Copyright © 2019 the authors

Research Articles: Development/Plasticity/Repair

Ocular dominance plasticity in binocularprimary visual cortex does not require C1q

https://doi.org/10.1523/JNEUROSCI.1011-19.2019

Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.1011-19.2019

Received: 2 May 2019Revised: 21 October 2019Accepted: 23 October 2019

This Early Release article has been peer-reviewed and accepted, but has not been throughthe composition and copyediting processes. The final version may differ slightly in style orformatting and will contain links to any extended data.

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Ocular dominance plasticity in binocular primary visual cortex does not 1 require C1q 2

Abbreviated title: Normal ocular dominance plasticity without C1q 3

Christina A. Welsh1, Céleste-Élise Stephany1, Richard W. Sapp2, Beth Stevens1, 3, 4 4 1 F. M. Kirby Neurobiology Center, Boston Children's Hospital and Harvard Medical School, 5 Boston, Massachusetts, 02115 6 2 Departments of Biology and Neurobiology, and Bio-X, James H. Clark Center, Stanford 7 University, Stanford, California, 94305 8 3 Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, 9 Massachusetts, 02139 10 4 Howard Hughes Medical Institute, Boston Children’s Hospital, Boston, MA, 02115 11

Corresponding author: Beth Stevens, beth.stevens@childrens.harvard.edu 12

Author contributions: CAW, CES and BS designed research, CAW and CES performed and 13 analyzed research, RWS contributed unpublished reagents/analytic tools, CAW wrote the paper. 14

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Length: 51 pages, 10 figures 16

Abstract: 229 words 17

Introduction: 605 words 18

Discussion: 1230 words 19

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Conflict of interest: B.S. serves on the scientific advisory board of Annexon LLC and is a minor 21 shareholder of Annexon LLC. All other authors declare no competing interests. 22

Acknowledgments: We thank Daniel Tom, Ryan Carelli, Michael Blanchard and Michelle 23 Ocana of the Neurobiology Imaging Facility at Harvard Medical School (NINDS P30 Core 24 Center Grant #NS072030) and Cassandra-Victoria Innocent of the IDDRC Cellular Imaging 25 Core at Boston Children’s Hospital (U54 HD090255) for help with imaging. For SIM imaging, 26 we thank Allie Muthukumar for technical help and the Harvard Center for Biological Imaging 27 for infrastructure and support; we also acknowledge NIH SIG award (1S10RR029237-01) which 28 was used to acquire the ELYRA microscope. We thank Nick Andrews of the 29 Neurodevelopmental Behavioral Core at Boston Children’s Hospital (CHB IDDRC, 30 1U54HD090255) for providing a dark room. Work was supported by the NINDS (R01-NS-31 071008). We thank Carla Shatz for scientific advice and for generously sharing a mouse Arc 32 probe. We would also like to thank Tim Hammond and Chinfei Chen for critical reading of the 33 manuscript, and Dorathy Vargas for help with mouse colony maintenance. 34

35

1

Abstract 36

C1q, the initiator of the classical complement cascade, mediates synapse elimination in the 37

postnatal mouse dorsolateral geniculate nucleus of the thalamus (dLGN) and sensorimotor 38

cortex. Here, we asked whether C1q plays a role in experience-dependent synaptic refinement in 39

the visual system at later stages of development. The binocular zone of primary visual cortex 40

(V1b) undergoes spine loss and changes in neuronal responsiveness following the closure of one 41

eye during a defined critical period (a process referred to as ocular dominance plasticity or 42

ODP). We therefore hypothesized that ODP would be impaired in the absence of C1q, and that 43

V1b development would also be abnormal without C1q-mediated synapse elimination. However, 44

when we examined several features of V1b development in mice lacking C1q, we found that the 45

densities of most spine populations on basal and proximal apical dendrites, as well as firing rates 46

and ocular dominance, were normal. C1q was only transiently required for the development of 47

spines on apical, but not basal, secondary dendrites. Dendritic morphologies were also 48

unaffected. Although we did not observe the previously described spine loss during ODP in 49

either genotype, our results reveal that the animals lacking C1q had normal shifts in neuronal 50

responsiveness following eye closure. Experiments were performed in both male and female 51

mice. These results suggest that the development and plasticity of the mouse V1b is grossly 52

normal in the absence of C1q. 53

54

Significance statement 55

These findings illustrate that the development and experience-dependent plasticity of V1b is 56

mostly normal in the absence of C1q, even though C1q has previously been shown to be required 57

for developmental synapse elimination in the mouse visual thalamus as well as sensorimotor 58

2

cortex. The V1b phenotypes in mice lacking C1q are more similar to the mild defects previously 59

observed in the hippocampus of these mice, emphasizing that the contribution of C1q to synapse 60

elimination appears to be dependent on context. 61

62

Introduction 63

Synapse formation and elimination occur continuously in subsets of synapses in the brain 64

(Grutzendler et al., 2002; Trachtenberg et al., 2002). Synapse elimination is prominent during 65

development in both mice and humans (Huttenlocher, 1979; Hong and Chen, 2011; Kano et al., 66

2018), and can be increased by changes in sensory experience (Trachtenberg et al., 2002; 67

Holtmaat et al., 2006; Keck et al., 2008). Excessive synapse elimination has been implicated in 68

neuropsychiatric and neurodegenerative disease (Mucke and Selkoe, 2012; Glausier and Lewis, 69

2013). Thus, understanding the cellular and molecular mechanisms that regulate this process is 70

of great importance to human health. 71

Synapse elimination is associated with the induction of ODP, a classic model of 72

experience-dependent plasticity in the visual system (Mataga et al., 2004; Coleman et al., 2010; 73

Zhou et al., 2017). In normal mice, the responsiveness of neurons in V1b is dominated by input 74

from the contralateral eye. However, depriving the contralateral eye of input for three to four 75

days by monocular deprivation (MD) during the critical period (approximately postnatal day 76

(P)19-32 in the mouse (Gordon and Stryker, 1996)), causes a shift in V1b responsiveness toward 77

the non-deprived ipsilateral eye. Synapse loss in V1b accompanies this shift (Mataga et al., 2004; 78

Coleman et al., 2010; Zhou et al., 2017). ODP can thus be used as a model to study the 79

mechanisms mediating experience-dependent synapse elimination. 80

3

The innate immune molecule C1q is a known regulator of synaptic development, and a 81

candidate for regulating synapse elimination in V1b. C1q is a secreted protein, which in the 82

immune system binds to pathogens and apoptotic cells and initiates a proteolytic cascade referred 83

to as the classical complement cascade (Ricklin et al., 2010; Thielens et al., 2017). In at least 84

some parts of the brain, C1q is thought to mediate synapse elimination by binding to synapses 85

and locally activating the classical complement cascade, which results in engulfment of 86

presynaptic inputs by microglia (Stevens et al., 2007; Schafer et al., 2012). Mice lacking C1q 87

show impaired anatomical and electrophysiological refinement of retinal ganglion cell (RGC) 88

inputs to the dLGN at one month of age, demonstrating sustained defects in connectivity 89

(Stevens et al., 2007). Furthermore, mice lacking C1q show increased densities of dendritic 90

spines in sensorimotor cortex, have increased pyramidal neuron dendritic branching, and are 91

prone to seizures (Chu et al., 2010; Ma et al., 2013). Whether C1q promotes synapse elimination 92

in V1b during development or in response to changing visual experience has, however, not been 93

examined. 94

Here, we investigated the role of C1q in the development and plasticity of V1b in mice. 95

In mice lacking C1q, we counted spines to examine synapse development and elimination, 96

performed in vivo electrophysiology to measure firing rates, and used electrophysiology and in 97

situ hybridization against the immediate early gene Arc to determine how loss of C1q affects 98

ODP. We found that although C1q is present in V1b during the critical period, it was not 99

required for the development of most spine populations on layer (L)2/3 pyramidal neurons. The 100

dendritic arbors of these neurons were also unaffected by loss of C1q. In vivo 101

electrophysiological recordings furthermore revealed normal spontaneous and visually evoked 102

firing rates in V1b in the absence of C1q. Spine loss following critical period MD has previously 103

4

been described on the apical dendrites of L2/3 pyramidal neurons (Mataga et al., 2004), but we 104

failed to observe MD-induced spine loss in either genotype. However, we found normal OD 105

shifts in mice lacking C1q when compared to their wild-type littermates. Together, these findings 106

thus indicate that in V1b of mice lacking C1q, the development and plasticity of neuronal 107

morphology and eye specific inputs are largely normal. 108

109

Materials and Methods 110

Mice: All procedures were approved by the Boston Children’s Hospital institutional animal care 111

and use committee in accordance with NIH guidelines for the humane treatment of animals. 112

C1qa-/- mice are on a C57BL/6J background and were a kind gift from M. Botto. Wild-type 113

animals used in Figures 1 and 7 were purchased from the Jackson Laboratory (Bar Harbor, ME, 114

stock number 000664), or derived from C1qa+/-x C1qa+/-crosses. All other animals were derived 115

from C1qa+/-x C1qa+/-crosses. 116

117

Monocular deprivation and enucleation: Monocular deprivation was performed under 118

isoflurane anesthesia. The eyelids were sutured together with a single mattress suture using nylon 119

sutures (Ethilon; Ethicon cat # G697G). Monocular enucleation (ME) was performed under 120

isoflurane anesthesia. If the eye to be enucleated had previously been sutured, the sutures were 121

removed and the eyelids opened. The eyelid margins were trimmed. The optic nerve was then cut 122

with scissors and the eye removed, and the eyelids sealed shut with cyanoacrylate. 123

124

5

C1q immunohistochemistry: Mice were anesthetized with avertin (IP injection, 240 mg/kg) and 125

transcardially perfused with PBS. Brains were dissected out and drop fixed in 4% PFA (Electron 126

Microscopy Sciences, cat # 15710) for 2 hours at room temperature. Brains were then washed in 127

PBS, transferred to 30% sucrose in PBS, and kept in sucrose at 4°C for 24-48 hours. Brains were 128

embedded in a 2:1 mixture of 30% sucrose in PBS and OCT (Sakura Finetek, cat # 4583), and 129

sectioned at 30 m on a cryostat onto X-tra slides (Leica Microsystems, cat # 3800200). Tissue 130

sections were dried, washed in PBS, and blocked for 2 hours at room temperature with slow 131

agitation in 10% normal goat serum (Sigma, cat # G9023-10ML) with 1% TX-100 (Sigma, cat # 132

T8787-100ML) in PBS. Primary antibody (rabbit anti-C1q, 1:500, Abcam cat# ab182451, 133

RRID:AB_2732849) was applied overnight, in 5% normal goat serum with 0.5% TX-100 in 134

PBS, at room temperature with slow agitation. Tissue sections were then washed 3x10 min with 135

PBS and incubated with the appropriate Alexa-conjugated secondary antibody (1:200, 136

Invitrogen/Thermo Scientific) for 2 hours, in 5% normal goat serum with 0.1% TX-100 in PBS, 137

at room temperature with slow agitation. Finally, tissue sections were mounted with Vectashield 138

with DAPI (Vector labs, cat # H-1000). Immunofluorescence intensity was calculated in ImageJ 139

(NIH) from images taken on an SPE confocal microscope (Leica). Layers were identified by 140

examining the density of DAPI-positive nuclei. Epifluorescence images were taken on a VS120 141

Virtual Slide Microscope (Olympus). 142

For C1q and spinophilin staining, tissue collection was performed as above. Brains were 143

sectioned at 14 m on a cryostat onto X-tra slides (Leica Microsystems, cat # 3800200). Tissue 144

sections were dried, washed in PBS, and blocked for 2 hours at room temperature in 5% donkey 145

serum (Sigma, cat # D9663-10ML) with 0.3% TX-100 (Sigma, cat # T8787-100ML) in PBS. 146

