red) and CaMKIIα (green) in layer V of the barrel cortex contralateral to whisker stimulation.(A) In SC mouse, Arc immunostaining is apparent in low intensity within the soma of some CaMKIIα+ neurons (arrowhead).(B) In SD2 mouse, Arc immunostaining is apparent in moderate to high intensity in the cytoplasm and nucleus of many CaMKIIα+ neurons (arrowheads).(C) In SD4 mouse, Arc is apparent in high intensity in many CaMKIIα+ neurons within the cytoplasm of soma and proximal dendrites and in the nucleus (arrowheads).(D) In SR mouse, Arc immunostaining is no longer apparent in CaMKIIα+-labeled neurons. Scale bar, 20 μm.
Figure 7
Figure 7
GABAARs on CaMKIIα+ pyramidal cells following SD and SR. Confocal images of sections dual-immunostained for (the β2-3 subunits of the) GABAAR (red) and CaMKIIα (green) in the layer V of the barrel cortex contralateral to the whisker stimulation.(A) In SC mouse, GABAAR immunostaining is apparent along portions of the plasma membrane in some CaMKIIα+ pyramidal cells (arrowhead).(B) In SD2 mouse, GABAAR immunostaining is apparent in high intensity along the entire plasma membrane of multiple CaMKIIα+ neurons (arrowheads).(C) In SD4 mouse, GABAAR immunostaining is still evident in high intensity along the entire membrane of CaMKIIα+ neurons (arrowheads).(D) In SR mice, GABAAR immunostaining is of relatively low intensity and apparent along portions of few CaMKIIα+ cells (arrowhead). Cells indicated with large arrowhead enlarged to right. Scale bars, 10 μm. Thickness, 1440 nm.
Figure 8
Figure 8
GluA1Rs on CaMKIIα+ pyramidal cells following SD and SR. Confocal images of dual-immunostaining for GluA1 (subunit of the AMPA) R (red) and CaMKIIα (green) in layer V of the barrel cortex contralateral to whisker stimulation.(A) In SC mouse, GluA1R immunostaining is apparent in relatively low intensity with a granular profile through the cytoplasm of few CaMKIIα+ cells (arrowhead).(B) In SD2 mouse, GluA1R immunostaining is apparent in medium intensity with prominent large granules in multiple CaMKIIα+ cells (arrowheads).(C) In SD4 mouse, GluA1R immunostaining appears less intense and concentrated within large granules within the cytoplasm near the plasma membrane in the CaMKIIα+ cells (arrowheads).(D) In SR mouse, GluA1R immunostaining is apparent in moderate to high intensity in small and large granules through the cytoplasm of soma and proximal dendrites of multiple CaMKIIα+ cells (arrowheads). Cells indicated with large arrowheads enlarged to right. Scale bars, 10 μm. Thickness, 3600 nm.
Figure 9
Figure 9
GluA1Rs in early endosomes following SD and SR. Confocal images of dual-immunostaining for GluA1 (subunit of the AMPA) R (red) and the marker of early endosomes, Rab5 (green), in layer V of the barrel cortex contralateral to the whisker stimulation.(A) In SC mouse, Rab5 immunostaining is just barely visible in a couple of endosomes which are lightly immunostained for GluA1R within a GluA1R+ cell (filled arrowhead), that was not judged to be Rab5+/GluA1R+ (unfilled arrowheads).(B) In SD2 mouse, intense Rab5 immunostaining is evident in multiple endosomes which are double-labeled for GluA1R (filled arrowheads) and are close to the plasma membrane in a GluA1R+ cell (filled arrowhead).(C) In SD4 mouse, Rab5 immunostaining is evident in multiple endosomes, which are double-labeled for GluA1R (filled arrowheads) in GluA1R+ cells (filled arrowheads).(D) In SR mouse, Rab5 immunostaining is not clearly visible in endosomes (unfilled arrowheads) within GluA1R+ cell (filled arrowhead). Cells indicated with large arrowhead enlarged to right. Scale bars, 10 μm. Thickness, 1440 nm.
Figure 10
Figure 10
GluA1Rs in recycling endosomes following SD and SR. Confocal images of dual-immunostained sections for GluA1 (subunit of the AMPA) R (red) and the marker of late recycling endosomes, Rab11 (green), in layer V of the barrel cortex contralateral to the whisker stimulation.(A) In SC mouse, Rab11 immunostaining is not clearly visible in endosomes (open arrowheads) within GluA1R+ cell (filled arrowhead).(B) In SD2 mouse, Rab11 immunostaining is evident in one large clump, likely to be over the golgi apparatus, and in a few endosomes which are double-labeled for GluA1R (filled arrowheads) within GluA1R+ cell (filled arrowhead).(C) In SD4 mouse, Rab11 immunostaining is evident in many endosomes, which are double-labeled for GluA1R (filled arrowheads) and some close to the plasma membrane within GluA1R+ cell (filled arrowhead).(D) In SR mouse, Rab11 immunostaining is apparent in many endosomes, which are double-labeled for GluA1R (filled arrowheads) and some close to the plasma membrane within GluA1R+ cells (filled arrowheads). Cells indicated with large arrowhead enlarged to right. Scale bars, 10 μm. Thickness, 1440 nm.
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