Figure 3. Increased cofilin activity in the hippocampus mediates the spine loss associated with sleep deprivation.

(A) Five hours of sleep deprivation leads to a reduction in cofilin phosphorylation at serine 3 in the hippocampus. A representative blot is shown. Each band represents an individual animal. (n = 13–14, Student’s t-test p=0.0090). (B) Mice were injected with pAAV₉-CaMKIIα0.4-eGFP or pAAV₉-CaMKIIα0.4-cofilin^S3D-HA into the hippocampus to drive expression of eGFP or the mutant inactive form of cofilin (cofilin^S3D) in excitatory neurons. This inactive mutant form of cofilin was made by substituting serine 3 for aspartic acid, which mimics a phosphoserine residue. An HA-tag was included to discriminate between mutant and endogenous cofilin. (C) A representative image showing that viral eGFP expression was restricted to the hippocampus. (D–F) Cofilin^S3D expression was excluded from astrocytes in area CA1 as indicated by a lack of co-labeling (F) between viral cofilin (D) and GFAP expression (E). Scale bar, 100 µM. (G) Virally delivered cofilin^S3D protein levels were approximately 75% (blue bar) of wild-type cofilin levels (green bar). Wild-type cofilin levels were not significantly affected by expression of cofilin^S3D. An HA-tag antibody was used to detect the mutant inactive form of cofilin. (n = 4). (H) Hippocampal cofilin^S3D expression prevents spine loss in apical/basal dendrites of CA1 neurons that is associated with sleep deprivation (n = 6, Student’s t-test, p>0.05). (I) Hippocampal cofilin^S3D expression prevents the decrease in apical/basal dendritic spine length in neurons of hippocampal that is caused by sleep deprivation (n = 6, Student’s t-test, p>0.05). (J) Sleep deprivation does not alter the number of any spine type in apical/basal dendrites of CA1 neurons in the hippocampus of mice expressing cofilin^S3D (n = 6, Student’s t-test, p>0.05). (K) Sleep deprivation does not attenuate apical/basal spine density at any distance from the soma in mice expressing cofilin^S3D (n = 6, Student’s t-test, p>0.05). NSD: non-sleep deprived, SD: sleep deprived. Values represent the mean ± SEM. **p=0.0090. Student’s t test. See also Figure 3—figure supplement 1. For separate analyses of apical and basal spine numbers see Figure 3—figure supplement 2.

DOI: http://dx.doi.org/10.7554/eLife.13424.008

Figure 3—figure supplement 1. Sleep deprivation does not alter cofilin phosphorylation in the prefrontal cortex.

Five hours of sleep deprivation does not lead to a reduction in cofilin phosphorylation at serine 3 in the prefrontal cortex. A representative blot is shown. Each band represents an individual animal. (Student’s t test p>0.5).

DOI: http://dx.doi.org/10.7554/eLife.13424.010

Figure 3—figure supplement 2. Cofilin^S3D expression prevents sleep deprivation-induced reductions in spine numbers and dendrite length in both basal and apical dendrites of CA1 neurons.

(A) In mice expressing cofilin^S3D, sleep deprivation does not decrease the spine density of basal and apical dendrite of CA1 neurons (6 animals per group, 4 neurons per animal). (B) In mice expressing cofilin^S3D, sleep deprivation does not cause a decrease in the length of basal and apical dendrite length of CA1 neurons (6 animals per group, 4 neurons per animal). (C) In mice expressing cofilin^S3D, sleep deprivation does not cause a reduction in the total number of spines for each subtype in basal dendrites of CA1 neurons with exception of the branched spines (6 animals per group, 4 neurons per animal, Student’s t test p>0.5, branched spines Student’s t test p<0.05). (D) In mice expressing cofilin^S3D, sleep deprivation does not cause a decrease in the total number of spines for each subtype in apical dendrites of CA1 neurons with exception of the branched spines (6 animals per group, 4 neurons per animal, Student’s t test p>0.5, branched spines Student’s t test p<0.05). (E) In mice expressing cofilin^S3D, sleep deprivation does not reduce the spine density of basal dendrites at any distance from the soma (6 animals per group, 4 neurons per animal, Student’s t test p>0.05). (F) In mice expressing cofilin^S3D, sleep deprivation reduces spine density of apical dendrites only at a 180 µm distance from the soma (6 animals per group, 4 neurons per animal, Student’s t test p>0.05). (G) In mice expressing cofilin^S3D, sleep deprivation does not decrease the spine density in any dendritic branch of the basal dendrites of CA1 neurons (6 animals per group, 4 neurons per animal, Student’s t test p>0.05). (H) In mice expressing cofilin^S3D, sleep deprivation does not reduce the spine density in any dendritic branch of apical dendrites of hippocampal CA1 neurons (6 animals per group, 4 neurons per animal, Student’s t test p>0.05). NSD: non-sleep deprived, SD: sleep deprived. Values represent the mean ± SEM. *p<0.05 by Student’s t test.

DOI: http://dx.doi.org/10.7554/eLife.13424.012