Primary antibody (rabbit anti-C1q, 1:500, Abcam cat # ab182451, RRID:AB_2732849; sheep 147

6

anti-spinophilin/PPP1R9B, 1:200, R&D Systems cat # AF6465, RRID:AB_10718703) was 148

applied overnight, in 5% donkey serum with 0.3% TX-100 in PBS, at 4°C. Tissue sections were 149

then washed 3x10 min with PBS and incubated with the appropriate Alexa-conjugated secondary 150

antibody (donkey anti-rabbit, 1:200, Invitrogen/Thermo Scientific; donkey anti-sheep, 1:200, 151

Jackson ImmunoResearch) for 2 hours, in 5% donkey serum with 0.3% TX-100 in PBS, at room 152

temperature. Finally, tissue sections were mounted with Vectashield with DAPI (Vector labs, cat 153

# H-1000). Images were taken on an SP8 confocal microscope (Leica). Layers were identified by 154

examining the density of DAPI-positive nuclei. 155

For structured illumination microscopy (SIM) imaging, staining was performed as above, 156

except Hoechst 33342 (Molecular Probes/Thermo Fisher Scientific, cat # H3570) was added to 157

the secondary antibody at 1:10000, and the sections were mounted with ProLong Glass Antifade 158

Mountant (Invitrogen/Thermo Scientific, cat # P36984). Images were then acquired with five 159

grating rotations using an ELYRA PS1 Superresolution Microscope (Zeiss), and processed using 160

the Zeiss SIM algorithms. 161

162

Colocalization analysis: Confocal images of C1q and spinophilin immunostaining were 163

acquired on an SP8 confocal microscope (Leica) as described above. Images were then processed 164

in Ilastik (Sommer et al., 2011), which is an image processing software that can be trained to 165

recognize various features in images, without being explicitly told which criteria to use for 166

feature recognition. Ilastik was trained to recognize C1q and spinophilin immunostaining using 167

separate training paradigms for each marker, and to output segmented images. The overlap of 168

C1q and spinophilin puncta was then calculated in ImageJ (NIH) using the Analyze Particles 169

7

function, and compared to the overlap obtained when the segmented C1q channel was rotated by 170

90°. 171

172

Spine counting: Spines were counted in animals aged P10, P20, and P29-30. Animals in the 173

oldest group were either normally reared, or had undergone four days of MD with MD starting at 174

P25-26. Spines were visualized using the FD Rapid GolgiStain Kit (FD NeuroTechnologies, Inc, 175

cat # PK401A), according to manufacturer’s instructions. In brief, animals were anesthetized, 176

decapitated, and brains were washed in double deionized water prior to immersion in solution 177

A+B. After 4 weeks’ incubation, brains were transferred to solution C for 3-7 days. They were 178

then embedded in ice and sectioned coronally at 100 m on a cryostat. Sections were placed on 179

gelatin-coated slides (FD NeuroTechnologies, Inc, cat # PO101), stained with solutions D+E, 180

dehydrated with ethanol and cleared with xylene. Sections were then mounted under coverslips 181

with Permount (Fisher Scientific, cat # SP15-500). 182

Golgi-Cox stained sections were imaged under brightfield on a Virtual Slide Microscope 183

VS120 (Olympus) and on an E800 microscope (Nikon). Using these images, cells were selected 184

for subsequent higher magnification imaging and spine counts. Cells that fit the following 185

selection criteria were chosen: located in L2/3 of V1b (preferably but not exclusively in deeper 186

L2/3); located superficially in the slice for better imaging quality; no highly clustered cells where 187

it would be difficult to determine to which cell the apical dendrite belonged. For the P10 and P20 188

age groups cells in both hemispheres were used, while cells in the P29-30 NR and MD groups 189

were only in the hemisphere contralateral to the deprived eye. Depth of cortical layers was 190

determined using the Allen Mouse Brain Atlas (http://portal.brain-map.org/)(Sunkin et al., 2012). 191

Identification was performed blind to genotype and experimental paradigm. 192

8

Image stacks of golgi-stained cells that passed the selection process were imaged on a 193

BX63 upright brightfield microscope (Olympus), using a 100X objective for the primary and 194

secondary apical dendrite spine quantification, and a 60X objective for the secondary basal 195

dendrite spine quantification and for Sholl analysis (see below). Light source intensity was 196

adjusted for each cell in order to account for differences in tissue clarity. When a cell was 197

partially obscured by other stained neurons or by background signal, it was only used for some 198

of the quantifications; the number of cells or mice used thus varies between the quantifications. 199

For measuring the distance between the soma and the first branch point, and to identify 200

the 25 m segment distal to the first branch point, cells were reimaged on an SP8 confocal 201

microscope (Leica) by detecting reflected light (Spiga et al., 2011). Measurements were then 202

made in the FilamentTracer module in Imaris (version 7.7.2). For measuring the length of 203

dendrite segments on secondary branches (apical and basal), dendrite lengths were measured 204

using the Simple Neurite Tracer plugin in ImageJ (Longair et al., 2011). Dendrite segments on 205

secondary branches used for spine quantification generally started at the first branch point, and 206

ended when the dendrite ended, branched again, moved out of the section, or was obscured by 207

other neurites or noise. Spines were then counted manually in ImageJ, with the experimenter 208

blind to genotype and experimental paradigm. For each animal, 2-8 cells were counted. 209

210

Sholl analysis: The arbors of the same neurons used for spine counting were reconstructed 211

manually using the Simple Neurite Tracer plugin in ImageJ (Longair et al., 2011), blind to 212

genotype. Sholl analysis was then performed on the traced apical and basal arbors using the Sholl 213

analysis plugin in ImageJ (Ferreira et al., 2014). 214

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215

Electrophysiological recordings in primary visual cortex: Recording methods were adapted 216

from previously published methods (Stephany et al., 2014; Stephany et al., 2016; Stephany et al., 217

2018). Mice that were either normally reared, or had undergone four days of MD, were 218

anesthetized with isoflurane (2%) and placed in a stereotaxic frame where body temperature was 219

maintained at 37°C using a homeostatically-regulated heat probe (TCAT-2LV, Physitemp). 220

Dexamethasone (4mg/kg; West-Ward Pharmaceuticals Corp.) was administered subcutaneously 221

to reduce cerebral edema. The eyes were flushed with saline, covered with a thin layer of silicone 222

oil (Sigma, cat # 378429), and then covered for the duration of craniotomy. The scalp was 223

resected and a titanium head bar was secured to the skull with cyanoacrylate glue and dental 224

cement (Ortho-Jet Liquid and Ortho-Jet Powder; Lang Dental Mfg. Co., Inc.). A small 225

craniotomy (2mm in diameter) was made over V1, and a small silver grounding wire (A-M 226

Systems, Inc.; cat # 782500) was inserted through a burr hole that was made over the 227

contralateral cerebellum. Then, a dose of chlorprothixene (0.5 mg/kg; s.c.; Sigma, cat #1671) 228

was administered prior to transferring the mouse to the recording set up. 229

Recordings were made with silicone multi-site electrodes (A1x16-5mm-50-177-A16, 230

Neuronexus Technologies, Inc.) inserted into V1 using a motorized drive (MP-225, Sutter 231

Instruments). Once the electrode was in place, warm agarose (2.5% in ACSF) was placed over 232

the craniotomy to protect the cortex and to reduce mechanical noise. The electrical signal was 233

sampled at 25kHz on a RZ5P Workstation (Tucker-Davis Technologies, Inc.), and filtered 234

between 300-5000Hz for spiking activity. Multi-unit activity was recorded on all 16 channels 235

(separated by 50 m) starting 100 m below the pial surface. In each mouse, there were 3-4 236

penetrations separated by at least 200 m across the binocular zone of V1. Subsequently, noise 237

10

artifacts were removed and single units were isolated from the multi-unit activity at each contact 238

site using Offline Sorter (Plexon). 239

Animals were immobilized using the titanium head bar at a distance of 25cm from a 240

video monitor (ASUS VG248QE 24" LED Backlit LCD Monitor) with a refresh rate of 144Hz, 241

and a mean luminance of 40cd/cm2. Visual responses were driven by custom stimuli generated in 242

Matlab Pyschophysics Toolbox. First, a sparse noise stimulus was presented to both eyes at the 243

beginning of each penetration, and the resulting activity map was used to determine receptive 244

field location. Full luminance (ON) and minimum luminance (OFF) squares covering 4° of 245

visual space were flashed (350ms) one at a time on a grey background in a pseudorandom order 246

along a 16x10 grid subtending 64° of visual space in azimuth, and 40° of visual space in 247

elevation. Activity from sites outside of the binocular zone, as defined by a receptive field >25° 248

in azimuth, were excluded from subsequent analysis. Then, responses were driven by sinusoidal 249

gratings (0.02cpd, 100% contrast, 2Hz temporal frequency) drifting in 12 directions separated by 250

30° for 2s ON followed by 2s of a luminance-matched grey screen. Each stimulus condition was 251

repeated at least 10 times in a pseudorandom order, interleaved by a grey screen to measure 252

spontaneous activity. Action potentials (APs) were identified online in recorded traces with 253

Synapse Software (Tucker Davis Technologies). Only waveforms extending beyond 4 standard 254

deviations above the average noise were included in subsequent analysis. For each contact site, 255

the number of APs in response to the grating stimuli was summed and averaged over the number 256

of presentations. If the average number of APs for the grating stimuli was not greater than 50% 257

above the blank, the contact site was discarded. 258

Single unit activity was used to calculate the average spontaneous and visually evoked 259

activity. The spontaneous activity was calculated from the average number of APs elicited by the 260

11

blank, while the visually evoked activity was calculated by subtracting the spontaneous activity 261

of that unit from the average number of APs elicited by the grating in the unit’s preferred 262

orientation. 263

The ocular dominance index (ODI) was calculated for the multi-unit activity on each 264

contact site within the binocular zone by comparing the number of APs elicited in a given unit 265

when showing the same visual stimulus to each eye independently. The order of presentation 266

(contra then ipsi vs ipsi then contra) was alternated between penetrations for each animal. Units 267

were assigned to one of seven ocular dominance (OD) categories (1-7) where units assigned to 268

category 1 are largely dominated by input from the contralateral eye, and units assigned to 269

category 7 are largely dominated by input from the ipsilateral eye (Wiesel and Hubel, 1963). To 270

categorize each contact-site, the average number of APs elicited by the blank (spontaneous 271

activity) was subtracted from the average number of APs elicited by the gratings (evoked 272

activity) for the contralateral eye (CE) and the ipsilateral eye (IE), across all grating orientations. 273

Next, the ocular dominance index (ODI), given by ODI = (IE - CE)/(IE + CE) was calculated for 274

each contact-site and assigned to OD categories 1-7 as follows: -1 to -0.6 = 1, -0.6 to -0.4 = 2, -275

0.4 to -0.1 = 3, -0.1 to 0.1 = 4, 0.1 to 0.4 = 5, 0.4 to 0.6 = 6, 0.6 to 1 = 7. Finally, the sum of the 276

number of cells in each category was used to calculate the CBI for each animal with the formula: 277

CBI = [(n1 –n7) + (2/3)(n2 – n6) + (1/3)(n3 – n5) + N]/2N where N is the total number of 278

contact sites and nx is the number of contact sites with OD scores equal to x (Gordon and 279

Stryker, 1996). 280

281

Measuring binocular zone expansion: Ocular dominance plasticity was assayed by measuring 282

binocular zone expansion similar to previous publications (Tagawa et al., 2005; Bochner et al., 283

