bioRxiv preprint doi: https://doi.org/10.1101/2021.02.10.430631; this version posted February 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

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5 Latrophilin GPCR Signaling Mediates Synapse Formation

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9 Richard Sando1,*and Thomas C. Südhof1

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12 1Dept. of Molecular & Cellular Physiology and Howard Hughes Medical Institute, Stanford 13 University School of Medicine, 265 Campus Dr. Room G1021, Stanford CA 94305 USA

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16 *To whom correspondence should be addressed at [email protected]

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21 ABSTRACT

22 Neural circuit assembly in the brain requires precise establishment of synaptic connections, 23 but the mechanisms of synapse assembly remain incompletely understood. Latrophilins are 24 postsynaptic adhesion-GPCRs that engage in trans-synaptic complexes with presynaptic 25 teneurins and FLRTs. In CA1-region neurons, Latrophilin-2 and Latrophilin-3 are essential 26 for formation of entorhinal-cortex-derived and Schaffer-collateral-derived synapses, 27 respectively. However, it is unknown whether latrophilins function as GPCRs in synapse 28 formation. Here, we show that Latrophilin-2 and Latrophilin-3 exhibit constitutive GPCR 29 activity that increases cAMP levels, which was blocked by a mutation interfering with G- 30 protein and arrestin interactions of GPCRs. The same mutation impaired the ability of 31 Latrophilin-2 and Latrophilin-3 to rescue the synapse-loss phenotype in Latrophilin-2 and 32 Latrophilin-3 knockout neurons in vivo. Our results suggest that Latrophilin-2 and Latrophilin- 33 3 require GPCR signaling in synapse formation, indicating that latrophilins promote synapse 34 formation in the hippocampus by activating a classical GPCR-signaling pathway.

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36 INTRODUCTION

37 Although many proteins that shape synaptic functions have been identified, little is known 38 about how synapses are generated. Few proteins are known to be essential for synapse 39 formation in vertebrates or invertebrates. Increasing evidence supports the notion that bi- 40 directional trans-synaptic signaling by adhesion molecules promotes synapse formation 41 (Sanes and Yamagata, 2009; Nusser, 2018; Ribic and Biederer, 2019; Arac and Li, 2019; 42 Lie et al., 2018; De Wit and Ghosh, 2016; Südhof, 2018). However, nearly all of the 43 candidate synaptic adhesion molecules that have been tested by rigorous genetic 44 approaches are not required for synapse formation per se. Instead, some of these candidate 45 synaptic adhesion molecules, such as SYG1 in C. elegans (Shen et al., 2004) or cadherins 46 in the retina (Duan et al., 2018), direct neuronal processes to the correct position of future 47 synapses. Other candidate synaptic adhesion molecules, such as neurexins and their 48 ligands (reviewed in Südhof, 2017) or the SALM/Lrfn family (reviewed in Lie et al., 2018), 49 specify the properties of synapses. A case in point are cerebellins, which connect 50 presynaptic neurexins to postsynaptic GluD and DCC/neogenin adhesion molecules. 51 Although deletion of cerebellin-1 or GluD2 causes robust parallel-fiber synapse loss in the 52 cerebellum, synapses are only eliminated secondarily after they were established. 53 Moreover, only a fraction of parallel-fiber synapses is lost, whereas all parallel-fiber 54 synapses exhibit altered synaptic plasticity (reviewed in Yuzaki, 2018). In other parts of the 55 brain, cerebellin-1 and -2 deletions also do not hinder initial synapse formation, but impair 56 synapse function and also lead to secondary synapse elimination (Rong et al., 2012; 57 Seigneur and Südhof, 2018; Seigneur et al., 2018). Only few adhesion molecules appear to 58 be actually required for the initial establishment of synapses. Among these molecules are 59 two families of adhesion-GPCRs, latrophilins and BAIs, that are essential for establishing 60 synapses in all brain regions in which they have been tested (Kakegawa et al., 2015; Sigoillot 61 et al., 2015; Anderson et al., 2017; Sando et al., 2019; Wang et al., 2020). Studying the 62 mechanism of action of these adhesion-GPCRs offers an avenue for understanding the 63 basic processes underlying initial synapse formation.

