Developmental plasticity in visual cortex is necessary for normal visuomotor integration and visuomotor skill learning

The experience of coupling between motor output and visual feedback is necessary for the development of visuomotor skills and shapes visuomotor integration in visual cortex. Whether these experience-dependent changes involve plasticity in visual cortex remains unclear. Here, we probed the role of NMDA receptor-dependent plasticity in mouse primary visual cortex (V1) during visuomotor development. Using a conditional knockout of NMDA receptors and a photoactivatable inhibitor of CaMKII, we locally perturbed plasticity in V1 during first visual experience, recorded neuronal activity in V1, and tested the mice in a visuomotor task. We found that perturbing plasticity before, but not after, first visuomotor experience reduces responses to unpredictable stimuli, diminishes the suppression of predictable feedback in V1, and impairs visuomotor skill learning later in life. Our results demonstrate that plasticity in the local V1 circuit during early life is critical for shaping visuomotor integration. *** Dear reader, please note this manuscript is formatted in a standard submission format, and all statistical information is provided in Table S1. ***


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Movement results in predictable sensory consequences. Through experience, the brain learns this 23 mapping from motor output to sensory feedback. Raised without coupling between movements and 24 visual feedback during visual development, kittens fail to use visual input to guide movements with 25 the impairment specific to the dimension in which the artificial uncoupling occurred (Hein and Held,26 1967; Held and Hein, 1963). The same coupling between locomotion and visual feedback is necessary 27 to integrate visual and motor-related signals in primary visual cortex (V1). Under normal conditions, 28 V1 has been shown to exhibit distinct and salient responses to unpredictable mismatches between 29 movement and visual feedback in both humans and mice (Keller et al., 2012;Stanley and Miall, 2007;30 Zmarz and Keller, 2016). In mice raised from birth without coupling between movement and visual 31 in V1 prior to first visuomotor experience. NMDA receptors are known to be involved in a wide variety 48 of different forms of plasticity (Paoletti et al., 2013;Rodriguez et al., 2019), and are necessary for 49 activity-dependent synaptic strengthening in cortex (Hasan et al., 2013;Kirkwood and Bear, 1994;Lo 50 et al., 2013). In a parallel approach, to impair plasticity in V1 in a cell type specific manner, we used a 51 photoactivatable inhibitor of the calcium/calmodulin-dependent protein kinase II (CaMKII). CaMKII 52 has been shown to be an essential element of NMDA receptor-dependent plasticity (Barria and 53 Malinow, 2005; Gambrill and Barria, 2011;Wang et al., 2011). NMDA receptors are thought to exert 54 their influence on synaptic plasticity by increasing calcium influx into the cell, where calmodulin binds 55 calcium and activates CaMKII. The idea that NMDA receptors and CaMKII are on the same plasticity 56 pathway is supported by a number of findings. For example, spine enlargement triggered by NMDA 57 receptor stimulation can be inhibited by blocking CaMKII (Herring and Nicoll, 2016). Additionally, 58 activated CaMKII and NMDA receptors directly interact (Leonard et al., 1999) to form CaMKII-NMDA 59 receptor complexes that are required for the induction of long-term potentiation (Barria and Malinow, 60 2005), and likely control synaptic strength (Lisman et al., 2012). Thus, we expected that NMDA 61 receptor knockout and CaMKII inhibition would have similar effects on the responses of L2/3 neurons. 62 We find that both types of manipulations systematically impair the development of normal 63 visuomotor integration in L2/3 neurons, commensurate with the impairment observed in mice that 64 virtual environment that provided closed-loop feedback between forward locomotion and backward 81 visual flow in a virtual corridor . Mice were trained in this setup for 2 hours every 82 other day for 12 days (for a total of 6 sessions), after which we then measured calcium activity in L2/3 83 neurons using two-photon imaging ( Figure 1C). We validated the method for the local knockout of 84 Grin1, using an in situ hybridization with a Grin1 mRNA probe in a subset of mice and found a marked 85 reduction at the injection site of the Cre vector ( Figure 1D). During the imaging experiments, mice 86 were first exposed to closed-loop visual flow feedback in a virtual corridor (see Methods). To measure 87 mismatch responses, we introduced brief (1 s) halts of visual flow at random times (Keller et al., 2012). 