12

2014). For MD experiments, animals underwent MD starting at P25-28. After four days of MD, 284

when the mice were P29-32, the suture was opened and the deprived eye enucleated. Age-285

matched littermates (NR in the Results section) that had not undergone MD were also 286

monocularly enucleated at this time (i.e. at age P29-32). The enucleated mice were placed in the 287

dark overnight (16 hours), then exposed to bright lights for 30 min and finally decapitated under 288

isoflurane anesthesia. Both MD and NR conditions thus received strong stimulation of the 289

remaining eye prior to sacrifice, thereby making it possible to visualize the binocular region in 290

the hemisphere ipsilateral to the remaining eye, but only the MD mice were given time to adapt 291

to the change in visual input. 292

Brains were flash frozen on liquid nitrogen and embedded in OCT (Sakura Finetek, cat # 293

4583). The brains were sectioned coronally at 16 m on a cryostat onto X-tra slides (Leica 294

Microsystems, cat # 3800200). Arc in situ hybridization was then performed using RNAscope 295

Fluorescent Multiplex kits (ACD, cat # 320850) with the probe against mouse Arc in color 1 296

(ACD, cat # 316911), using alt B in the Amp 4 amplification step. The in situ hybridization was 297

performed according to manufacturer’s instructions, except for the protease treatment being 298

shortened to 3-10 minutes (shorter times for lower quality tissue). 299

The hemisphere ipsilateral to the remaining eye (contralateral to the deprived eye) was 300

imaged on a TiEclipse microscope (Nikon). The binocular zone of V1 was imaged from Bregma 301

-3.15 to -3.87. Sections without signal in L2/3 of V1b were discarded. The length of the Arc 302

positive zone in L4 and L2/3 was calculated in ImageJ (NIH) as the full width at half maximum 303

(FWHM), as has been done previously (William et al., 2017). To measure the FWHM, the half-304

maximum was first determined. The maximum intensity was defined as the average intensity in a 305

121.5 x 121.5 m square placed in the center of V1b of the layer analyzed, while the minimum 306

13

was defined as the average intensity in a same-sized square placed in the monocular section of 307

V1 (V1m) of the layer analyzed. The half-maximum was calculated as the value halfway 308

between these two intensities. The Arc intensity was then measured across V1 in the following 309

manner. A polygon was drawn around the layer of interest in V1b and surrounding areas. The 310

Arc signal was filtered inside this polygon using a mean filter with radius 50 pixels (=81 m). A 311

line of width 50 pixels (=81 m) was drawn through the filtered region, parallel to the cortical 312

layer and the surface of the brain. The intensity profile along the line was plotted, and the 313

FWHM, and thus the length of the binocular zone, was defined as the length for which the Arc 314

intensity was above the half-maximum. If there were multiple peaks above the half-maximum, 315

the original image was examined to determine whether the binocular zone spanned the peaks or 316

consisted of only one of them. V1b Arc fluorescence intensity was quantified by dividing the 317

maximum intensity by the minimum intensity, using the same measurements as the ones used to 318

determine the FWHM. Analysis was performed blind to genotype and experimental condition. 319

For ME experiments, mice were monocularly enucleated at P28. After four days, at P32, 320

the ME mice were placed in the dark overnight (16 hours). Age-matched littermates (NR in the 321

Results section) that had not previously undergone enucleation were monocularly enucleated at 322

this time and also placed in the dark overnight. The next morning, at P33, mice were then light 323

exposed, and tissue collected and sectioned, as described above for the MD animals. For the ME 324

group, Arc transcript was detected using colorimetric in situ hybridization, similar to previous 325

studies (Lein and Shatz, 2000; William et al., 2012). The full-length mouse Arc probe was 326

generously shared with us by C. Shatz (Stanford University), who had received it from P. 327

Whorley (Lyford et al., 1995). DNA was digested with NotI, transcribed from the T7 promoter, 328

and labeled with digoxigenin using a kit (Sigma, cat # 11175025910). Sections were allowed to 329

14

thaw at room temperature immediately before use, then fixed for 10 min in 4% PFA (Electron 330

Microscopy Sciences, cat # 15710) in PBS. Sections were permeabilized with Proteinase K for 5 331

minutes, fixed for another 5 min in 4% PFA (Electron Microscopy Sciences, cat # 15710), and 332

then acetylated for 10 minutes. Sections were blocked with yeast RNA for 1hr, then incubated 333

overnight 72 °C with an anti-sense digoxigenin-labeled Arc probe under coverslips. Coverslips 334

were removed in 5X SSC. Slides were then washed in 0.2X SSC and blocked with 10% normal 335

goat serum (Sigma, cat # G9023-10ML) at room temperature. Slides were incubated overnight at 336

4°C in sheep anti-digoxigenin-AP (1:3000, Fab fragments, Sigma, cat # 11093274910) in 1% 337

normal goat serum (Sigma, cat # G9023-10ML). Finally, the signal was developed using 338

NBT/BCIP solution (Sigma, cat # 11681451001). 339

The hemisphere ipsilateral to the remaining eye was imaged under brightfield on a 340

TiEclipse microscope (Nikon). Images were inverted and then analyzed as described above. 341

342

Statistics/experimental design: Both males and females were used for all experiments. Data 343

analysis was performed in Graphpad Prism 8 (La Jolla), except for the already mentioned 344

analyses performed using Matlab. More information about statistical design can be found in the 345

Results section and in the figure legends. α=0.05 unless otherwise stated. 346

347

Results 348

C1q is present in juvenile V1b 349

15

C1q is a large complex, consisting of peptides encoded by the three genes C1qa, C1qb, and 350

C1qc. All three genes are highly expressed by microglia and macrophages across the brain, and 351

at negligible levels by other cell types (Fonseca et al., 2017; Saunders et al., 2018; Zeisel et al., 352

2018; Hammond et al., 2019). We therefore expected C1q protein to be present also in V1b. We 353

performed immunohistochemistry using an antibody raised against the entire C1q complex 354

(Stephan et al., 2013), and examined the staining patterns at P10, P20 and P32; the latter two 355

ages approximately mark the beginning and the end of the critical period for ODP in the mouse 356

(Gordon and Stryker, 1996). At low magnification, C1q was present across cortical layers at all 357

three ages (Figure 1A). Since ODP has primarily been studied in L2/3 and in L4, we used 358

confocal imaging to specifically examine these layers. Loss of the C1qa gene is sufficient to 359

disrupt the protein complex and abolish classical complement cascade activation (Botto et al., 360

1998), and immunoreactivity of the C1q antibody has previously been shown to be absent in 361

C1qa-/- animals (Stephan et al., 2013). We therefore used these mice as negative controls for the 362

immunohistochemistry. 363

At all three ages, C1q protein was present above C1qa-/- levels in both L2/3 and L4 364

(Figure 1A-C). The amount of C1q was low at P10, but the immunofluorescence intensities 365

increased with age, including during the critical period (Figure 1D-E). This finding applied both 366

to L2/3 and L4 (one-way ANOVA with Tukey’s multiple comparisons test. L2/3: F(2, 9)=17.04, 367

p=0.0009, P10 vs P32, p=0.0007, P20 vs P32, p=0.0123; L4: F(2, 9)=12.12, p=0.0028, P10 vs 368

P32, p=0.0023, P20 vs P32, p=0.0297). Thus, C1q protein is present in the developing V1b, with 369

overall C1q levels increasing with age. 370

Many previous studies have found that C1q localizes to subsets of synapses (Stevens et 371

al., 2007; Stephan et al., 2013; Hong et al., 2016; Lui et al., 2016; Dejanovic et al., 2018). We 372

16

confirmed these observations in L2/3 of V1b at P29-32, by co-staining for C1q and spinophilin, a 373

protein enriched at spines (Allen et al., 1997). Using confocal imaging, we found that a subset of 374

spinophilin puncta overlapped with, or were closely apposed to, C1q puncta (Figure 1F, left). To 375

confirm that this colocalization was not solely due to chance, we rotated the C1q channel by 90°, 376

and compared the number of colocalized C1q and spinophilin puncta when the C1q channel was 377

in its original configuration to when it was rotated. We found that the number of colocalized 378

puncta went down slightly but significantly when the C1q channel was rotated (original number 379

of puncta per field of view: 2152±116.7, rotated number of puncta per field of view: 380

2007±112.5, error represents SEM; paired two-tailed t-test, p=6.2x10-7, n=30 sections from 3 381

mice), suggesting that C1q is mildly enriched at spinophilin puncta. We furthermore used super-382

resolution SIM imaging to examine the C1q and spinophilin staining patterns, and identified 383

examples of colocalization also at this higher resolution (Figure 1F, right). These results suggest 384

that C1q puncta are present near dendritic spines in V1b, and thus could bind to synaptic 385

structures to promote their elimination by microglia. 386

387

C1q is not required for the development of a spine population implicated in ODP 388

All previous studies on the role of C1q in nervous system development have been performed in 389

mice lacking C1qa. One phenotype described in C1qa-/- mice is increased spine densities in L5 of 390

somatosensory cortex at P23 (Ma et al., 2013). Thus, we next asked if loss of C1qa impacts spine 391

development in V1b. MD during the critical period induces spine loss on the apical dendrites of 392

V1b L2/3 pyramidal neurons, which is most pronounced in the 25 m segment distal to the first 393

branch point of the apical dendrite (Mataga et al., 2004). We therefore limited our analysis to this 394

specific segment, and counted spines in wild-type (WT) and C1qa-/- mice at P10, P20 and P29-395

17

30. We labeled neurons using Golgi-Cox staining, and used brightfield microscopy to image 396

pyramidal neurons in L2/3 of V1b (Figure 2A-C). In z-stacks from P10, P20 and P29-30 animals, 397

we counted the number of spines in the 25 m segment following the first branch point. At all 398

three ages, the spine numbers in the C1qa-/- mice were indistinguishable from their WT 399

littermates (Figure 2D). This finding applied both when spine numbers were analyzed per 400

animal, and when they were analyzed per cell (unpaired two-tailed t-test. Per mouse: P10: 401

t(7)=0.7854, p=0.4580; P20: t(8)=1.665, p=0.1345; P29-30: t(10)=1.54, p=0.1546; Per cell: P10: 402

t(41)=0.2109, p=0.8340; P20: t(52)=1.504, p=0.1386; P29-30: t(46)=1.511, p=0.1377; Figure 403

2E-F). 404

Spine densities on apical dendrites of L2/3 pyramidal neurons increase with distance 405

from the soma (Mataga et al., 2004). Indeed, we found that the number of spines on a dendrite 406

segment was significantly correlated with the distance between the soma and the first branch 407

point at the two older ages (P10 WT: rs=0.2616, p=0.3081, P10 C1qa-/-: rs=0.5366, p=0.0047, 408

P20 WT: rs=0.6199, p=0.0003, P20 C1qa-/-: rs=0.5634, p=0.0041, P29-30 WT: rs=0.7321, 409

p=0.0001, P29-30 C1qa-/-: rs=0.7085, p<0.0001; Figure 3A). A difference in the position of the 410

first branch point between the genotypes could thus obscure any absolute changes in spine 411

numbers between WT and C1qa-/- mice. We therefore confirmed that the position of the first 412

branch point was unchanged in the C1qa-/- animals, by measuring the distance between the soma 413

and the first branch point in all cells used for spine counting. For all three ages, the average 414

distance in the C1qa-/- animals was the same as in the WT mice (unpaired two-tailed t-test. P10: 415

t(7)=0.2727, p=0.7929; P20: t(8)=0.5198, p=0.6173; P29-30: t(10)=0.3156, p=0.7588; Figure 416

3B). Together, these results suggest that the number of spines in this apical dendrite segment 417

develops normally in C1qa-/- mice. 418

18

419

C1q is transiently required for the development of spines on apical, but not basal, secondary 420

dendrites 421

The proximal spines on the apical dendrites of L2/3 pyramidal neurons constitute only one out of 422

many spine populations in V1b. To determine whether other spine populations were also 423

unaffected in mice lacking C1q, we therefore examined spine densities on secondary apical and 424

basal dendrites of V1b L2/3 pyramidal neurons. We again used Golgi-Cox staining to visualize 425

the neurons. 426

We first examined spine densities on apical dendrite secondary branches at P10, P20 and 427