64 Adhesion-GPCRs characteristically contain large extracellular sequences with multiple 65 potential ligand-binding domains and a canonical GAIN domain that is coupled to the 7- 66 transmembrane region module characteristic of GPCRs (reviewed in Vizurraga et al., 2020;

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67 Morgan et al., 2019; Folts et al., 2019; Purcell and Hall, 2018). Most adhesion-GPCRs 68 additionally have a long cytoplasmic region. The three mammalian latrophilins (Lphn1-3) 69 follow the same design principle as other adhesion-GPCRs. They feature N-terminal lectin- 70 and olfactomedin-like ligand-binding domains, a serine-threonine rich and ‘hormone binding’ 71 domain of unknown function, and a canonical GAIN domain (Figure 1A; Sugita et al., 1998). 72 These domains are attached to a classical 7-transmembrane region GPCR module, followed 73 by a long cytoplasmic region that interacts at the C-terminus with the PDZ-domain of 74 SHANKs (Kreienkamp et al., 2000; Tobaben et al., 2000). The N-terminal lectin- and 75 olfactomedin-like domains of latrophilins engage in trans-cellular interactions with multiple 76 presynaptic ligands, including teneurins and FLRTs (Sugita et al., 1998; O’Sullivan et al., 77 2012; Silva et al., 2011; Boucard et al., 2014). Multiple latrophilin isoforms are generally co- 78 expressed in the same neurons. CA1 pyramidal neurons of the hippocampus co-express 79 Lphn2 and Lphn3 that are targeted to distinct dendritic domains of the same neuron and are 80 essential for formation of different excitatory synaptic inputs (Anderson et al. 2017; Sando 81 et al., 2019). Moreover, Lphn2 and Lphn3 are redundantly required in the cerebellum for 82 parallel-fiber synapse formation (Zhang et al., 2020). The function of postsynaptic Lphn3 in 83 hippocampal synapse formation requires intact binding sites for both presynaptic teneurins 84 and FLRTs, indicating that latrophilins may mediate synapse specificity by acting as 85 coincidence detectors that respond to two input signals (Sando et al., 2019).

86 The central role of latrophilins as postsynaptic adhesion-GPCRs in synapse formation raises 87 the question whether latrophilins actually function as GPCRs in synapse formation. An 88 intrinsic GPCR signaling activity was suggested for several adhesion-GPCRs, including 89 latrophilins (Purcell and Hall, 2018; Nazarko et al., 2018; Müller et al., 2015; Ovando- 90 Zambrano et al., 2019; Scholz et al., 2017; Arac et al., 2016; Liebscher et al., 2014; 91 Vizarraga et al., 2020). However, few data exist for the physiological role of mammalian 92 adhesion-GPCRs in general, and for their GPCR activity in particular. Whether latrophilins 93 physiologically act as GPCRs remains unclear. Given the ubiquity of GPCRs in all cells, 94 especially in neurons, and the relative non-specificity of GPCR signaling, a GPCR-signaling 95 mechanism in synapse formation would be surprising and imply a tight spatial segregation 96 of such signals in neurons. Indeed, other potential mechanisms for the function of latrophilins 97 in synapse formation are equally plausible, for example a pure adhesion mechanism. To 98 address this question, we here examined the GPCR signaling functions of latrophilins in

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99 cultured neurons and in vivo, testing this question using multiple independent approaches 100 for two different latrophilins to ensure validity. Our data show that latrophilin-dependent 101 synapse formation requires their GPCR-signaling activity that may have been adapted to 102 synapses as a unique intercellular junction by use of a high degree of spatial 103 compartmentalization.