88 To estimate the contributions of visual flow and locomotion separately, mice were then presented 89 with a playback of the visual flow they had previously self-generated in the closed-loop session (we 90 will refer to this as the open-loop session). To measure visual responses, mice were presented with 91 full-field drifting gratings of different orientations. Finally, to isolate motor-related signals, we 92 measured locomotion-related activity in complete darkness. 93 We found that visuomotor mismatch responses in the knockout hemisphere were reduced compared 94 to the control hemisphere ( Figure 1E), commensurate with the response reduction in mice that never 95 experienced coupling between locomotion and backward visual flow ( Figure S1A). We also found a 96 reduction in grating onset responses ( Figure 1F), but no evidence of a reduction in motor-related 97 hemisphere ( Figure 1J). Thus, NMDA receptor knockout prior to first visual experience prevents the 131 development of normal visual and visuomotor mismatch responses in V1. 132 These results would be consistent with either a role of the NMDA receptor in the plasticity necessary 133 for the establishment of visuomotor integration in V1, or a direct involvement of NMDA receptors in 134 generating neuronal calcium responses. The latter could be driven by an influence of NMDA receptors 135 on the overall excitability of the neurons, or, given that NMDA receptors conduct calcium, by directly 136 reducing the calcium response. To disambiguate this, we repeated the same NMDA receptor knockout 137 experiments in a second group of mice that had been reared in a normal light-dark cycle (ΔGrin1adult; 138 Figure 1B). We found that in these mice there was no difference in any of the responses between 139 those in the control hemisphere and those in the knockout hemisphere (Figures 2A-2C correlations with activity in control and knockout hemispheres was very similar ( Figure 2E). Lastly, as 145 in the juvenile knockout, we found an increase in the average correlation between neurons ( Figure  146 2F). This increase in correlation is likely specific to L2/3 neurons, as a similar knockout in layer 4 (L4) 147 neurons results in a decrease in correlation between neurons that lack NMDA receptors (Mizuno et 148 al., 2021). This demonstrates that NMDA receptors are not necessary to maintain mismatch and visual 149 responses once V1 is fully trained by visuomotor experience. 150 Both a visuomotor mismatch and the sudden appearance of a visual stimulus are unpredictable events 151 and can be interpreted as negative and positive prediction errors, respectively. Assuming there is 152 indeed a deficit in the development of prediction error responses induced by the NMDA receptor 153 knockout, we would also expect a similar deficit in the suppression of predictable responses. To 154 investigate this, we quantified the suppression of running onset responses by visual flow in the closed-155 loop session. In normally reared mice, a running onset with closed-loop visual feedback is typically 156 associated with an increase in activity that is transient, whereas the response to the same running 157 onset in darkness results in a sustained change in mean activity ( Figure 3A). One interpretation of this 158 is that the visual flow coupled to locomotion in the closed-loop session triggers a suppression of the 159 running-related responses. We can quantify the suppression in the closed-loop session by taking the 160 difference between the running onset response in darkness and that in the closed-loop session ( Figure  161 3A). Computing this difference for control mice, ΔGrin1juv mice, and ΔGrin1adult mice, we found that 162 this suppression was absent only in the knockout hemisphere of the ΔGrin1juv mice (Figures 3B and  163  3C). This is consistent with an impairment in the suppression of predictable responses in L2/3 neurons 164 by an NMDA receptor knockout prior to first visual experience. 