P29-30 (Figure 4A). Surprisingly, these did not behave as the primary apical dendrite segments 428

analyzed in Figure 2. While the C1qa-/- mice showed normal spine densities at both P10 and P29-429

30, the densities were increased in the C1qa-/- animals at P20 when the data was quantified per 430

mouse (unpaired two-tailed t-test. P10: t(7)=0.04222, p=0.9675; P20: t(8)=2.371, p=0.0452; 431

P29-30: t(8)=0.2614, p=0.8004; Figure 4B). The results were the same when data was quantified 432

per cell (unpaired two-tailed t-test. P10: t(40)=0.4146, p=0.6807; P20: t(47)=2.251, p=0.0291; 433

P29-30: t(32)=0.8172, p=0.4199; Figure 4C). In contrast, the spine densities on the basal 434

secondary dendrites did not change at any of the ages (Figure 4D). This finding applied both 435

when densities were quantified per mouse (unpaired two-tailed t-test. P10: t(7)=0.6858, 436

p=0.5149; P20: t(8)=0.5260, p=0.6132; P29-30: t(9)=0.5023, p=0.6275; Figure 4E), as well as 437

per cell (unpaired two-tailed t-test. P10: t(41)=0.7537, p=0.4553; P20: t(47)=1.104, p=0.2750; 438

P29-30: t(42)=0.4044, p=0.6879; Figure 4F). These results thus suggest that loss of C1qa can 439

cause increased spine numbers, but only in certain spine populations and at certain times. 440

19

However, before the critical period ends, all spine populations in C1qa-/- mice examined here are 441

indistinguishable from those of their WT littermates. 442

443

Dendritic morphology is unaffected by loss of C1q 444

An increase in the number or length of dendrites could cause elevated overall numbers of 445

synapses, even without a dramatic change in spine densities on each neuron. Indeed, increased 446

dendrite branching and length has been reported on L5 pyramidal neurons in sensorimotor cortex 447

of C1qa-/- mice (Ma et al., 2013). We therefore traced the apical and basal dendritic arbors of 448

Golgi-Cox stained neurons in L2/3 of V1b, and performed Sholl analysis on the tracings (Figure 449

5A). When we calculated the number of dendrite intersections with concentric shells placed 10, 450

30, 50, 70, 90, and 110 m away from the soma, we found no difference between the genotypes 451

at any of the ages, either in the apical or the basal arbor (apical: unpaired two-tailed t-test, Holm–452

Sidak's method with α=0.05 was used to correct for multiple comparisons. P10: adjusted p-453

value=0.8744 (10μm), 0.9343 (30μm), 0.9683 (50μm), 0.9508 (70μm), 0.9508 (90μm), 0.9683 454

(110μm). P20: adjusted p-value=0.8869 (10μm), 0.8869 (30μm), 0.8869 (50μm), 0.4866 (70μm), 455

0.8869 (90μm), 0.8869 (110μm). P29-30: adjusted p-value=0.8618 (10μm), 0.7560 (30μm), 456

0.8618 (50μm), 0.7560 (70μm), 0.6894 (90μm), 0.7560 (110μm); basal: unpaired two-tailed t-457

test, Holm–Sidak's method with α=0.05 was used to correct for multiple comparisons. P10: 458

adjusted p-value=0.6602 (10μm), 0.6602 (30μm), 0.8774 (50μm), 0.6602 (70μm), 0.6172 459

(90μm), NA (110μm; all values are 0). P20: adjusted p-value=0.9969 (10μm), 0.9969 (30μm), 460

0.9969 (50μm), 0.9969 (70μm), 0.9969 (90μm), 0.9969 (110μm). P29-30: adjusted p-461

value=0.9720 (10μm), 0.9720 (30μm), 0.6167 (50μm), 0.5629 (70μm), 0.3536 (90μm), 0.3119 462

20

(110μm); Figure 5B-D). Thus, the morphologies of V1b L2/3 pyramidal neuron apical and basal 463

dendritic arbors are not perturbed in C1qa-/- mice. 464

465

Firing rates in V1b of C1qa-/- mice are normal 466

Although we found mostly normal spine numbers and dendritic morphology in developing C1qa-467

/- mice, visual processing in these animals may still be different from that of WT mice, since it is 468

possible that other synapse populations in V1b are affected by loss of C1qa. To assess the 469

general properties of visual responsiveness in animals lacking C1qa, we performed in vivo 470

extracellular electrophysiological recordings in P28-31 WT and C1qa-/- littermates. Neuronal 471

activity was recorded across cortical layers in V1b of anesthetized mice, using multi-site silicone 472

probes. Units with receptive fields outside of the binocular zone (further than 25° from the center 473

of the visual field) were excluded from further analysis (see Materials and Methods). 474

Using these recordings, we examined how loss of C1qa impacts firing rates of single 475

units in V1b. Visual activity was elicited using sinusoidal gratings drifting in twelve different 476

directions interleaved by a grey screen, presented to each eye independently. Each stimulus was 477

presented a minimum of seven times (Figure 6A). The spontaneous activity was measured as the 478

average firing rate in response to the grey screen, while the evoked activity was the average 479

firing rate in response to the direction that elicited the most activity. The average spontaneous 480

activity was the same when compared between eyes and genotypes (one-way ANOVA, F(3, 481

181)=0.08636, p=0.9674; Figure 6B). Similarly, although the evoked firing rate was higher for 482

the contralateral eye, reflecting normal contralateral bias (Gordon and Stryker, 1996), there was 483

no difference in the evoked firing rate between genotypes (one-way ANOVA with Sidak’s 484

21

multiple comparisons test, F(3, 181)=4.788, p=0.0031, WT ipsi vs WT contra, p=0.0428, C1qa-/- 485

ipsi vs C1qa-/- contra, p=0.0424, WT ipsi vs C1qa-/- ipsi, p=0.9413, WT contra vs C1qa-/- contra, 486

p=0.9165; Figure 6C). Thus, C1qa does not influence baseline firing rates in V1b. 487

488

C1q protein levels in V1b are not experience-dependent 489

Our results suggest that several features of V1b development proceed normally in the absence of 490

C1qa. However, it is possible that the initial development of V1b is independent of C1q, but that 491

C1q instead is required for the synaptic remodeling that occurs in response to visual deprivation. 492

We therefore investigated whether C1q contributes to ODP. We first examined how MD impacts 493

C1q levels in L2/3 and L4 of V1b. An increase could suggest that C1q is upregulated in order to 494

promote synapse elimination during ODP. We evaluated C1q levels in normally reared P32 mice 495

(NR), as well as in mice that had undergone two or four days of MD starting at P28 (2D MD and 496

4D MD, respectively). ODP is complete after four days of MD (Gordon and Stryker, 1996); two 497

days was chosen as an intermediate timepoint during which remodeling is still in progress. As in 498

Figure 1, we measured C1q levels by quantifying immunofluorescence intensities (Figure 7A). 499

We found that MD did not impact C1q levels in either layer (one-way ANOVA. L2/3: F(2, 500

15)=0.1803, p=0.8368; L4: F(2, 15)=0.2338, p=0.7943; Figure 7B-C). Immunofluorescence 501

intensities are a fairly crude measurement of protein levels, so we cannot conclude that MD has 502

no effect on the amounts of C1q in V1b. However, MD does not appear to drive dramatic 503

changes in the levels of C1q. 504

505

MD does not reduce spine numbers on L2/3 pyramidal neurons 506

22

C1q could contribute to synapse elimination following MD even if C1q levels are unchanged. 507

Subtle changes in C1q localization to synapses or local activation of the classical complement 508

cascade may be sufficient to promote increased synapse engulfment. Alternatively, reduced 509

levels of signals that inhibit synaptic engulfment, such as CD47, could allow increased C1q-510

dependent synapse phagocytosis to occur (Lehrman et al., 2018). We therefore investigated 511

whether the absence of C1qa prevents MD-induced spine loss on L2/3 pyramidal neuron apical 512

dendrites (Mataga et al., 2004). We executed the experiment as previously described (Mataga et 513

al., 2004). However, one notable difference was that we used Golgi-Cox staining to label the 514

neurons, while the previous study used diolistic labeling. We performed MD on WT and C1qa-/- 515

animals at P25-26 (in the middle of the critical period) and collected brains four days later. The 516

controls consisted of NR WT and C1qa-/- mice aged P29-30. For all four groups, we counted 517

spines in the 25 m segment following the first branch point, on the apical dendrites of L2/3 518

pyramidal neurons. The NR data points were already included in Figure 2, but were collected 519

together with the MD cohort and analyzed blind to both genotype and experimental condition; as 520

described above, there was no difference between the genotypes in the number of spines for the 521

NR group. When we compared the WT NR and MD conditions, we surprisingly also found no 522

change in spine numbers (two-way ANOVA with Tukey’s multiple comparisons test, F(1, 523

21)=3.034 and p=0.0962 for MD, F(1, 21)=0.344 and p=0.5638 for genotype, and F(1, 524

21)=2.906 and p=0.1030 for interaction; WT NR vs WT MD, p>0.9999; Figure 8A-B). This 525

finding, in stark contrast to the previously reported 40% reduction (Mataga et al., 2004), held 526

true also when spine numbers were analyzed per cell (two-way ANOVA with Tukey’s multiple 527

comparisons test, F(1, 110)=3.543 and p=0.0625 for MD, F(1, 110)=0.2824 and p=0.5962 for 528

genotype, and F(1, 110)=2.695 and p=0.1035 for interaction; WT NR vs WT MD, p=0.9986; 529

23

Figure 8C). In addition, the C1qa-/- mice did not show a spine reduction following MD, and 530

instead displayed a trend towards an increase in spine numbers in the MD condition compared to 531

NR, when spine number were analyzed per mouse (C1qa-/- NR vs C1qa-/- MD, p=0.0920). When 532

we quantified spine numbers per cell, this trend became statistically significant (C1qa-/- NR vs 533

C1qa-/- MD, p=0.0411); however, since MD, genotype and their interaction all lacked 534

statistically significant effects on spine numbers, it is not clear whether the difference between 535

C1qa-/- NR and C1qa-/- MD is relevant. As above, we quantified the distance between the soma 536

and first branch point for all cells used for spine counting, and they were the same across all four 537

conditions (two-way ANOVA, F(1, 21)=0.02282 and p=0.8814 for MD, F(1, 21)=0.2156 and 538

p=0.6472 for genotype, and F(1, 21)=0.0069 and p=0.9346 for interaction; Figure 8D). Our 539

observations therefore suggest that MD has no strong effect on spine numbers on this particular 540

part of L2/3 apical dendrites in WT or C1qa-/- animals. 541

542

C1qa is not required for binocular zone expansion following MD and monocular enucleation 543

Our failure to detect a spine loss following MD in either genotype makes this experiment 544

difficult to interpret. We therefore proceeded to determine whether ODP can occur in the 545

absence of C1q, using two distinct techniques. We first measured how loss of C1qa impacts 546

ODP using in situ hybridization against the immediate early gene Arc (Tagawa et al., 2005). 547

Acute light stimulation results in Arc labeling of V1b. During the critical period, four days of 548

monocular enucleation (ME) or MD of the contralateral eye, prior to light stimulation, causes a 549

widening of the binocular Arc zone (William et al., 2012; Bochner et al., 2014). This observation 550

suggests that Arc in situs can be used as a proxy to measure ODP. Indeed, both enhanced and 551

reduced ODP detected using this method has been confirmed using alternative techniques (Syken 552

24

et al., 2006; Kanold et al., 2009; William et al., 2012; Djurisic et al., 2013; Bochner et al., 2014; 553

