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. 2023:2:1145203.
doi: 10.3389/frsle.2023.1145203. Epub 2023 Apr 3.

Regulation of dendritic spines in the amygdala following sleep deprivation

Lindsay Rexrode  1 , Matthew Tennin  1 , Jobin Babu  1   2 , Caleb Young  1 , Ratna Bollavarapu  1 , Lamiorkor Ameley Lawson  1 , Jake Valeri  1   2 , Harry Pantazopoulos  1   2 , Barbara Gisabella  1   2
Affiliations

Regulation of dendritic spines in the amygdala following sleep deprivation

Lindsay Rexrode et al. Front Sleep. 2023.

Abstract

The amygdala is a hub of emotional circuits involved in the regulation of cognitive and emotional behaviors and its critically involved in emotional reactivity, stress regulation, and fear memory. Growing evidence suggests that the amygdala plays a key role in the consolidation of emotional memories during sleep. Neuroimaging studies demonstrated that the amygdala is selectively and highly activated during rapid eye movement sleep (REM) and sleep deprivation induces emotional instability and dysregulation of the emotional learning process. Regulation of dendritic spines during sleep represents a morphological correlate of memory consolidation. Several studies indicate that dendritic spines are remodeled during sleep, with evidence for broad synaptic downscaling and selective synaptic upscaling in several cortical areas and the hippocampus. Currently, there is a lack of information regarding the regulation of dendritic spines in the amygdala during sleep. In the present work, we investigated the effect of 5 h of sleep deprivation on dendritic spines in the mouse amygdala. Our data demonstrate that sleep deprivation results in differential dendritic spine changes depending on both the amygdala subregions and the morphological subtypes of dendritic spines. We observed decreased density of mushroom spines in the basolateral amygdala of sleep deprived mice, together with increased neck length and decreased surface area and volume. In contrast, we observed greater densities of stubby spines in sleep deprived mice in the central amygdala, indicating that downscaling selectively occurs in this spine type. Greater neck diameters for thin spines in the lateral and basolateral nuclei of sleep deprived mice, and decreases in surface area and volume for mushroom spines in the basolateral amygdala compared to increases in the cental amygdala provide further support for spine type-selective synaptic downscaling in these areas during sleep. Our findings suggest that sleep promotes synaptic upscaling of mushroom spines in the basolateral amygdala, and downscaling of selective spine types in the lateral and central amygdala. In addition, we observed decreased density of phosphorylated cofilin immunoreactive and growth hormone immunoreactive cells in the amygdala of sleep deprived mice, providing further support for upscaling of dendritic spines during sleep. Overall, our findings point to region-and spine type-specific changes in dendritic spines during sleep in the amygdala, which may contribute to consolidation of emotional memories during sleep.

Keywords: amygdala; dendritic spines; growth hormone; memory consolidation; sleep.

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Conflict of interest statement

Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Dendritic spines in the basolateral amygdala (BL) are lower in sleep-deprived mice. AAV viral vector was used to label dendritic processes (A). Decreased density of dendritic spines was observed in the BL amygdala in sleep-deprived mice (nmice = 6; ndendrites = 383) compared to control mice (nmice = 6; ndendrites = 450) (B). In contrast, density of dendritic spines in the LA of sleep-deprived mice (nmice = 5; ndendrites = 167) was increased in comparison to control mice (nmice = 5; ndendrites = 105) (B). A similar increase in density was observed in the CEA of sleep-deprived mice (nmice = 5; ndendrites = 222) compared to control mice (nmice = 5; ndendrites = 239). Representative confocal micrograph of a 10 μm branch segment from the BL of a sleep-deprived mouse (D), with fewer spines than a representative branch segment from a control mouse (C). (E) Representative confocal image depicting examples of thin, mushroom, and stubby spines. Dendritic spine density was decreased in mushroom spines in the BL of sleep-deprived mice (nmice = 6; ndendrites = 383) compared to control mice (nmice = 6; ndendrites = 450) (F). Mushroom spines from the LA of sleep-deprived mice (nmice = 5; ndendrites = 167) were not significantly different from control mice (nmice = 5; ndendrites = 105) (F). Similarly, no differences were observed in CEA mushroom spines from sleep deprived mice (nmice = 5; ndendrites = 222) compared to control mice (nmice = 5; ndendrites = 239). No difference in density of thin spines was observed in the LA of sleep-deprived mice (nmice = 5; ndendrites = 167) compared to control mice (nmice = 5; ndendrites = 105) (G), or in thin spines from the CEA of sleep-deprived mice (nmice = 5; ndendrites = 222) compared to control mice (nmice = 5; ndendrites = 239) (G). Similarly, no difference was observed in the density of thin spines in the BL of sleep deprived mice (nmice = 6; ndendrites = 383) compared to control mice (nmice = 6; ndendrites = 450). Increased density of stubby spines was observed in the LA of sleep-deprived mice (nmice = 5; ndendrites = 167) compared to control mice (nmice = 5; ndendrites = 105) (H). No difference was observed in the CEA of sleep-deprived mice (nmice = 5; ndendrites = 222) compared to control mice (nmice = 5; ndendrites = 239), or in the density of stubby spines in the BL of sleep deprived mice (nmice = 6; ndendrites = 383) compared to control mice (nmice = 6; ndendrites = 450) (G). Box plots depict values for each group, statistical significance was determined using the Wilcoxon-Mann-Whitney test with Bonferroni correction for multiple comparisons.
FIGURE 2
FIGURE 2
Differential changes in neck backbone and head backbone length dendritic spines in sleep deprived mice. Neurolucida 360 was used to obtain measures of dendritic spine neck backbone length and head backbone length from confocal images. The diagram (A, B) depicts neck backbone length measured as the distance from the insertion point to the center of the spine head minus the head radius and anchor radius (A). Head backbone length is measured as the distance from the insertion point to the center of the spine head minus the anchor radius (B). Neck backbone length was significantly greater in mushroom spines from the LA of sleep-deprived mice (nmice = 5; nspines = 1,339) compared to control mice (nmice = 5; nspines = 1,217) (C). A similar increase was observed in the BL of sleep-deprived mice (nmice = 6; nspines = 3,143) compared to control mice (nmice = 6; nspines = 5,385) for mushroom spine backbone length (C). No significant difference was observed for neck backbone length of mushroom spines in the CEA of sleep deprived mice (nmice = 5; nspines = 1,543) compared to control mice (nmice = 5; nspines = 1,768). In comparison, neck backbone length in thin spines was not altered in the LA of sleep-deprived mice (nmice = 5; nspines = 2,525) compared to control mice (nmice = 6; nspines = 2,046) or in the BL of sleep-deprived mice (nmice = 5; nspines = 5,645) compared to control mice (nmice = 5; nspines = 9,079). Neck backbone length was increased in thin spines from the CEA in sleep-deprived mice (nmice = 5; nspines = 3,207) compared to control mice (nmice = 5; nspines = 3,449) (D). Neck backbone length was greater in stubby spines from the BL of sleep deprived mice (nmice = 6; nspines = 1,343) compared to control mice (nmice = 6; nspines = 2,230). No difference in neck backbone length was observed in the CEA of sleep-deprived mice (nmice = 5; nspines = 878) compared to control mice (nmice = 5; nspines = 871) (E), or in stubby spines in the LA of sleep-deprived mice (nmice = 5; nspines = 618) compared to control mice (nmice = 5; nspines = 469). Similar changes were observed for head backbone length measures, with increased length in mushroom spines from sleep-deprived mice vs. controls in the LA and BL areas and for stubby spines in the CEA (F–H). Box plots depict values for each group, statistical significance was determined using the Wilcoxon-Mann-Whitney test with Bonferroni correction for multiple comparisons.
FIGURE 3
FIGURE 3