165 Local NMDA receptor dysfunction during development resulted in impaired visuomotor skill 166 learning later in life. 167 Assuming developmental plasticity is necessary for the establishment of normal visuomotor 168 integration, we expected that the ΔGrin1juv mice would exhibit behavioral impairments in cortex-169 dependent visuomotor tasks. To test this, we trained 6 ΔGrin1juv mice in a visuomotor task later in life. 170 For these experiments we used two control groups. The first was composed of 13 ΔGrin1adult mice, and 171 the second was composed of 6 control mice (Controljuv) that did not receive a Grin1 knockout but were 172 dark reared from birth. The ΔGrin1juv and Controljuv groups were dark reared until P32. All three groups 173 were initially exposed to closed-loop experience in a virtual reality setup as described above and 174 subsequently trained to perform a virtual navigation task (Heindorf et al., 2018) (Figures 4A and 4B). 175 In this task, mice had control over movement in a virtual environment through rotation and forward 176 locomotion on a spherical treadmill and were trained to reach the end of a virtual corridor for a water 177 reward. Training lasted for 7 days, 1 hour per day. We quantified performance using an index that is 178 based on the fraction of distance traveled toward the target normalized by the total distance traveled 179 (see Methods). The dark reared Controljuv mice and the adult knockout ΔGrin1adult mice both learned 180 to perform the task over the course of the training. The ΔGrin1juv mice, however, failed to show 181 evidence of increased performance over the course of the 7 days of training, and exhibited 182 significantly reduced performance compared to the two control groups late in training ( Figure 4C). To 183 test for the mice's ability to induce a behavioral response to an unexpected perturbation of visual 184 feedback, we introduced sudden offsets of the current heading direction at random times by 30° 185 either to the left or to the right. With training, mice learned to correct for these offset perturbations 186 with a turn that corrected for the offset. Both Controljuv and ΔGrin1adult mice corrected for offset 187 perturbations with a compensatory turn in the correct direction by the end of training ( Figure 4D). 188 However, the ΔGrin1juv mice failed to correct for these offsets. Quantifying this as the learning related 189 change in offset perturbation response, we found that Controljuv and ΔGrin1adult mice both exhibit 190 larger learning related changes than the ΔGrin1juv mice ( Figure 4E). Thus, consistent with the 191 dependence of normal visuomotor integration on NMDA receptors during first visuomotor 192 experience, we found that mice that lack NMDA receptors during first visuomotor experience are 193 impaired in learning certain cortex-dependent visually guided motor tasks later in life. 194

CaMKII-dependent plasticity in SST interneurons was necessary for feed-forward visual inhibition. 195
Central to the subtractive computation of prediction error responses are inhibitory interneurons. By 196 implementing the opposing influence of visual and locomotion related input in L2/3 neurons (Jordan 197 and Keller, 2020), they allow for a subtraction of a bottom-up sensory input and a top-down prediction 198 to compute prediction errors (Keller and Mrsic-Flogel, 2018). Based on measurements of calcium 199 responses to visuomotor mismatches and artificial manipulations of activity in different interneuron 200 subtypes, we have previously speculated that a subset of somatostatin (SST) positive interneurons 201 mediates the visually driven inhibition necessary for negative prediction error responses in V1 L2/3 202 excitatory neurons . We thus set out to test whether an impairment of plasticity 203 selectively in SST interneurons in V1 during first visuomotor experience would result in a failure to 204 establish visually driven inhibition in L2/3 neurons. To do this, we turned to a method that would allow 205 us to target the intervention to SST interneurons selectively in V1. We used a photoactivatable 206 autocamtide inhibitory peptide 2 (paAIP2) (Murakoshi et al., 2017) to inhibit calcium/calmodulin-207 dependent kinase II (CaMKII) using blue light illumination. 208 We repeated the experiments we performed with the NMDA receptor knockout (Figures 1-3) using 209 paAIP2 in three groups of mice to target CaMKII inhibition either to excitatory neurons, SST 210 interneurons ( Figure 5A), or parvalbumin (PV) positive interneurons ( Figure S3A). Again, all mice were 211 dark reared from birth and received 2 hours of visuomotor experience in virtual reality environment 212 every other day for 12 days (Figures 5B and S3B). The first group consisted of 6 C57/Bl6 mice that 213 received an injection of an AAV to express paAIP2 under a CaMKIIα(1.3kb)-promoter (AAV2/1-214 CaMKIIα-mEGFP-P2A-paAIP2) in right V1. The other two groups consisted of 7 SST-Cre mice and 6 PV-215 Cre mice that each received an injection of AAV2/1-DIO-mEGFP-P2A-paAIP2 unilaterally in V1. At P30, 216 prior to first visuomotor experience, mice were injected with an AAV to express a red-shifted calcium 217 indicator (AAV2/1-Ef1α-NES-jRGECO1a) in both visual cortices. To activate paAIP2 during visuomotor 218 exposure while mice were on the virtual reality setup, we illuminated V1 bilaterally using a blue (473 219 nm) laser, through the glass windows implanted for subsequent two-photon imaging (see Methods). 