William et al., 2017). We therefore used the Arc in situ method to examine ODP in mice lacking 554

C1qa. 555

We performed MD on WT and C1qa-/- mice during the critical period, with MD starting 556

at P25-28. After four days of MD, the animals were light stimulated and their brains collected. 557

We then performed RNAscope fluorescence in situ hybridization against Arc, and quantified the 558

length of the binocular zone in L2/3 and L4 by measuring the full width at half maximum 559

(FWHM) (William et al., 2017) (Figure 9A). The MD group was compared to age-matched NR 560

control mice that had undergone the same light stimulation paradigm as the MD animals. In both 561

layers, MD induced a robust expansion of the binocular zone in WT as well as in C1qa-/- animals 562

(two-way ANOVA with Tukey’s multiple comparisons test. L2/3: F(1, 21)=35.19 and p<0.0001 563

for MD, F(1, 21)=2.717 and p=0.1142 for genotype, and F(1, 21)=0.02172 and p=0.8843 for 564

interaction, WT NR vs WT MD, p=0.0014, C1qa-/- NR vs C1qa-/- MD, p=0.0032; L4: F(1, 565

21)=69.53 and p<0.0001 for MD, F(1, 21)=0.9028 and p=0.3528 for genotype, and F(1, 566

21)=1.029, and p=0.3219 for interaction, WT NR vs WT MD, p=0.0002, C1qa-/- NR vs C1qa-/- 567

MD, p<0.0001; Figure 9B-C). Furthermore, the C1qa-/- binocular zone lengths were not different 568

from those of WT animals in either the NR or the MD condition (L2/3: WT NR vs C1qa-/- NR, 569

p=0.6052, WT MD vs C1qa-/- MD, p=0.7042; L4: WT NR vs C1qa-/- NR, p>0.9999, WT MD vs 570

C1qa-/- MD, p=0.5039). These results suggest that the binocular zone is not grossly abnormal in 571

C1qa-/- mice under baseline conditions, and that in the absence of C1qa the binocular zone 572

expands normally following MD. 573

When examining the Arc in situ images, we noticed that the fluorescence intensity of the 574

Arc signal in the binocular zone appeared brighter in the MD than in the NR animals. This 575

25

observation is consistent with an increase in open-eye responsiveness at the center of V1b 576

following MD (Tagawa et al., 2005). We therefore quantified the Arc fluorescence intensity in 577

the center of V1b, normalizing the signal to the fluorescence intensity in the monocular section 578

of V1 (V1m) to correct for variability in the background signal. In both L2/3 and L4, the 579

quantification of the Arc fluorescence intensity was consistent with the quantification of the 580

binocular zone length. MD led to an increase in Arc signal in both genotypes compared to the 581

NR animals (two-way ANOVA with Tukey’s multiple comparisons test. L2/3: F(1, 21)=82.53 582

and p<0.0001 for MD, F(1, 21)=0.02663 and p=0.8719 for genotype, and F(1, 21)=0.2541 and 583

p=0.6195 for interaction, WT NR vs WT MD, p<0.0001, C1qa-/- NR vs C1qa-/- MD, p<0.0001; 584

L4: F(1, 21)=55.72 and p<0.0001 for MD, F(1, 21)=0.3273 and p=0.5733 for genotype, and F(1, 585

21)=0.3465, and p=0.5624 for interaction, WT NR vs WT MD, p<0.0001, C1qa-/- NR vs C1qa-/- 586

MD, p=0.0006; Figure 9E-F). Additionally, in both NR and MD animals there was no significant 587

difference between genotypes (L2/3: WT NR vs C1qa-/- NR, p=0.9952, WT MD vs C1qa-/- MD, 588

p=0.9625; L4: WT NR vs C1qa-/- NR, p>0.9999, WT MD vs C1qa-/- MD, p=0.8366). Thus, ODP 589

in C1qa-/- animals is normal using two methods for quantifying the visually induced Arc signal. 590

To confirm these observations with an even more dramatic visual manipulation, we 591

measured binocular zone expansion following ME in WT and C1qa-/- animals (Bochner et al., 592

2014). In this experiment, we enucleated, rather than sutured shut, the contralateral eye at P28. 593

After four days of ME, the mice were light stimulated and their brains collected. Age-matched 594

NR animals were also light stimulated prior to tissue collection. We detected Arc transcript using 595

standard colorimetric, rather than RNAscope, in situ hybridization. When we examined the 596

length of the binocular zone in L4 by measuring the FWHM, we again observed a robust 597

expansion in both genotypes, and no difference between WT and C1qa-/- animals in either 598

26

condition (two-way ANOVA with Tukey’s multiple comparisons test, F(1, 16)=29.77 and 599

p<0.0001 for ME, F(1, 16)=0.5278 and p=0.4780 for genotype, and F(1, 16)=0.8672 and 600

p=0.3656 for interaction, WT NR vs C1qa-/- NR, p=0.6521, WT NR vs WT ME, p=0.0018, 601

C1qa-/- NR vs C1qa-/- ME, p=0.0258, WT ME vs C1qa-/- ME, p=0.9989; Figure 9D). We also 602

measured the signal intensity of the Arc signal in the four conditions, but did not see any 603

differences across the groups (two-way ANOVA with Tukey’s multiple comparisons test, F(1, 604

16)=0.9614 and p=0.3414 for ME, F(1, 16)=0.6366 and p=0.4366 for genotype, and F(1, 605

16)=0.4253 and p=0.5236 for interaction, WT NR vs C1qa-/- NR, p=0.9996, WT NR vs WT ME, 606

p=0.6626, C1qa-/- NR vs C1qa-/- ME, p=0.9954, WT ME vs C1qa-/- ME, p=0.7374; Figure 9G). 607

This contrast to the RNAscope results is consistent with RNAscope providing a highly sensitive 608

and quantitative measurement of transcripts levels (Wang et al., 2012; Wang et al., 2013). 609

Together, these experiments thus suggest that critical period ODP is normal C1qa-/- mice. 610

611

In vivo electrophysiology reveals normal ODP in C1qa-/- mice 612

The Arc in situ method is an indirect measurement of ocular dominance. Therefore, we used in 613

vivo extracellular recordings to measure ODP directly from neuronal activity in WT and C1qa-/- 614

mice. We recorded from P28-31 WT and C1qa-/- animals that had either been reared normally or 615

undergone four days of MD, and evaluated the eye dominance from the multi-unit activity on all 616

visually responsive contact sites. To calculate the relative contribution of each eye to the firing 617

rate measured at each contact site along the silicone probe, we averaged the response across all 618

directions of the sinusoidal grating stimulus for the contralateral and ipsilateral eye, 619

independently (see Materials and Methods). In the NR mice, we found no difference in eye-620

dominance between WT and C1qa-/- littermates (Figures 10A-C). Both genotypes had a normal 621

27

contralateral bias shown by ODI histograms with high proportions of units in the lower (contra-622

dominated) OD categories (chi-squared test, chi-square (5)=7.502, p=0.1859, α=0.0125 to 623

account for multiple comparisons; Figure 10A), similar ODI distributions (Kolmogorov-Smirnov 624

test, D=0.1272, p=0.0646, α=0.0125 to account for multiple comparisons; Figure 10B), and 625

average CBIs around 0.65 (two-way ANOVA with Tukey’s multiple comparisons test, F(1, 626

23)=32.57 and p<0.0001 for MD, F(1, 23)=0.06254 and p=0.8047 for genotype, and F(1, 627

23)=0.3021 and p=0.5879 for interaction, WT NR vs C1qa-/- NR, p=0.9491; Figure 10C). C1qa-/- 628

animals thus have normal ocular dominance under baseline conditions. 629

Next we examined ODP in WT and C1qa-/- mice following four days of MD during the 630

critical period starting at P25-26. We found that the WT MD mice had robust OD shifts 631

compared to the WT NR controls (OD categories: chi-squared test, chi-square (6)=114.5, 632

p<0.0001, α=0.0125 to account for multiple comparisons; ODI distributions: Kolmogorov-633

Smirnov test, D=0.4620, p<0.0001, α=0.0125 to account for multiple comparisons; CBI: WT NR 634

vs WT MD, p=0.0009). Among the C1qa-/- animals, the results were less clear: the C1qa-/- MD 635

animals showed a significant OD shift compared to the C1qa-/- NR group by OD categories and 636

ODI distribution, but not by CBI (OD categories: chi-squared test, chi-square (6)=39.74, 637

p<0.0001; ODI distributions: Kolmogorov-Smirnov test, D=0.2450, p<0.0001; CBI: C1qa-/- NR 638

vs C1qa-/- MD, p=0.0860; data not shown). Additionally, the C1qa-/- MD mice were also 639

significantly different from the WT MD animals by OD categories and ODI distribution, but 640

again not by CBI (OD categories: chi-squared test, chi-square (6)=26.14, p=0.0002; ODI 641

distributions: Kolmogorov-Smirnov test, D=0.1929, p<0.0001; CBI: WT MD vs C1qa-/- MD, 642

p=0.7962; data not shown). These results could indicate that while the C1qa-/- animals undergo 643

an OD shift in response to MD, they do not shift to the same degree as WT mice. 644

28

However, we noticed that one C1qa-/- MD animal (marked in grey in Figure 10C) had an 645

unusually high CBI value of 0.8158, which was almost two standard deviations above the mean 646

of the C1qa-/- MD group, as well as of the WT and C1qa-/- NR groups. Since we were concerned 647

that this one extreme animal could be skewing our results, we examined the dataset without this 648

mouse. Among the remaining seven C1qa-/- MD animals, OD was significantly different from 649

that of the C1qa-/- NR mice (OD categories: chi-squared test, chi-square (6)=52.64, p<0.0001, 650

α=0.0125 to account for multiple comparisons; ODI distributions: Kolmogorov-Smirnov test, 651

D=0.2994, p<0.0001, α=0.0125 to account for multiple comparisons; CBI: C1qa-/- NR vs C1qa-/- 652

MD, p=0.0078). Furthermore, these C1qa-/- MD animals were not different from the WT MD 653

animals (OD categories: chi-squared test, chi-square (6)=10.16, p=0.1179, α=0.0125 to account 654

for multiple comparisons; ODI distributions: Kolmogorov-Smirnov test, D=0.1336, p=0.0178, 655

α=0.0125 to account for multiple comparisons; CBI: WT MD vs C1qa-/- MD, p=0.9959). Under 656

the assumption that the high CBI value is atypical for C1qa-/- MD mice, our results thus suggest 657

that C1qa-/- animals undergo normal ODP. 658

659

Discussion 660

In this study, we investigated the contribution of C1q, the initiator of the classical complement 661

cascade, to V1b development and plasticity. We found that C1q protein was present in V1b 662

during the critical period, but that bulk levels were not affected by MD. When we quantified 663

spine numbers on apical and basal dendrites of L2/3 pyramidal neurons, we observed mostly 664

normal spine numbers in C1qa-/- mice during development. The dendritic arbors of these neurons 665

were also unaffected by loss of C1qa. We furthermore found that neuronal firing rates in V1b 666

were unchanged in C1qa-/- compared to WT mice. Moreover, we did not observe a reduction in 667

29

spine numbers in WT or C1qa-/- mice following critical period MD, and when we measured 668

ODP, we detected robust plasticity in C1qa-/- mice using both in situ hybridization against the 669

immediate early gene Arc, and in vivo electrophysiology. Taken together, our results suggest that 670

V1b is generally normal in the absence of C1qa. 671

In most experiments performed in this study, the C1qa-/- animals were similar to their WT 672

counterparts. These observations are in agreement with previous work performed in the 673

hippocampus, where C1qa-/- mice were generally found to have no or mild synaptic phenotypes, 674

depending on the experiment and the age of the animal (Stephan et al., 2013). In addition, this 675

previous study found that whole-brain C1q levels increase with age, similar to what we report 676

here. The phenotype that we do observe in the C1qa-/- animals is that they have increased spine 677

densities on apical secondary dendrites at P20. Previous dLGN phenotypes in C1qa-/- mice have 678

been attributed to impaired microglial phagocytosis of retinogeniculate synapses (Stevens et al., 679

