Figures and data in Ultrastructural effects of sleep and wake on the parallel fiber synapses of the cerebellum

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Sophia S Loschky

Giovanna Maria Spano

William Marshall

Andrea Schroeder

Kelsey Marie Nemec

Shannon Sandra Schiereck

Luisa de Vivo

Michele Bellesi

Sebastian Weyn Banningh

Giulio Tononi

Chiara Cirelli

(2022)
Ultrastructural effects of sleep and wake on the parallel fiber synapses of the cerebellum

eLife 11:e84199.

https://doi.org/10.7554/eLife.84199
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Figures
Tables
Additional files

7 figures, 4 tables and 3 additional files

Figures

Figure 1

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Experimental design.

(A) The three experimental groups. W, spontaneous wake at night; EW, extended wake during the day; S, sleep during the day. Black and yellow horizontal bars indicate the 12 hr dark and 12 hr light …

Figure 2

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Reconstruction of all dendritic segments used in the study.

W, spontaneous wake (4 mice, 29 dendrites); EW, extended wake (4 mice, 24 dendrites); S, sleep (6 mice, 43 dendrites). Each mouse is identified by a number.

Figure 3

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Spines and synapses across groups.

(A) 3D reconstruction of a dendritic branch (spines and dendrite in gray, axon-spine interfaces [ASIs] in red). (**B, C**) Absolute density of spines and synapses (N per dendrite surface area in µm²) in …

Figure 4

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Branched spines and synapses across groups.

(A) 2D image showing an example of two branched synapses, that is, housed in two spines that share the same spine neck, called branched spines (synapses in blue, axon-spine interfaces [ASIs] in …

Figure 5

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The size of the axon-spine interfaces (ASI) across groups.

(A) Examples of spines with a synapse (ASI in red). Scale bars = 1 µm. (B) Distribution of ASI sizes in cerebellum, cerebral cortex (replotted from de Vivo et al., 2017), and hippocampus (CA1, …

Figure 6

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Summary of the main findings: increase in branched synapses in wake compared to sleep and its putative functional consequences.

Author response image 1

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Tables

Table 1

Parameter estimates for the linear mixed effect (LME) models used to assess changes in dendrite diameter and length.

Residual plots showed no evidence against the assumptions of constant variance and normality.

LME model – dendrite diameter
Random effects	Standard error
Mouse (intercept)	0.0680
Residual	0.1500
Fixed effects	Level	Estimate	Standard error
Intercept		1.3133	0.0459
Condition	EW (reference)	0	0
	W	–0.0024	0.0637
	S	0.0698	0.0584
LME model – dendrite length
Random effects	Standard error
Mouse (intercept)	0.8024
Residual	3.2883
Fixed effects	Level	Estimate	Standard error
Intercept		8.7228	0.7841
Condition	EW (reference)	0	0
	W	–0.4763	1.0734
	S	0.0007	0.9878

Table 2

Summary of ultrastructural measures in the molecular layer of the cerebellum.

Previously reported measures in the hippocampus CA1 region (Spano et al., 2019) and cerebral cortex (de Vivo et al., 2017) are also shown to facilitate the comparison across regions. All protrusions …