220 As before (Figures 1E-1G and Figures 2A-2C), we then proceeded to measure mismatch, grating, and 221 running onset responses in L2/3 neurons at P44. Similar to the responses observed in ΔGrin1juv mice, 222 we found that with paAIP2 expressed under the CaMKIIα(1.3kb)-promoter, the strongest changes 223 were in mismatch and visual responses, while running onset responses were less affected (Figures 5C-224   5E). Mismatch responses were again reduced in the inhibited hemisphere compared to the control 225 hemisphere ( Figure 5C). Intriguingly, CaMKII inhibition resulted in a massive increase in visually driven 226 activity of L2/3 neurons ( Figure 5D). We speculate that this difference can be explained by the fact 227 that the power of light used to activate paAIP2 falls off exponentially with cortical depth (Figure S2A) 228 and that CaMKII inhibition predominantly influences superficial synapses, which preferentially carry 229 top-down signals (see Discussion). Despite the difference in the effect of the manipulation on visually 230 driven responses, the effect of the CaMKII inhibition on correlations between the activity of L2/3 231 neurons was an increase, similar to that observed in the ΔGrin1juv mice ( Figure S2B). 232 Given the differences between ΔGrin1juv and the CaMKII inhibition in excitatory neurons, we compared 233 the effect of CaMKII inhibition in inhibitory interneurons to that observed when inhibiting CaMKII in 234 excitatory neurons. Inhibiting CaMKII in SST interneurons had an effect similar to the one we found 235 when inhibiting CaMKII in excitatory neurons, decreasing mismatch responses and increasing visual 236 responses (Figures 5F-5H). Interestingly, the running onset responses during the closed-loop session 237 were much larger in the inhibited hemisphere ( Figure 5H). This could be explained by an increased 238 motor-related excitatory input, a decreased bottom-up visual inhibition, or a combination of both. in life. We find that preventing this process from occurring in V1 during visuomotor development 284 impairs the ability of mice to learn visuomotor tasks later in life. Thus, we speculate that the ability of 285 V1 to compute visuomotor prediction errors is an essential component of the computational strategy 286 the brain uses to guide movement by visual feedback in complex behavioral tasks. Interestingly, later 287 in life, plasticity in V1 is no longer necessary for visuomotor skill learning, indicating that most of the 288 learning related plasticity occurs outside of V1 or independent of NMDA receptors. 289 When interpreting our results, it should be kept in mind that our strategy to knock out NMDA 290 receptors in V1 is not specific to L2/3 neurons, and we cannot be certain if the effects we observed in 291 Another potential confound of these experiments is that we are using intracellular calcium 299 concentration changes to measure neuronal activity, when the NMDA receptor channel is permeable 300 to calcium and constitutes the main source of calcium in dendritic spines (Sabatini et al., 2002). 301 However, given that we are measuring calcium signals at the soma where the main source of calcium 302 is voltage-gated calcium channels (Grienberger and Konnerth, 2012), the direct effect of the NMDA 303 receptor knockout on intracellular calcium is unlikely to interfere with our conclusions. Moreover, an 304 overall reduction in calcium would influence all responses equally and would not explain why after 305 NMDA receptor knockout, we find a strong reduction in mismatch and visual responses but only a 306 small reduction in mean activity levels in juvenile mice (Figure 1), while in adult mice the converse is 307 true (Figure 2). 