2007; Schafer et al., 2012), and it is conceivable that the elevated spine densities on P20 apical 680

secondary dendrites in the mice lacking C1qa could be due to a lack of synapse engulfment. It is, 681

however, only a transient phenotype, as spine densities return to normal at P29-30. 682

In contrast to V1b and the hippocampus, sensorimotor cortex and the dLGN show 683

reduced synapse elimination in the absence of C1qa. Sensorimotor cortex of juvenile C1qa-/- 684

animals have increased spine and bouton numbers, as well as increased frequencies of mEPSCs 685

and seizures, and refinement in the P30 dLGN is impaired in C1qa-/- mice (Stevens et al., 2007; 686

Chu et al., 2010; Ma et al., 2013). These differences in phenotypes are surprising for two 687

reasons: firstly that the phenotypes seem to be quite variable depending on the brain region 688

examined, and secondly that the dLGN shows improper refinement at the same time as V1b is 689

largely normal, even though V1b receives input from the dLGN (Stevens et al., 2007). There are 690

30

possible explanations for why V1b does not appear to inherit the refinement defects of the 691

dLGN. Defects in eye-specific segregation can represent changes in axon arborization that do not 692

necessarily result in changed synaptic connectivity. Furthermore, while electrophysiological 693

studies performed in the part of the dLGN that is targeted by the contralateral eye found that loss 694

of C1qa prevents the elimination of weak inputs in the dLGN, at least some RGC inputs seem to 695

strengthen normally in P30 C1qa-/- mice (Stevens et al., 2007). dLGN relay neurons are only 696

driven by strong RGC inputs (Liu and Chen, 2008). It is therefore possible that the presence of 697

many weak inputs, as is found in the dLGN of C1qa-/- mice at P30, does not impact the 698

transmission of visual information to V1b. Why different brain regions show different 699

phenotypes is less clear. It does not appear to be dependent on the overall levels of C1q protein. 700

C1q levels are generally low in cortex and high in the hippocampus (Stephan et al., 2013), 701

indicating that both regions with high and low levels of C1q, such as the hippocampus and V1b, 702

respectively, can have mild phenotypes. It is possible that expression or localization of 703

downstream complement proteins required for microglial engulfment is context-dependent. In 704

addition, negative regulators of C1q activity, such as complement inhibitors or signaling 705

pathways that block phagocytosis, including CD47, may be heterogeneously distributed across 706

brain regions (Ricklin et al., 2010; Lehrman et al., 2018). Such heterogeneity could explain why 707

phenotypes in C1qa-/- mice are so diverse. 708

Several previous studies have described synapse loss following critical period MD 709

(Mataga et al., 2004; Coleman et al., 2010; Zhou et al., 2017). Here we attempted to replicate the 710

finding that MD induces a reduction in spine numbers on the apical dendrites of L2/3 pyramidal 711

neurons (Mataga et al., 2004). Surprisingly, we found no loss of spines in WT animals following 712

MD. Why we fail to replicate published work is not clear. One explanation may be the use of 713

31

different techniques to label spines: while the previous study used diolistic labeling to mark the 714

neurons, we used Golgi-Cox staining. It is possible that the two methods label spines differently, 715

so that spines detected using Golgi-Cox staining are not visualized with diolistic labeling, or vice 716

versa. Alternatively, the techniques may be labeling different subpopulations of neurons. Finally, 717

there may be strain-specific differences in responses to ODP between the two transgenic lines. 718

Although we lack a conclusive answer to the contradictory results, it seems reasonable to assume 719

from our results that MD does not induce a robust spine loss on the apical dendrites of all mouse 720

L2/3 pyramidal neurons. 721

Our results suggest that neither C1q, nor spine loss on proximal apical dendrites of L2/3 722

pyramidal neurons, is required for ODP, but this does not mean that synapse loss in general has 723

no impact on ODP. Several previous studies have described loss of different synaptic populations 724

following brief (three to four days) and chronic MD in mice and rats (Coleman et al., 2010; 725

Montey and Quinlan, 2011; Zhou et al., 2017). Furthermore, microglial engulfment has been 726

implicated in ODP. Levels of the microglial lysosomal marker CD68 increase in V1b following 727

MD (Schecter et al., 2017), and microglial engulfment of postsynaptic markers is elevated in 728

V1b during ODP (Sipe et al., 2016). Additionally, the microglial gene P2ry12 contributes to 729

ODP (Sipe et al., 2016), although the microglial chemokine receptor CX3CR1 does not (Lowery 730

et al., 2017; Schecter et al., 2017). Thus, in the circumstances where MD-induced spine loss has 731

been previously described, microglia could contribute to ODP by eliminating synapses through 732

other mechanisms than C1q. Future work will be necessary to determine whether microglial 733

synapse engulfment does indeed help promote synapse loss during ODP, and whether the many 734

observed examples of MD-induced synapse loss are dependent on microglia. 735

32

Although the phenotypes that we observe in the C1qa-/- animals in this study are mild, the 736

classical complement cascade has previously been compellingly implicated in several brain 737

disorders involving excess synapse elimination. One connection is to schizophrenia: the 738

downstream classical complement cascade member C4A is genetically linked to that disease 739

(Sekar et al., 2016). Whether C1q contributes to the role of C4A in schizophrenia is, however, 740

not yet clear. By far the strongest connection between C1q and disease instead lies in 741

neurodegeneration. C1q is significantly upregulated and mediates pathological synapse loss in 742

models of Alzheimer’s and neurodegenerative disease (Hong et al., 2016; Lui et al., 2016; Vasek 743

et al., 2016; Dejanovic et al., 2018). Depending on the context, C1q can thus have significant 744

effects on brain function. Elucidating the circumstances that determine the impact of C1q on 745

synapses will be highly helpful for understanding its role in disease. 746

747

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907

Figure legends 908

Figure 1: C1q is present in the developing V1b, and levels increase with age. A. 909

Representative images of immunohistochemistry against C1q (white) and DAPI (blue) across 910

cortical layers in V1b at P10 (top), P20 (middle), and P32 (bottom), of wild-type (WT) (left) and 911

C1qa-/- (right) animals. Approximate locations of the cortical layers are noted to the left of the 912

images. Scale bar=100μm. B-C. Representative confocal images of immunohistochemistry 913

against C1q (white) and DAPI (blue) in L2/3 (B) and L4 (C) in V1b of WT and C1qa-/- animals, 914

42

at P10 (top), P20 (middle), and P32 (bottom). The C1q signal is absent in C1qa-/- tissue, 915

demonstrating specificity of the antibody. Scale bar=20μm. D. Quantification of C1q confocal 916

immunofluorescence intensities in L2/3 of V1b across ages. C1q levels are significantly higher at 917

P32 than at P20 or P10 (one-way ANOVA, F(2, 9)=17.04, p=0.0009, n=4 animals/age. P10 vs 918

P20, p=0.1538, P10 vs P32, p=0.0007, P20 vs P32, p=0.0123, Tukey’s multiple comparisons 919

test). E. Quantification of C1q confocal immunofluorescence intensities in L4 of V1b across 920

ages shows significantly higher C1q levels at P32 than at P20 or P10 (one-way ANOVA, F(2, 921

9)=12.12, p=0.0028, n=4 animals/age. P10 vs P20, p=0.2468, P10 vs P32, p=0.0023, P20 vs P32, 922

p=0.0297, Tukey’s multiple comparisons test). F. Left: Representative confocal image of 923

immunohistochemistry against C1q (magenta) and spinophilin (green) in L2/3 of V1b at P32, in 924

a WT animal. Images are representative of n=3 animals. White circles mark examples of 925

spinophilin puncta that overlap with, or are closely apposed to, C1q puncta. Scale bar=2μm. 926

Right: Two examples of superresolution SIM images of partially overlapping C1q and 927

spinophilin puncta (not from the same sections as the confocal image to the left) from P29-30 928

WT mice. Images are representative of n=3 animals. Scale bar=0.3μm. All error bars represent 929

SEM. *p<0.05, **p<0.01, ***p<0.001. 930

931

Figure 2: A spine population implicated in ODP develops normally in C1qa-/- mice. A. 932

Brightfield image of a Golgi-Cox stained brain from a critical period mouse. The black box 933

indicates the approximate location of V1b. Scale bar=1000μm. B. Enlarged image of the region 934

marked in A. A L2/3 pyramidal neuron used for spine analysis is circled. Scale bar=200μm. C. 935

Higher magnification image of the cell circled in B. The soma is at the bottom of the image, 936

while the 25μm segment used for spine counting is marked with red arrowheads. Scale 937

43

bar=10μm. D. Representative single plane images of L2/3 pyramidal neuron apical dendrites, 938

from P10 (left), P20 (middle) and P29-30 (right) WT (top in each age group) and C1qa-/- (bottom 939

in each age group) animals. Images show part of the 25μm segment used for spine counting. Red 940

arrowheads mark spines, while a red arrow marks a dendrite branch point. Scale bar=2μm. E. 941

Quantification of spine numbers in P10 (left), P20 (middle) and P29-30 (right) animals, analyzed 942

per mouse. There is no difference between spine numbers in WT and C1qa-/- animals at any of 943

the ages (P10: unpaired two-tailed t-test, t(7)=0.7854, p=0.4580, n=4-5 animals/genotype; P20: 944

unpaired two-tailed t-test, t(8)=1.665, p=0.1345, n=4-6 animals/genotype; P29-30: unpaired two-945

tailed t-test, t(10)=1.54, p=0.1546, n=6 animals/genotype). F. Quantification of spine numbers in 946

P10 (left), P20 (middle) and P29-30 (right) animals, analyzed per cell. There is no difference 947

between spine numbers in WT and C1qa-/- animals at any of the ages (P10: unpaired two-tailed t-948

test, t(41)=0.2109, p=0.8340 n=17 WT, 26 C1qa-/- cells; P20: unpaired two-tailed t-test, 949

t(52)=1.504, p=0.1386, n=30 WT, 24 C1qa-/- cells; P29-30: unpaired two-tailed t-test, 950

t(46)=1.511, p=0.1377, n=22 WT, 26 C1qa-/- cells). All error bars represent SEM. 951

952

Figure 3: The distance between the soma and first branch point of L2/3 pyramidal neuron 953

apical dendrites is normal in C1qa-/- mice. A. Quantification of the correlation between spine 954

numbers and distance between soma and first branch point in P10 (left), P20 (middle) and P29-955

30 (right) animals, analyzed per cell. The spine numbers and distance are significantly correlated 956

at the two older ages, using Spearman’s rank correlation coefficient rs (P10 WT: rs=0.2616, 957

p=0.3081, n=17 cells, P10 C1qa-/-: rs=0.5366, p=0.0047, n=26 cells, P20 WT: rs=0.6199, 958

p=0.0003, n=30 cells, P20 C1qa-/-: rs=0.5634, p=0.0041, n=24 cells, P29-30 WT: rs=0.7321, 959

p=0.0001, n=22 cells, P29-30 C1qa-/-: rs=0.7085, p<0.0001, n=26 cells). B. Quantification of the 960

44

distance between the soma and the first branch point in P10 (left), P20 (middle) and P29-30 961

(right) animals. This distance is not different between WT and C1qa-/- animals at any of the ages 962

(P10: unpaired two-tailed t-test, t(7)=0.2727, p=0.7929, n=4-5 animals/genotype; P20: unpaired 963

two-tailed t-test, t(8)=0.5198, p=0.6173, n=4-6 animals/genotype; P29-30: unpaired two-tailed t-964

test, t(10)=0.3156, p=0.7588, n=6 animals/genotype). All error bars represent SEM. 965