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105 RESULTS

106 Design and validation of Lphn2 and Lphn3 mutations. To examine whether latrophilins 107 act as GPCRs in synapse formation, we generated three types of mutants (Fig. 1A). The 108 Lphn3-ECD mutant deletes all transmembrane regions and cytoplasmic sequences of 109 Lphn3 and attaches its extracellular domains to the membrane using a GPI-anchor, thereby 110 preserving only the Lphn3 adhesion function and deleting all of its potential signaling 111 capacity. The Lphn2-T4L and Lphn3-T4L mutants contain an insertion of a T4 lysozyme 112 (T4L) sequence into the 3rd intracellular loop of Lphn2 or Lphn3, which abolishes G-protein 113 coupling and impairs arrestin signaling to these GPCRs (Rosenbaum et al., 2007; Cherezov 114 et al., 2007; Thorsen et al., 2014). The Lphn3-Ct mutant truncates most of the cytoplasmic 115 C-terminal sequences, thereby ablating cytoplasmic interactions (Fig. 1A). Of these three 116 types of mutants, the Lphn3-ECD mutant asks whether Lphn3 might simply act as an 117 adhesion molecule, the Lphn2-T4L and Lphn3-T4L mutants test whether GPCR-signaling is 118 involved in latrophilin function, and the Lphn3-Ct mutant examines the possible role of 119 binding partners for the cytoplasmic tail, such as Shanks (Kreienkamp et al., 2000; Tobaben 120 et al., 2000). All wild-type (WT) and mutant Lphn2 and Lphn3 proteins were encoded with 121 an N-terminal FLAG epitope tag to enable their detection on the cell surface (Fig. 1A).

122 We first investigated whether the Lphn2 and Lphn3 mutants are transported properly to the 123 cell surface of transfected HEK293 cells, and whether they are competent to engage in the 124 normal trans-cellular adhesion interactions of Lphn2 and Lphn3 with FLRTs and teneurins. 125 To test this question, we measured the ability of the mutants to support cell-aggregation 126 mediated by these interactions. We generated two populations of HEK293 cells that co- 127 expressed either FLRT3 or teneurin-2 (Ten2) with EGFP, or WT or mutant Lphn2 or Lphn3 128 with tdTomato, using transfections with empty plasmid DNA as a control. We then mixed the 129 two cell populations of HEK293 cells and measured cell aggregation (Boucard et al., 2014).

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130 The mutations had no effect on the surface transport of Lphn2 or Lphn3, nor did they impair 131 the ability of Lphn2 or Lphn3 to form trans-cellular adhesion complexes with FLRT3 or Ten2, 132 suggesting that the mutant latrophilins are folded and functional in protein-protein 133 interactions (Fig. 1B; S1).

134 We next expressed the WT and mutant Lphn2 and Lphn3 proteins in cultured hippocampal 135 neurons by lentiviral delivery. Using immunocytochemistry for the surface FLAG-epitope 136 present in all latrophilin proteins and for the dendritic marker MAP2, we visualized the 137 surface transport and subcellular localization of WT and mutant Lphn2 and Lphn3 proteins 138 in the neurons (Fig. 1C, 1D). All Lphn2 and Lphn3 proteins were efficiently transported to 139 the surface of dendritic spines, with no apparent difference in localization between WT and 140 mutant proteins. Quantifications documented that all mutant proteins were as well expressed 141 on the surface as WT proteins (Fig. 1C, 1D). Only the levels of the GPI-anchored Lphn3- 142 ECD protein were slightly lower on the cell surface than those of the other proteins, although 143 the difference was not statistically significant (Fig. 1C, 1D). Viewed together, these results 144 show that the various Lphn2 and Lphn3mutations do not impair their surface transport, 145 ligand interactions, and dendritic spine targeting.

146 Lphn2 and Lphn3 are constitutively active GPCRs whose signaling is blocked by the 147 T4L mutation. To test whether latrophilins exhibit GPCR activity and how such activity is 148 altered by the Lphn2 and Lphn3 T4L mutations, we examined whether latrophilins alter 149 cellular cAMP levels. We co-transfected HEK293T cells with the fluorescent cAMP indicator 150 (‘pink Flamindo’; Harada et al., 2017), EGFP as an internal fluorescence standard, Gs (a 151 G-protein) together with Gβ1γ2, and various WT and mutant Lphn2 and Lphn3 constructs. 152 In addition, we co-transfected subsets of cells with the high affinity cAMP-specific 153 phosphodiesterase PDE7b as a negative control (Hetman et al., 2000). Overexpressed 154 PDE7b should eliminate all cytoplasmic cAMP, enabling us to estimate the background 155 fluorescence signal. Imaging revealed that without latrophilins, PDE7b had no effect on pink 156 Flamindo fluorescence, suggesting that the observed signal represents background and that 157 the basal levels of cAMP in HEK293T cells are undetectably low at these acquisition settings 158 (Fig. 2A; Fig. S2). Expression of WT Lphn2 and Lphn3 caused a constitutive elevation of 159 pink Flamindo fluorescence. This fluorescence increase was blocked by PDE7b, 160 demonstrating that the pink Flamindo signal is specific for cAMP and that Lphn2 and Lphn3