308 There is a marked difference between the NMDA receptor knockout results and the CaMKII inhibition 309 results in that the latter led to a massive increase in visual responses. There are a number of possible 310 explanations that could account for this difference. First, despite the fact that NMDA receptors and 311 CaMKII are closely linked in many forms of synaptic plasticity, there could be a systematic difference 312 in the dependence of plasticity on the two molecules as a function of neuron or synapse type. Thus, the differences in effect on grating responses between the NMDA receptor knockout and the 326 CaMKII inhibition could be explained by a differential influence on top-down and bottom-up pathways. 327 While we cannot exclude the involvement of the other potential explanations discussed above, it is 328 not immediately clear why they would result in a differential effect with regards to positive and 329 negative prediction errors. Thus, we speculate that the CaMKII inhibition results in a differential 330 impairment of plasticity in top-down and bottom-up pathways. 331 It is important to note that the within animal control suffers from the confound that the two 332 hemispheres are directly connected. For instance, the fact that visual responses are also massively 333 increased in the control hemisphere of mice in which we inhibited CaMKII in excitatory neurons 334 relative to the level of responses one would expect normally (e.g., compare Figure 5D with Figure   testing, see Table S1.  Table S1.  Table S1. 445    Table S1. Comparison against adult control data: n.s.: p > 0.05, **: p < 0.01, ***: p < 0.001. For all details of 504 statistical testing, see Table S1. control hemisphere were similar to those in CT mice. Note, the data shown in this figure includes all 528 data, while the data shown in Figure 1E includes only those data from recording sites for which we 529 also had sufficient grating and running onset data (see Methods). Here and in subsequent panels, n.s.: 530 p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001. For all details of statistical testing, see Table S1. Here and in subsequent panels, n.s.: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001. For all details of 553 statistical testing, see Table S1. For all details of statistical testing, see Table S1. details of statistical testing, see Table S1. 583 (B) As in A, but for responses to the onset of a drifting grating stimulus (see Methods). Green shading 584 and bar indicate presence of grating stimulus. 585 (C) As in A, but for running onset responses in closed-loop sessions. 586 (D) As in A, but for inhibition of CaMKII in SST interneurons using paAIP2. 587 (E) As in B, but for inhibition of CaMKII in SST interneurons using paAIP2.

Virtual reality environment and virtual navigation task 614
In all experiments involving the virtual reality system, mice were head-fixed and mounted on a 615 spherical treadmill, as described previously (Leinweber et al., 2014). In brief, mice were free to run on For the virtual navigation experiments (Figure 4), rotation of the ball was not restricted, and mice 634 could control forward and backward motion, as well as rotation in the virtual environment. To 635 incentivize mice to engage in the visuomotor skill learning task, they were water-restricted with access 636 to 1 mL water daily 3 days before the start of the behavioral experiments. Care was taken to prevent 637 a drop in body weight to below 80% throughout training. During the experiment, mice could obtain 638 water rewards by reaching the end of the virtual corridor, after which they were presented with a 5 639 second gray screen and teleported to the beginning of the corridor again. Task difficulty was increased 640 with increasing performance of the mice by expanding the length of the virtual corridor to keep the 641 rate of water rewards roughly constant. At the beginning of training the length-to-width ratio of the 642 corridor was 5. Every 4 trials, the length of the corridor would be updated by a factor between 1 (no 643 change) and 1.5 (50% increase in length), where the factor was determined as 20 s divided by the 644 mean duration of those 4 trails. Maximum corridor length was restricted to 400% of the length on the 645 first day. Visual offset perturbations were introduced once per trial, presented at a random position 646 within 20% and 80% of the total corridor length and consisted of 30° heading offsets introduced 647 randomly, either to the left or to the right. The task performance index (PI) was calculated as follows: 648 = ∫ cos( ( )) * ( ) ∫ ( ) * 649 Where θ(t) is the direction of running relative to the target, and v(t) is the running speed of the mouse. 650 The intuition behind this index is to quantify performance as the fraction of distance traveled in the 651 direction of the target, normalized by the total distance traveled. The second factor is added to reduce 652 variability driven by a short time spent running, as is typical in early training sessions. 653