966

Figure 4: Spine densities in C1qa-/- mice are elevated at P20 on apical secondary dendrites, 967

but are otherwise normal. A. Left: Brightfield image of a L2/3 Golgi-Cox stained pyramidal 968

neuron from a critical period mouse. The soma is at the bottom of the image. An example of a 969

secondary apical dendrite segment used for spine counting is marked with red arrowheads. Scale 970

bar=10μm. Right: Representative single plane images of apical dendrite secondary branches 971

from L2/3 pyramidal neurons, from P10 (left), P20 (middle) and P29-30 (right) WT (top in each 972

age group) and C1qa-/- (bottom in each age group) animals. Red arrowheads mark spines. Scale 973

bar=2μm. B. Quantification of spine densities on apical dendrite secondary branches in P10 974

(left), P20 (middle) and P29-30 (right) animals, analyzed per mouse. There is no difference 975

between spine densities in WT and C1qa-/- animals at P10 or P29-30, but P20 C1qa-/- mice show 976

elevated spine densities compared to WT (P10: unpaired two-tailed t-test, t(7)=0.04222, 977

p=0.9675, n=4-5 animals/genotype; P20: unpaired two-tailed t-test, t(8)=2.371, p=0.0452, n=4-6 978

animals/genotype; P29-30: unpaired two-tailed t-test, t(8)=0.2614, p=0.8004, n=4-6 979

animals/genotype). C. Quantification of spine densities on apical dendrite secondary branches in 980

P10 (left), P20 (middle) and P29-30 (right) animals, analyzed per cell. There is no difference 981

between spine densities in WT and C1qa-/- animals at P10 or P29-30, but P20 C1qa-/- mice show 982

elevated spine densities compared to WT (P10: unpaired two-tailed t-test, t(40)=0.4146, 983

45

p=0.6807, n=17 WT, 25 C1qa-/- cells; P20: unpaired two-tailed t-test, t(47)=2.251, p=0.0291, 984

n=28 WT, 21 C1qa-/- cells; P29-30: unpaired two-tailed t-test, t(32)=0.8172, p=0.4199, n=14 985

WT, 20 C1qa-/- cells). D. Left: Brightfield image of a L2/3 Golgi-Cox stained pyramidal neuron 986

from a critical period mouse. The soma is in the center of the image. An example of a secondary 987

basal dendrite segment used for spine counting is marked with red arrowheads. Scale bar=10μm. 988

Right: Representative single plane images of basal dendrite secondary branches from L2/3 989

pyramidal neurons, from P10 (left), P20 (middle) and P29-30 (right) WT (top in each age group) 990

and C1qa-/- (bottom in each age group) animals. Red arrowheads mark spines. Scale bar=2μm. E. 991

Quantification of spine densities on basal, secondary branches in P10 (left), P20 (middle) and 992

P29-30 (right) animals, analyzed per mouse. There is no difference between spine densities in 993

WT and C1qa-/- animals at any of the ages (P10: unpaired two-tailed t-test, t(7)=0.6858, 994

p=0.5149, n=4-5 animals/genotype; P20: unpaired two-tailed t-test, t(8)=0.5260, p=0.6132, n=4-995

6 animals/genotype; P29-30: unpaired two-tailed t-test, t(9)=0.5023, p=0.6275, n=5-6 996

animals/genotype). F. Quantification of spine densities on basal, secondary branches in P10 997

(left), P20 (middle) and P29-30 (right) animals, analyzed per cell. There is no difference between 998

spine densities in WT and C1qa-/- animals at any of the ages (P10: unpaired two-tailed t-test, 999

t(41)=0.7537, p=0.4553, n=17 WT, 26 C1qa-/- cells; P20: unpaired two-tailed t-test, t(47)=1.104, 1000

p=0.2750, n=26 WT, 23 C1qa-/- cells; P29-30: unpaired two-tailed t-test, t(42)=0.4044, 1001

p=0.6879, n=23 WT, 21 C1qa-/- cells). All error bars represent SEM. *p<0.05. 1002

1003

Figure 5: Sholl analysis reveals normal L2/3 pyramidal neuron dendritic arbors in C1qa-/- 1004

mice. A. Representative tracings of V1b L2/3 pyramidal neuron dendritic arbors from P29-30 1005

WT (left) or C1qa-/- (right) mice. Circles in the center of the arbors represent the soma. Scale 1006

46

bar=50μm. B-D. Sholl analysis does not find a difference between WT and C1qa-/- V1b L2/3 1007

pyramidal neurons in the number of dendrite intersections at 10, 30, 50, 70, 90, or 110μm from 1008

the soma in either the apical (left) or the basal (right) arbor. This applies at P10 (B), P20 (C), and 1009

P29-30 (D). B: unpaired two-tailed t-test, n=4-5 mice/genotype. Holm–Sidak's method with 1010

α=0.05 was used to correct for multiple comparisons. Apical: adjusted p-value=0.8744 (10μm), 1011

0.9343 (30μm), 0.9683 (50μm), 0.9508 (70μm), 0.9508 (90μm), 0.9683 (110μm). Basal: adjusted 1012

p-value=0.6602 (10μm), 0.6602 (30μm), 0.8774 (50μm), 0.6602 (70μm), 0.6172 (90μm), NA 1013

(110μm; all values are 0). C: unpaired two-tailed t-test, n=4-6 mice/genotype. Holm–Sidak's 1014

method with α=0.05 was used to correct for multiple comparisons. Apical: adjusted p-1015

value=0.8869 (10μm), 0.8869 (30μm), 0.8869 (50μm), 0.4866 (70μm), 0.8869 (90μm), 0.8869 1016

(110μm). Basal: adjusted p-value=0.9969 (10μm), 0.9969 (30μm), 0.9969 (50μm), 0.9969 1017

(70μm), 0.9969 (90μm), 0.9969 (110μm). D: unpaired two-tailed t-test, n=5-6 mice/genotype. 1018

Holm–Sidak's method with α=0.05 was used to correct for multiple comparisons. Apical: 1019

adjusted p-value=0.8618 (10μm), 0.7560 (30μm), 0.8618 (50μm), 0.7560 (70μm), 0.6894 1020

(90μm), 0.7560 (110μm). Basal: adjusted p-value=0.9720 (10μm), 0.9720 (30μm), 0.6167 1021

(50μm), 0.5629 (70μm), 0.3536 (90μm), 0.3119 (110μm). All error bars represent SEM. 1022

1023

Figure 6: Firing rates are normal in C1qa-/- mice. A. Raster plots for representative single 1024

units from WT and C1qa-/- mice in response to drifting sinusoidal gratings in twelve different 1025

directions interleaved by a grey screen (ø) shown to each eye independently. Each circle 1026

represents one action potential. Scale bar=1s. B-C. Spontaneous (B) and evoked (C) activity of 1027

all isolated, visually responsive single units from WT and C1qa-/- mice. Spontaneous firing rates 1028

are not different across stimulated eye or genotype (one-way ANOVA, F(3, 181)=0.08636, 1029

47

p=0.9674, n=42-50 single units/condition, from 6 animals/genotype). Evoked firing rates are 1030

higher when the contralateral eye rather than the ipsilateral eye is stimulated, but are not different 1031

between the genotypes (one-way ANOVA, F(3, 181)=4.788, p=0.0031, n=42-50 single 1032

units/condition, from 6 animals/genotype, WT ipsi vs WT contra, p=0.0428, C1qa-/- ipsi vs 1033

C1qa-/- contra, p=0.0424, WT ipsi vs C1qa-/- ipsi, p=0.9413, WT contra vs C1qa-/- contra, 1034

p=0.9165, Sidak’s multiple comparisons test). All error bars represent SEM. *p<0.05. 1035

1036

Figure 7: C1q levels in V1b do not change with monocular deprivation. A. Representative 1037

images of C1q immunohistochemistry in L2/3 (top) and L4 (bottom) of V1b, from P32 mice that 1038

were normally reared (NR), or from animals that had undergone two days (2D MD) or four days 1039

(4D MD) of MD starting at P28. Images are from the hemisphere contralateral to the deprived 1040

eye. Scale bar=20μm. B. Quantification of C1q immunofluorescence intensities in L2/3 of V1b 1041

in NR, 2D MD and 4D MD animals. The conditions are not statistically different from one 1042

another (one-way ANOVA, F(2, 15)=0.1803, p=0.8368, n=6 animals/condition). C. 1043

Quantification of C1q immunofluorescence intensities in L4 of V1b in NR, 2D MD and 4D MD 1044

animals. There is no statistically significant difference in immunofluorescence intensities 1045

between the conditions (one-way ANOVA, F(2, 15)=0.2338, p=0.7943, n=6 animals/condition). 1046

All error bars represent SEM. 1047

1048

Figure 8: MD does not reduce spine numbers on L2/3 pyramidal neuron apical dendrites. 1049

A. Representative single plane images of L2/3 pyramidal neuron apical dendrites from WT and 1050

C1qa-/- littermates that were either NR, or that had undergone four days of MD. All cells come 1051

48

from the hemisphere contralateral to the deprived eye. Images show part of the 25μm segment 1052

used for spine counting. Red arrowheads mark spines. Scale bar=2μm. B. Quantification of spine 1053

numbers in WT and C1qa-/- NR and MD animals, per mouse. Data points in the NR condition are 1054

the same as the P29-30 group in Figure 2E. MD does not impact spine numbers, and there is no 1055

difference between spine numbers in WT and C1qa-/- animals in either condition (two-way 1056

ANOVA, F(1, 21)=3.034 and p=0.0962 for MD, F(1, 21)=0.344 and p=0.5638 for genotype, and 1057

F(1, 21)=2.906 and p=0.1030 for interaction, n=6-7 animals/group. WT NR vs C1qa-/- NR, 1058

p=0.4049, WT NR vs WT MD, p>0.9999, C1qa-/- NR vs C1qa-/- MD, p=0.0920, WT MD vs 1059

C1qa-/- MD, p=0.8510, Tukey’s multiple comparisons test). C. Quantification of spine numbers 1060

in WT and C1qa-/- NR and MD animals, per cell. Data points in the NR condition are the same as 1061

the P29-30 group in Figure 2F. MD does not impact spine numbers in the WT group, but slightly 1062

increases spine numbers in the C1qa-/- mice (two-way ANOVA, F(1, 110)=3.543 and p=0.0625 1063

for MD, F(1, 110)=0.2824 and p=0.5962 for genotype, and F(1, 110)=2.695 and p=0.1035 for 1064

interaction, n=22-40 cells/group. WT NR vs C1qa-/- NR, p=0.4774, WT NR vs WT MD, 1065

p=0.9986, C1qa-/- NR vs C1qa-/- MD, p=0.0411, WT MD vs C1qa-/- MD, p=0.8323, Tukey’s 1066

multiple comparisons test). D. Quantification of the distance between the soma and the first 1067

branch point in WT and C1qa-/- NR and MD animals. Data points in the NR condition are the 1068

same as the P29-30 group in Figure 3B. There is no difference in distance between WT and 1069

C1qa-/- animals in either condition (two-way ANOVA, F(1, 21)=0.02282 and p=0.8814 for MD, 1070

F(1, 21)=0.2156 and p=0.6472 for genotype, and F(1, 21)=0.0069 and p=0.9346 for interaction, 1071

n=6-7 animals/group. WT NR vs C1qa-/- NR, p=0.9808, WT NR vs WT MD, p>0.9999, C1qa-/- 1072

NR vs C1qa-/- MD, p=0.9982, WT MD vs C1qa-/- MD, p=0.9925, Tukey’s multiple comparisons 1073

test). All error bars represent SEM. *p<0.05. 1074

49

1075

Figure 9: Binocular zone expansion is normal in critical period C1qa-/- mice. A. 1076