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161 increase cAMP levels (Fig. 2A). The T4L mutation completely abolished the specific pink 162 Flamindo signal, suggesting that it blocks the GPCR activity of Lphn2 and Lphn3 (Fig. 2A).

163 A potential problem of the cAMP measurements with pink Flamindo is the rather high 164 background signal of pink Flamindo that is cAMP-independent (Fig. 2A). To further ensure 165 specificity of the measurements, we designed an indicator construct for measuring the 166 activity of cAMP-dependent protein kinase A (PKA). In this construct, a PKA-phosphorylation 167 sequence for which a phospho-specific-antibody is available is fused N-terminally to 168 tdTomato, which functions as an internal fluorescent control (Fig. 2B). Immunocytochemical 169 analysis of the PKA-indicator protein in HEK293T cells as a function of stimulation with 170 forskolin and IBMX revealed a strong cAMP-dependent signal with a much better signal-to- 171 noise ratio than the fluorescent pink Flamindo indicator (Fig. 2C).

172 We then used the newly constructed PKA-indicator to examine the effect of WT and mutant 173 Lphn2 and Lphn3 proteins on PKA activity. Co-expression of the PKA indicator with Gs 174 and various Lphn2 and Lphn3 proteins, without or with PDE7b, confirmed that Lphn2 and 175 Lphn3 both activate cAMP signaling (Fig. 2D). The Lphn2- and Lphn3-induced PKA- 176 indicator signal was greatly attenuated by PDE7b, confirming that it is due to cAMP. The 177 T4L-mutations of Lphn2 and Lphn3 similarly suppressed the PKA-indicator signal induced 178 by Lphn2 or Lphn3, and PDE7b had no additional effect (Fig. 2D). Together, these data 179 suggest that Lphn2 and Lphn3 stimulate cAMP production in cells in a manner dependent 180 on a GPCR transduction mechanism.

181 Lphn3 function in cultured hippocampal neurons requires both its GPCR activity and 182 its cytoplasmic sequence. We infected hippocampal neurons cultured from Lphn3 183 conditional KO mice with lentiviruses encoding Cre (control) or Cre, without or with addition 184 of Lphn3 rescue constructs. We measured synapse density by immunolabeling the neurons 185 for vGluT1 and PSD-95, which are pre- and postsynaptic markers of excitatory synapses, 186 respectively (Fig. 3A). As shown previously (Sando et al., 2019), the Lphn3 deletion robustly 187 decreased (~40%) the excitatory synapse density in cultured hippocampal neurons. This 188 decrease was fully rescued by WT Lphn3 but not by any of the three Lphn3 mutants (Lphn3- 189 ECD, Lphn3-T4L, and Lphn3-Ct) (Fig. 3A). This result suggests that Lphn3 functions as a 190 GPCR in synapse formation and that this function depends additionally on the cytoplasmic 191 sequences of Lphn3.

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192 To confirm this conclusion, we performed whole-cell patch-clamp recordings of mEPSCs 193 (Fig. 3B). The Lphn3 deletion caused a ~40% decrease in mEPSC frequency, which 194 corroborates the decrease in synapse density, but had only minor effects on the mEPSC 195 amplitude (Fig. 3C, 3D). Again, this phenotype was fully rescued by WT Lphn3 but not by 196 the three Lphn3 mutants tested, which were inactive even though they were fully transported 197 to the surface of dendritic spines (Fig. 1, 3C, 3D). These results suggest that Lphn3 functions 198 as a GPCR in synapse formation in cultured neurons, and that this function additionally 199 requires their long cytoplasmic tail region.