	CA1 (stratum radiatum)			Cortex (M1+S1, layer 2)			Cerebellum
	W	EW	S	W	EW	S	W	EW	S
Total N of dendrites	37	26	38	54	57	57	29	24	43
Total N of spines	2764	2139	3148	2919	2616	2892	1944	2050	3394
Total N of spines with synapse (some incomplete/oblique)	2702	2080	3037	2467	2267	2415	1861	1926	3066
Total N spines with synapse with measured ASI	2552	1949	2840	2180	1989	2136	1766	1848	3004
ASI (µm², mean ± std) range (µm²)	0.128±0.122 0.003–1.033	0.130±0.122 0.008–0.764	0.120±0.108 0.006–0.672	0.299±0.323 0.012–4.019	0.290±0.305 0.005–3.543	0.248±0.278 0.006–2.038	0.187±0.109 0.010–0.702	0.158±0.095 0.006–0.569	0.174±0.104 0.005–1.113
Dendrite diameter (µm, mean ± std)	0.60±0.10	0.56±0.06	0.57±0.09	0.89±0.24	0.82±0.20	0.86±0.25	1.31±0.14	1.32±0.16	1.38±0.19
Dendrite length (µm, mean ± std)	23.3±5.4	23.2±6.9	21.9±6.8	26.8±5.9	23.8±7.5	23.7±8.0	8.19±2.97	8.68±3.00	8.66±3.92
Spine density (with/without synapse) by dendrite surface area (#/µm², mean ± std)	1.72±0.30	2.17±0.33	2.00±0.34	0.76±0.22	0.78±0.23	0.84±0.23	1.83±0.30	2.16±0.44	1.97±0.51
Process density (spines without synapse) by dendrite surface area (#/µm², mean ± std)	0.04±0.03	0.06±0.06	0.07±0.08	0.12±0.07	0.10±0.06	0.15±0.09	0.08±0.06	0.13±0.07	0.19±0.10
Synapse density (spines with synapse) by dendrite surface area (#/µm², mean ± std)	1.69±0.30	2.12±0.34	1.93±0.32	0.64±0.20	0.68±0.22	0.69±0.19	1.76±0.28	2.04±0.41	1.78±0.46
Nonperforated synapse density by dendrite surface area (#/µm², mean ± std)	1.27±0.26	1.72±0.29	1.47±0.29	–	–	–	–	–	–
Perforated synapse density by dendrite surface area (#/µm², mean ± std)	0.41±0.13	0.36±0.10	0.44±0.18	–	–	–	–	–	–
Synapse density (spines with synapse) by dendrite length (#/µm, mean ± std)	3.14±0.60	3.69±0.50	3.41±0.50	–	–	–	7.86±1.39	9.07±1.73	8.29±1.82
Nonperforated synapse density by dendrite length (#/µm, mean ± std)	2.38±0.52	3.00±0.43	2.59±0.35	–	–	–	–	–	–
Perforated synapse density by dendrite length (#/µm, mean ± std)	0.77±0.23	0.63±0.16	0.78±0.31	–	–	–	–	–	–
Perforated synapses % of synapses per mouse (mean ± std)	24.5 ± 1.3%	17.6 ± 1.9%	23.2 ± 5.3%	–	–	–	–	–	–
Oblique spines % of synapses per mouse (mean ± std)	4.8 ± 1.7%	4.7 ± 0.9%	5.2 ± 2.8%	8.5 ± 2.2%	10.8 ± 3.3%	10.0 ± 1.7%	7.0 ± 6.0%	5.6 ± 3.5%	1.8 ± 2.1%
Incomplete synapses (go off the image) % of spines with synapse per mouse (mean ± std)	1.0 ± 0.9%	1.7 ± 0.9%	1.5 ± 1.5%	2.2 ± 2.2%	2.8 ± 5.2%	4.0 ± 4.4%	0.8 ± 1.5%	1.2 ± 0.3%	1.2 ± 0.7%
Spines with spine apparatus % of spines with synapse per mouse (mean ± std)	8.2 ± 0.7%	10.1 ± 3.1%	13.5 ± 3.5%	30.1 ± 4.0%	29.2 ± 2.5%	26.1 ± 4.5%	2.2 ± 3.6%	6.4 ± 9.8%	1.8 ± 3.1%
Spines with endosome/s % of spines with synapse per mouse (mean ± std)	34.8 ± 9.4%	42.9 ± 12.4%	41.3 ± 9.5%	46.4 ± 11.1%	67.2 ± 8.0%	57.5 ± 10.0%	96.3 ± 7.4%	98.9 ± 1.5%	99.8 ± 0.5%
Synapses with coated vesicle % of spines with synapse per mouse (mean ± std)	2.8 ± 2.2%	2.9 ± 1.3%	1.8 ± 1.1%	2.5 ± 1.8%	4.3 ± 3.2%	2.6 ± 1.7%	1.5 ± 1.9%	0.2 ± 0.2%	0.1±0.2%
Synapses with spinula % of spines with synapse per mouse (mean ± std)	19.4 ± 1.9%	10.1 ± 3.8%	14.0 ± 3.2%	1.7 ± 2.0%	1.3 ± 1.4%	1.1 ± 0.5%	1.6 ± 1.0%	10.3 ± 13.5%	22.7 ± 16.6%
Synapses with MVB % of spines with synapse per mouse (mean ± std)	5.3 ± 2.3%	2.7 ± 3.2%	3.4 ± 3.2%	6.4 ± 3.5%	6.6 ± 2.9%	3.3 ± 2.2%	3.4 ± 4.1%	5.7 ± 8.0%	2.8 ± 3.9%
Synapses with presynaptic mitochondrion % of spines with synapse per mouse (mean ± std)	30.3 ± 2.1%	30.3 ± 3.7%	31.6 ± 3.6%	34.7 ± 4.2%	35.1 ± 2.1%	33.7 ± 3.3%	58.0 ± 1.9%	61.8 ± 7.5%	57.3 ± 4.4%
Branched synapses % of spines with synapse per mouse (mean ± std)	15.0 ± 3.7%	15.8 ± 4.9%	13.5 ± 1.6%	13.9 ± 6.1%	12.2 ± 3.2%	13.7 ± 3.1%	17.8 ± 3.3%	21.0 ± 7.4%	14.1 ± 2.4%