Differential changes of head and neck diameter across dendritic spine types during sleep. Mushroom spine head diameter was not significantly different between sleep-deprived and control mice in the LA (Control: nmice = 5; nspines = 1,217; SD: nmice = 5; nspines = 1,339), BL (Control: nmice = 6; nspines = 5,385; SD: nmice = 6; nspines = 3,143) or CEA (Control: nmice = 5; nspines = 1,768; SD: nmice = 5; nspines = 1,543) (A). Thin spine head diameter was selectively decreased in the CEA of sleep-deprived mice (Control: nmice = 5; nspines = 3,449; SD: nmice = 5; nspines = 3,207) (B), with no significant differences in the LA (Control: nmice = 5; nspines = 2,046; SD: nmice = 5; nspines = 2,525) or BL (Control: nmice = 6; nspines = 9,079; SD: nmice = 6; nspines = 5,645) (B). A similar selective decrease was observed for stubby spines in the CEA (Control: nmice = 5; nspines = 871; SD: nmice = 5; nspines = 878), with no significant changes in the LA (Control: nmice = 5; nspines = 469; SD: nmice = 5; nspines = 618) or BL (Control: nmice = 6; nspines = 2,230; SD: nmice = 6; nspines = 1,343) (C). Spine neck diameter was significantly lower in mushroom spines in the BL of sleep deprived mice, with no changes in mushroom spines in the LA or CEA (D). In contrast, neck diameter for thin spines was significantly greater in the LA and BL of sleep-deprived mice and lower in the CEA (E). Neck diameter for stubby spines was not altered (F). Box plots depict values for each group, statistical significance was determined using the Wilcoxon-Mann-Whitney test with Bonferroni correction for multiple comparisons.
FIGURE 4
FIGURE 4
Dendritic spine surface area and volume differences between sleep-deprived and control mice. Dendritic spine surface area was significantly lower in mushroom spines from sleep-deprived mice in the BL compared to control mice (Control: nmice = 6; nspines = 5,385; SD: nmice = 6; nspines = 3,143) (A). Spine surface area was significantly greater in mushroom spines from sleep-deprived mice in the CEA (Control: nmice = 5; nspines = 1,768; SD: nmice = 5; nspines = 1,543), and no differences were observed for mushroom spine surface area in the LA (Control: nmice = 5; nspines = 1,217; SD: nmice = 5; nspines = 1,339) (A). Thin spine surface area was not significantly altered in the LA (Control: nmice = 5; nspines = 2,046; SD: nmice = 5; nspines = 2,525), CEA (Control: nmice = 5; nspines = 3,449; SD: nmice = 5; nspines = 3,207) or BL (Control: nmice = 6; nspines = 9,079; SD: nmice = 6; nspines = 5,645) (B). The surface area of stubby spines was significantly greater in spines from the CEA (Control: nmice = 5; nspines = 871; SD: nmice = 5; nspines = 878) of sleep-deprived mice, with no differences observed from stubby spines in the LA (Control: nmice = 5; nspines = 469; SD: nmice = 5; nspines = 618) or BL (Control: nmice = 6; nspines = 2,230; SD: nmice = 6; nspines = 1,343) (C). Spine volumes displayed largely similar changes, with significantly lower volume in the BL and greater volume in the CEA for mushroom spines in sleep-deprived mice (D), no significant difference in thin (E), and greater volume of stubby spines in the CEA of sleep-deprived mice (F). Box plots depict values for each group, statistical significance was determined using the Wilcoxon-Mann-Whitney test with Bonferroni correction for multiple comparisons.
FIGURE 5
FIGURE 5
Lower density of pCofilin and GH immunoreactive cells in the amygdala of sleep deprived mice. Sleep-deprived mice displayed significantly reduced numerical density of pCofilin immunoreactive cells in the lateral and basolateral amygdala nuclei, but not in the central nucleus compared to control mice (A). Representative 5x and 40x images of pCofilin labeling in the amygdala from control (B) and SD mice (C). 40x magnification inserts depict pCofilin labeling in the BL. Decreased density of GH immunoreactive cells was also detected in the LA of sleep deprived mice (D), but not in the BL or CEA. Representative 5x and 40x images of GH labeling in the amygdala from control (E) and SD mice (F). 40x magnification inserts depict GH labeling in the LA. Scale bars in yellow = 100 μm for 5x images, 50 μm for 40x inserts. All graphs reflect the mean for each group with n = 6 control and n = 6 sleep-deprived animals. Bar graphs depicting mean and 95% confidence interval of the density of immunoreactive cells. Each dot represents the value for an individual mouse calculated as density of immunoreactive cells per area (cells/mm2).
FIGURE 6
FIGURE 6
Summary of dendritic spine changes during sleep in the mouse amygdala. The diagram represents a summary of the working hypothesis of dendritic spine changes during sleep in the mouse amygdala based on our data from sleep deprived mice. Spine densities for each region are indicated by the branch with multiple spines in the upper part of each panel, and spine morphological subtypes are indicated by corresponding shapes (thin, subby, mushroom) in each panel. Mushroom, thin, and stubby spines in the BL are upscaled during sleep through increased spine density and corresponding decreases in neck diameter, neck length, and increased volume. In comparison, thin and stubby dendritic spines in the LA and CEA are largely downscaled during sleep through decreased spine density along with more selective changes in neck and head properties.
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