200 Lphn2 and Lphn3 function as GPCRs in the hippocampus in vivo. Do Lphn3 –and by 201 extension, Lphn2– function as GPCRs in vivo? To address this question, we sparsely 202 infected pyramidal neurons in the CA1 region of the hippocampus of newborn Lphn2 or 203 Lphn3 conditional KO mice with lentiviruses encoding Cre, without or with co-expression of 204 WT or G-protein-binding deficient Lphn2 (Lphn2-T4L) or Lphn3 (Lphn3-T4L). Three weeks 205 later, we measured synaptic connectivity via Schaffer-collateral and entorhinal inputs in 206 infected CA1 pyramidal neurons using whole-cell patch-clamp recordings in acute slices 207 (Fig. 4 and 5). We monitored AMPA-receptor-mediated synaptic responses in input-output 208 curves to control for differences in stimulus strength, using a dual stimulation approach that 209 enables separate measurements of Schaffer collateral- and entorhinal cortex-derived 210 synapses (Anderson et al., 2017; Sando et al., 2019). As a control, we measured uninfected 211 neurons in acute slices from the contralateral side of the same mice.

212 The Lphn2 deletion selectively impaired entorhinal cortical synaptic inputs but not Schaffer- 213 collateral synaptic inputs, confirming previous conclusions (Anderson et al., 2017). This 214 phenotype was rescued by WT but not by T4L-mutant Lphn2 that is unable to mediate GPCR 215 signaling (Fig. 4). The Lphn3 deletion, conversely, selectively impaired Schaffer collateral- 216 but not entorhinal cortex-derived synaptic inputs, again confirming previous findings (Sando 217 et al., 2019). This phenotype was also rescued by WT but not by T4L-mutant Lphn3 (Fig. 5), 218 confirming that GPCR signaling is essential for latrophilin function in synapse formation.

219 The fact that the input-output curves in the electrophysiological recordings (Fig. 4, 5) are 220 superimposable in these experiments attests to the solidity of the findings and the validity of 221 the approach for measuring synaptic connectivity. However, a decrease in synaptic strength 222 might be due to other causes than a loss of synaptic connectivity. To independently test

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223 these results, we used retrograde tracing of synaptic connections with pseudo-rabies viruses 224 (Callaway, 2008; Fig. 6A). Quantitative retrograde monosynaptic rabies tracing allows in vivo 225 analyses of synaptic connections formed by a specific postsynaptic neuron (Sando et al., 226 2019). Again, the Lphn3 conditional KO impaired synaptic connectivity (Fig. 6B, 6C). This 227 phenotype of rescued by expression of WT Lphn3, but not by the T4L-mutant Lphn3 unable 228 to mediate GPCR signaling (Fig. 6B, 6C). Viewed together, these data indicate that 229 latrophilins function as postsynaptic GPCRs that control input-specific excitatory synapse 230 formation in the hippocampus via a GPCR-dependent mechanism.

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232 DISCUSSION

233 Here, we demonstrate that Lphn2 and Lphn3 are constitutively active GPCRs that enhance 234 cAMP levels when expressed in HEK293 cells, and that the GPCR activity of Lphn2 and 235 Lphn3 is essential for their ability to promote synapse formation in vivo. In addition, we show 236 that Lphn3 requires its cytoplasmic sequences for supporting synapse formation. Based on 237 these findings, we propose that latrophilins act in synapse formation by transmitting a 238 GPCR-mediated signal, possibly by elevating cAMP. This is a surprising finding given that 239 GPCRs are present in many neuronal subcompartments where they mediate multifarious 240 regulatory functions. Thus, the latrophilin GPCR signal most likely is translated into specific 241 organizational activities during synapse formation in a context- dependent manner, either 242 via interactions of latrophilins with postsynaptic proteins such as SHANKs that are mediated 243 by their cytoplasmic sequences, or via parallel signals induced by other trans-synaptic 244 adhesion complexes.