Two-photon calcium imaging 654
Two-photon imaging of L2/3 neurons in V1 was performed as described previously (Leinweber et al., 655 2014(Leinweber et al., 655 , 2017. In brief, two-photon imaging was done using a modified Thorlabs Bergamo I or II 656 microscope. Excitation light source was a tunable, femtosecond-pulsed laser (Insight, Spectra Physics, 657 tuned to 910 nm or 980 nm for GCaMP6f excitation, and 1030 nm for jRGECO1a excitation). The scan 658 head was based on an 8 kHz or 12 kHz resonant scanner (Cambridge Technology). We used a piezo Instruments) and band-pass filtered at 80 MHz using digital Fourier transform on a field-664 programmable gate array (NI5772, National Instruments, loaded with custom-designed logic). Images 665 were acquired at 750 by 400 pixels using custom-written LabView software (available on a public  666   SourceForge repository, see Key Resources table), with 10 or 15 frames per z-plane per second and a 667 field of view of approximately 375 µm by 300 µm. Whenever possible, imaging was performed in both 668 control and intervention hemisphere in each mouse. In a subset of mice, (see Table S2) imaging was 669 only possible in one hemisphere as imaging quality did not meet our minimum quality standards in 670 the other (clear image visible in single frame at less than 60 mW total laser power). 671 Conditional Grin1 knockout, histology, and pharmacological NMDA receptor inhibition 672 All ΔGrin1 knockout experiments were performed using the Grin1 tm2Stl (also known as fNR1 or NR1 flox ) 673 mouse line (Tsien et al., 1996), which has a pair of loxP sites flanking the transmembrane domain and 674 C-terminal region of the Grin1 gene that codes for GluN1 (also referred to as NR1), a subunit essential 675 to the NMDA receptor (Monyer et al., 1994). We confirmed the knockout using mRNA in situ 676 hybridization (RNAscope, Ventana) in a separate cohort of 3 mice, 14 days after injection of an AAV 677 vector expressing Cre recombinase in both juvenile and adult mice (ΔGrin1juv, adult). We followed a 678 standardized formaldehyde-fixed paraffin-embedded protocol. In brief, mice were transcardially 679 perfused with phosphate buffered saline (PBS), followed by perfusion with a solution of 4% 680 paraformaldehyde (PFA) in PBS. Brains were isolated, post-fixed overnight in 4% PFA, paraffinized for 681 24 h, and cut at 5 µm using a microtome (ThermoFisher). Slices were stained using hematoxylin to 682 mark cell bodies, and Mm-Grin1-O1 (#473079, target region 2892 -4127, ACDBio) to label Grin1 mRNA. 683 To ease identification of the knockout area in two-photon microscopy, a vector co-expressing a red 684 fluorophore (mCherry) and Cre was used to induce the Grin1 knockout in most experiments. Due to a 685 shortage of the correct vector, a subset (6 of 14) of the ΔGrin1adult experiments were performed 686 without the mCherry fluorophore. For pharmacological NMDA receptor inhibition experiments 687 ( Figures S1C-D), adult wild-type mice were injected with 0.1 mg/kg MK-801 intraperitoneally and 688 neuronal activity was recorded before and after injection. 689

Optogenetic activation of paAIP2 and laser attenuation measurements 690
We used a photoactivatable autocamtide inhibitory peptide 2 (paAIP2) (Murakoshi et al., 2017) to 691 inhibit calcium/calmodulin-dependent kinase II (CaMKII) for the entire duration of the visuomotor 692 exposure in the virtual reality environment. We directed a blue laser (OBIS 473 nm LX 75 mW, 693 Coherent) onto V1 in both hemispheres using a galvo-galvo system and a set of mirrors and lenses 694 (GVSM002-EC/M, Thorlabs). Beam diameter on the cortical surface was 3 mm. Light was triggered at 695 0.2 Hz with a duty cycle of 20% (1 s on, 4 s off). During illumination periods, we alternated between 696 the two hemispheres at 50 Hz. Peak laser power was 20 mW, which resulted in a time-averaged power 697 density at the cortical surface of 0.28 mW/mm 2 . To measure the laser attenuation through tissue 698 ( Figure S2A), we prepared slices of fresh brain tissue of 100 µm, 200 µm, 300 µm, 400 µm and 500 µm 699 thickness. We then illuminated slices with the blue laser used for optogenetic inhibition of CaMKII set 700 to 20 mW power and measured the fraction of power transmitted through each slice using a power 701 meter (PM100D, Thorlabs). 702