Representative images of RNAscope Arc in situ hybridization in V1b of critical period WT or 1077

C1qa-/- mice that were either NR or had undergone four days of MD. Approximate locations of 1078

cortical layers are noted at the far left of the images. Red stars mark the extent of the binocular 1079

zone in L2/3 and L4, as measured using the FWHM. Scale bar=300μm. B-D. During the critical 1080

period, binocular zone expansion as measured by the FWHM of the Arc positive signal increases 1081

in L2/3 following MD (B), in L4 following MD (C), and in L4 following ME (D), compared to 1082

NR mice. However, loss of C1qa has no effect on the width of the binocular zone under any of 1083

these conditions. B: two-way ANOVA, F(1, 21)=35.19 and p<0.0001 for MD, F(1, 21)=2.717 1084

and p=0.1142 for genotype, and F(1, 21)=0.02172 and p=0.8843 for interaction, n=6-7 1085

animals/group. WT NR vs C1qa-/- NR, p=0.6052, WT NR vs WT MD, p=0.0014, C1qa-/- NR vs 1086

C1qa-/- MD, p=0.0032, WT MD vs C1qa-/- MD, p=0.7042, Tukey’s multiple comparisons test. 1087

C: two-way ANOVA, F(1, 21)=69.53 and p<0.0001 for MD, F(1, 21)=0.9028 and p=0.3528 for 1088

genotype, and F(1, 21)=1.029 and p=0.3219 for interaction, n=6-7 animals/group. WT NR vs 1089

C1qa-/- NR, p>0.9999, WT NR vs WT MD, p=0.0002, C1qa-/- NR vs C1qa-/- MD, p<0.0001, WT 1090

MD vs C1qa-/- MD, p=0.5039, Tukey’s multiple comparisons test. D: two-way ANOVA, F(1, 1091

16)=29.77 and p<0.0001 for ME, F(1, 16)=0.5278 and p=0.4780 for genotype, and F(1, 1092

16)=0.8672 and p=0.3656 for interaction, n=5 animals/group. WT NR vs C1qa-/- NR, p=0.6521, 1093

WT NR vs WT ME, p=0.0018, C1qa-/- NR vs C1qa-/- ME, p=0.0258, WT ME vs C1qa-/- ME, 1094

p=0.9989, Tukey’s multiple comparisons test. E-G. During the critical period, binocular zone 1095

Arc signal intensity, normalized to the signal intensity in V1m, increases in L2/3 following MD 1096

(E), in L4 following MD (F), but not in L4 following ME (G), compared to NR mice. Loss of 1097

50

C1qa has no effect on the Arc signal strength under any of these conditions. The lack of an effect 1098

of ME on signal intensity is likely due to the use of colorimetric, rather than RNAscope, in situ 1099

hybridization for this experiment. E: two-way ANOVA, F(1, 21)=82.53 and p<0.0001 for MD, 1100

F(1, 21)=0.02663 and p=0.8719 for genotype, and F(1, 21)=0.2541 and p=0.6195 for interaction, 1101

n=6-7 animals/group. WT NR vs C1qa-/- NR, p=0.9952, WT NR vs WT MD, p<0.0001, C1qa-/- 1102

NR vs C1qa-/- MD, p<0.0001, WT MD vs C1qa-/- MD, p=0.9625, Tukey’s multiple comparisons 1103

test. F: two-way ANOVA, F(1, 21)=55.72 and p<0.0001 for MD, F(1, 21)=0.3273 and p=0.5733 1104

for genotype, and F(1, 21)=0.3465 and p=0.5624 for interaction, n=6-7 animals/group. WT NR 1105

vs C1qa-/- NR, p>0.9999, WT NR vs WT MD, p<0.0001, C1qa-/- NR vs C1qa-/- MD, p=0.0006, 1106

WT MD vs C1qa-/- MD, p=0.8366, Tukey’s multiple comparisons test. G: two-way ANOVA, 1107

F(1, 16)=0.9614 and p=0.3414 for ME, F(1, 16)=0.6366 and p=0.4366 for genotype, and F(1, 1108

16)=0.4253 and p=0.5236 for interaction, n=5 animals/group. WT NR vs C1qa-/- NR, p=0.9996, 1109

WT NR vs WT ME, p=0.6626, C1qa-/- NR vs C1qa-/- ME, p=0.9954, WT ME vs C1qa-/- ME, 1110

p=0.7374, Tukey’s multiple comparisons test. All error bars represent SEM. *p<0.05, **p<0.01, 1111

***p<0.001, ****p<0.0001. 1112

1113

Figure 10: ODP is largely normal in critical period C1qa-/- mice. A. OD histograms for NR 1114

and 4D MD WT and C1qa-/- animals. MD shifts the histograms in both WT and C1qa-/- mice, but 1115

the histograms are not affected by loss of C1qa (WT NR vs C1qa-/- NR: chi-squared test, chi-1116

square (5)=7.502, p=0.1859, n=197-230 multi-units/genotype, from 6 animals/genotype. Note: 1117

categories 6 and 7 were combined for calculating the chi-squared test since there was only one 1118

unit in category 7. WT NR vs WT MD: chi-squared test, chi-square (6)=114.5, p<0.0001, n=197-1119

297 multi-units/genotype, from 6-8 animals/genotype. C1qa-/- NR vs C1qa-/- MD: chi-squared 1120

51

test, chi-square (6)=52.64, p<0.0001, n=230-239 multi-units/genotype, from 6-7 1121

animals/genotype. WT MD vs C1qa-/- MD: chi-squared test, chi-square (6)=10.16, p=0.1179, 1122

n=239-297 multi-units/genotype, from 7-8 animals/genotype. To account for multiple 1123

comparisons, the Bonferroni correction is used in this panel to set α=0.0125). The extreme C1qa-1124

/- MD mouse is excluded from this panel. B. MD shifts the distribution of ODIs in both WT and 1125

C1qa-/- mice, but the distributions are not affected by loss of C1qa (WT NR vs C1qa-/- NR: 1126

Kolmogorov-Smirnov test, D=0.1272, p=0.0646, n=197-230 multi-units/genotype, from 6 1127

animals/genotype. WT NR vs WT MD: Kolmogorov-Smirnov test, D=0.4620, p<0.0001, n=197-1128

297 multi-units/genotype, from 6-8 animals/genotype. C1qa-/- NR vs C1qa-/- MD: Kolmogorov-1129

Smirnov test, D=0.2994, p<0.0001, n=230-239 multi-units/genotype, from 6-7 animals/genotype. 1130

WT MD vs C1qa-/- MD: Kolmogorov-Smirnov test, D=0.1336, p=0.0178, n=239-297 multi-1131

units/genotype, from 7-8 animals/genotype. To account for multiple comparisons, the Bonferroni 1132

correction is used in this panel to set α=0.0125). The extreme C1qa-/- MD mouse is excluded 1133

from this panel. C. Quantification of CBI in WT and C1qa-/- mice. The CBI is not different 1134

between the two genotypes (two-way ANOVA, F(1, 23)=32.57 and p<0.0001 for MD, F(1, 1135

23)=0.06254 and p=0.8047 for genotype, and F(1, 23)=0.3021 and p=0.5879 for interaction, 1136

n=6-8 animals/group. WT NR vs C1qa-/- NR, p=0.9491, WT NR vs WT MD, p=0.0009, C1qa-/- 1137

NR vs C1qa-/- MD, p=0.0078, WT MD vs C1qa-/- MD, p=0.9959, Tukey’s multiple comparisons 1138

test). The grey circle marks the excluded C1qa-/- MD datapoint. All error bars represent SEM. 1139

**p<0.01, ***p<0.001. 1140

1141

1142

1143

1144

A B C

1

2/3

4

5

6

1

2/3

4

5

6

1

2/3

4

5

6

P10

P20

P32

WT

C1qa-/-

WT

C1qa-/-

WT

C1qa-/-

L2/3 L4

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10

20

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L4 C

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(AU

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P10 P20 P320

10

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L2/3

C1q

IF in

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DAPIC1q

WT C1qa-/-DAPIC1q

F C1q spinophilin C1q spinophilin SIM

WT C1qa-/-0

10

20

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Num

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f spi

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WT C1qa-/-0

10

20

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Num

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f spi

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WT C1qa-/-0

10

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Num

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WT C1qa-/-0

5

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Num

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f spi

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WT C1qa-/-0

5

10

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Num

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WT C1qa-/-0

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P10 P20 P29-30

P10 P20 P29-30

A C

D

WT

C1qa-/-

E

F

ns

nsns

nsns

ns

B

0 10 20 300

10

20

30

40

Number of spines

Dist

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(μm

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0 5 10 15 200

10

20

30

40

Number of spines

Dist

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(μm

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0 10 20 300

20

40

60

80

Number of spines

Dist

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(μm

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WT C1qa-/-0

10

20

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Dist

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(μm

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WT C1qa-/-0

10

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Dist

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(μm

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WT C1qa-/-0

10

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Dist

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(μm

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P10 P20 P29-30A

Bns

nsns

WTC1qa-/-

rs=0.2616rs=0.5366

WTC1qa-/-

rs=0.6199rs=0.5634

WTC1qa-/-

rs=0.7321rs=0.7085

WT C1qa-/-0.0

0.3

0.6

0.9

1.2

Spines/μm

WT C1qa-/-0.0

0.3

0.6

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Spines/μm

WT C1qa-/-0.0

0.3

0.6

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Spines/μmns

ns nsWT C1qa-/-

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0.6

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WT C1qa-/-0.0

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Spines/μm

WT C1qa-/-0.0

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Spines/μmns

ns ns

P10 P20 P29-30

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WT C1qa-/-0.0

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WT C1qa-/-0.0

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WT C1qa-/-0.00.30.60.91.21.5

Spines/μm

WT C1qa-/-0.00.30.60.91.21.5

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WT C1qa-/-0.00.30.60.91.21.5

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ns*C

D

A

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P10 P20 P29-30

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WT

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P10 P20 P29-30

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Distance from soma (μm)

Num

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Distance from soma (μm)

Num

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Distance from soma (μm)

Num

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5

10

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Distance from soma (μm)

Num

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2

4

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Distance from soma (μm)

Num

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ns

ns****

****

C

NR MD0

400

800

1200

L4 A

rc le

ngth

(μm

)

*******

ns

ns

WT NR C1qa-/- NR WT MD C1qa-/- MD

2/3456

1 Arc

** ** ******* *

****

A

B

**

NR MD0

400

800

1200

L2/3

Arc

leng

th (μ

m)

WT

C1qa-/-

**

ns

ns

E F

NR MD0.0

0.5

1.0

1.5

2.0

L4 A

rc fl

uore

scen

ce in

tens

ity

(nor

mal

ized

to V

1m) ns

ns****

***

D

NR ME0

400

800

1200

L4 A

rc le

ngth

(μm

)

***

ns

ns

G

NR ME0.0

0.5

1.0

1.5

L4 A

rc s

igna

l inte

nsity

(n

orm

alize

d to

V1m

)

ns

-1.0 -0.5 0.0 0.5 1.00

50

100

ODI

Rel

ative

freq

uenc

y (%

)

WT NR

C1qa-/- NR

WT MD

C1qa-/- MD

1 2 3 4 5 6 7OD Category

0

10

20

30

% o

f MU

s

C1qa-/- NR

1 2 3 4 5 6 70

10

20

30

40

% o

f MU

s

WT NR

OD Category

% o

f MU

s

1 2 3 4 5 6 7OD Category

0

10

20WT MD

1 2 3 4 5 6 7

% o

f MU

s

OD Category

0

10

20 C1qa-/- MD

NR MD0.0

0.2

0.4

0.6

0.8

1.0

CBI

WT

C1qa-/-ns

ns

*****

A

B C

C1qa-/- excluded

contra ipsi contra ipsi contra ipsi contra ipsi