245 Three lines of evidence support our conclusions. First, analysis of WT and mutant Lphn2 246 and Lphn3 in HEK293 cells demonstrated that WT Lphn2 and Lphn3 exhibit constitutively 247 active GPCR activity that elevates cAMP levels, and that this activity is blocked by the T4L 248 mutation, which is known to abolish GPCR signaling. Second, analysis of cultured neurons 249 demonstrated that the Lphn2 and Lphn3 mutations do not impair surface transport of Lphn2 250 and Lphn3 or targeting to dendritic spines, but that the Lphn3 mutations abolish its ability to 251 rescue the decrease in synapse formation induced by conditional KO of Lphn3. Lphn2 was 252 not tested in these experiments because they are labor-intensive and it was more relevant 253 to examine its function in vivo. Third, analysis of the in vivo activity of WT and mutant Lphn2

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254 and Lphn3 using sparse postsynaptic deletions of these molecules in hippocampal CA1 255 neurons in conditional KO mice demonstrated that WT Lphn2 and Lphn3 restored only the 256 loss of synaptic connectivity of their cognate synapses, but had no effect on those synapses 257 for which these latrophilins normally are not required. More importantly, these experiments 258 established that mutant Lphn2 and Lphn3 that is unable to mediate GPCR signaling is 259 incompetent to rescue the conditional KO phenotype. Together, these lines of evidence 260 show that Lphn2 and Lphn3 function in synapse formation as GPCRs.

261 However, the present study has inherent limitations. Most importantly, our results do not 262 identify the nature of the GPCR signal that is activated by latrophilins in synapse formation. 263 Latrophilins have been reported to increase or decrease cAMP levels in cells in a constitutive 264 manner (Nazarko et al., 2018; Müller et al., 2015; Ovando-Zambrano et al., 2019; Scholz et 265 al., 2017). Our results support a function in increasing cAMP (Fig. 2), but the physiological 266 signaling activities of GPCRs are complex and what the precise type of signaling of 267 latrophilins is in vivo may depend on the specific context. Although our results establish that 268 latrophilins act as GPCRs in synapse formation, they do not reveal what type of GPCR 269 signaling drives synapse formation. The notion that this signaling may consist of increases 270 in cAMP is attractive since pharmacological blockage of cAMP signaling impairs induction 271 of spine synapses by glutamate (Kwon and Sabatini, 2011), but no other evidence currently 272 shows that cAMP signaling –or any other GPCR signaling– mediates synapse formation. 273 Thus, this question remains a challenge for future studies that will require new tools and 274 approaches.

275 Moreover, the role of the cytoplasmic sequences of latrophilins remains unclear. These long 276 sequences contain multiple highly conserved regions, including a C-terminal PDZ-domain 277 binding motif (Sugita et al., 1998). What the function of these sequences is remains 278 uncharacterized, although it is tempting to speculate that these sequences confer context 279 specificity to the general GPCR activity of latrophilins by binding to specific interacting 280 partners. Such partners remain to be identified, although SHANKs are prime candidates 281 since they are postsynaptic scaffolding proteins that bind to latrophilins (Kreienkamp et al., 282 2000; Tobaben et al., 2000).

283 In summary, we here establish that Lphn2 and Lphn3 function as GPCRs in synapse 284 formation, suggesting the possibility that compartmentalization of a canonical signaling

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285 pathway widely used in biology and controlled by GPCRs may be instrumental in driving the 286 assembly of synaptic connections into circuits and control their specificity.

287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319

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320 FIGURES and FIGURE LEGENDS