Table 3

Parameter estimates for the linear mixed effect (LME) models used to assess changes in spine densities and proportions.

A square-root transformation was applied to each model. Residual analysis showed no evidence against normality or constant variance assumption.

Spine density (all spines) − sqrt(#/area)
Random effects	Standard error
Mouse (intercept)	0.0618
Residual	0.1426
Fixed effects	Level	Estimate	Standard error
Intercept		1.4681	0.0426
Condition	EW (reference)	0	0
	W	–0.1140	0.0590
	S	–0.0757	0.0542
Spine density (spines with synapse) − sqrt(#/area)
Random effects	Standard error
Mouse (intercept)	0.0605
Residual	0.1349
Fixed effects	Level	Estimate	Standard error
Intercept		1.4241	0.0412
Condition	EW (reference)	0	0
	W	–0.0994	0.0569
	S	–0.1002	0.0522
Spine density (branched synapses) − sqrt(#/area)
Random effects	Standard error
Mouse (intercept)	0.0350
Residual	0.1760
Fixed effects	Level	Estimate	Standard error
Intercept		0.5920	0.0401
Condition	EW (reference)	0	0
	W	–0.0934	0.0547
	S	–0.2281	0.0503
Spine density (spines without synapse) − sqrt(#/area)
Random effects	Standard error
Mouse (intercept)	0.0387
Residual	0.1027
Fixed effects	Level	Estimate	Standard error
Intercept		0.3443	0.0286
Condition	EW (reference)	0	0
	W	–0.0791	0.0395
	S	0.0760	0.0363
Proportion of spines without synapse − sqrt(# without/# total)
Random effects	Standard error
Mouse (intercept)	0.0270
Residual	0.0642
Fixed effects	Level	Estimate	Standard error
Intercept		0.2331	0.0189
Condition	EW (reference)	0	0
	W	–0.0386	0.0240
	S	0.0674	0.0262
Proportion of synapses with spinula – sqrt(# with spinula/# total)
Random effects	Standard error
Mouse (intercept)	0.1285
Residual	0.0898
Fixed effects	Level	Estimate	Standard error
Intercept		0.4276	0.0669
Condition	EW (reference)	0	0
	W	–0.1730	0.0944
	S	0.0733	0.0862

Table 4

Parameter estimates for the linear mixed effect (LME) models used to assess changes in axon-spine interfaces (ASI).

A square-root transformation was applied to the ASI values to ensure the residuals had an approximate Gaussian distribution. Residual analysis showed only minor departures from normality and …

ASI − sqrt(ASI)
Random effects	Standard error
Dendrite (intercept)	0.0134
Mouse (intercept)	0.0164
Residual	0.1219
Fixed effects	Level	Estimate	Standard error
Intercept		0.3797	0.0227
Dendrite diameter	Continuous (linear)	–1e-7	2e-5
Condition	EW (reference)	0	0
	W	0.0331	0.0129
	S	0.0197	0.0118
ASI (with branched) – sqrt(ASI)
Random effects	Standard error
Dendrite (intercept)	0.0132
Mouse (intercept)	0.0168
Residual	0.1217
Fixed effects	Level	Estimate	Standard error
Intercept		0.3812	0.0227
Dendrite diameter	Continuous (linear)	7e-7	2e-5
Condition	EW (reference)	0	0
	W	0.0333	0.0132
	S	0.0184	0.0120
Branched	No (reference)	0	0
	Yes	–0.0217	0.0052
ASI density (sum ASI/surface area)
Random effects	Standard error
Mouse (intercept)	0.0425
Residual	0.0608
Fixed effects	Level	Estimate	Standard error
Intercept		0.3159	0.0247
Condition	EW (reference)	0	0
	W	–0.0042	0.0349
	S	–0.0131	0.0316