321 322 Figure 1: Latrophilin-2 (Lphn2) and Latrophilin-3 (Lphn3) signal transduction mutants 323 engage in teneurin- and FLRT-mediated intercellular interactions on the cell surface. 324 A. Domain structures of wild-type and mutant Lphn2 and Lphn3. Mutants contain either only the 325 extracellular Lphn3 domains attached to the membrane via a GPI-anchor (Lphn3-ECD), block G- 326 protein coupling (Lphn2-T4L & Lphn3-T4L), or truncate the intracellular C-terminal tail of Lphn3 327 (Lphn3-ΔCt). B. Wild-type and mutant Lphn2 and Lphn3 proteins are robustly expressed on the cell 328 surface of HEK293 cells and efficiently mediate trans-cellular adhesion by binding to FLRT3 and 329 Ten2 ligands (left, representative images of trans-cellular aggregation assays as indicated (Ctrl = 330 empty plasmid); right, quantification of the aggregation efficiency for Lphn2 and Lphn3 constructs). 331 C & D. Wild-type and mutant Lphn2 (C) and Lphn3 proteins (D) are robustly expressed on the cell 332 surface of hippocampal neurons (left, representative images of dendritic segments of surface-labeled

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333 cultured hippocampal neurons infected with lentiviruses expressing the indicated proteins; right, 334 summary graphs of the surface levels of FLAG-tagged Lphn3 forms relative to MAP2 signal, as well 335 as the FLAG puncta area. Data in B-D are means ± SEM (n = 3 independent experiments).

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336 337 Figure 2: Lphn2- and Lphn3-induced cAMP signaling is blocked by insertion of T4- 338 lysozyme into the 3rd cytoplasmic loop. A. Expression of wild-type Lphn2 or Lphn3 in 339 HEK293T cells together with Gαs and Gβγ constitutively induces cAMP accumulation. The T4L

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340 mutation and co-expression of the cAMP-specific phosphodiesterase PDE7b prevent cAMP 341 increases by Lphn2 or Lphn3. The Pink Flamindo cAMP reporter (Odaka et al., 2014; Harada et al., 342 2017) was used to measure cAMP levels in live cells, and was quantified relative to the GFP signal 343 as an internal standard. B. Design of a new phospho-PKA indicator to measure PKA activation (top, 344 plasmid design; bottom, sequences of the N-terminal PKA phospho-peptide substrate (blue) that was 345 fused to the N-terminus of tdTomato (red) via a glycine-rich linker (green)). C. Characterization of 346 the phospho-PKA indicator in HEK293T cells. Treatment of cells with 100 µM Forskolin (FSK)/100 347 µM IBMX induces rapid phosphorylation of the phospho-PKA substrate (left, representative images; 348 right, quantification of the immunocytochemical phospho-PKA signal relative to the tdTomato signal 349 as an internal standard as a function of FSK/IBMX). D. Lphn2 and Lphn3 constitutively activate PKA 350 signaling in a manner blocked by the T4L-mutation. Experiments were performed as in A, but with 351 the PKA-indicator described in B & C (left, representative images; right, summary graph). Data are 352 means ± SEM (numbers of analyzed cells/experiments are indicated in bars). Statistical significance 353 was assessed by one-way ANOVA with post hoc Tukey tests for multiple comparisons (** = p<0.01; 354 * = p<0.05). For more representative images, see Fig. S2.

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355 356 Figure 3: Mutations that disrupt Lphn3 signal transduction abolish the ability of Lphn3 357 to rescue the synapse formation in Lphn3-deficient cultured hippocampal neurons. A. 358 Mutations of Lphn3 that delete its transmembrane regions (Lphn3-ECD), block GPCR signal 359 transduction (Lphn3-T4L), or delete its cytoplasmic tail (Lphn3-ΔCt) abolish the ability of Lphn3 to 360 rescue the decreased excitatory synapse density of Lphn3-deficient neurons despite engaging in 361 surface-ligand interactions. Hippocampal neurons cultured from Lphn3 cKO mice were infected at 362 DIV3 with lentiviruses encoding Cre (control) or Cre without or with co-expression of the indicated 363 Lphn3 rescue proteins, and were analyzed at DIV14-16. B-D. The same mutations as analyzed in A 364 also abolish the ability of Lphn3 to rescue the decreased mEPSC frequency of Lphn3-deficient 365 cultured hippocampal neurons. Data are means ± SEM (numbers of analyzed cells/experiments are 366 indicated in bars). Statistical significance was assessed by one-way ANOVA with post hoc Tukey 367 tests for multiple comparisons (** = p<0.01; * = p<0.05).

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368

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369 370 Figure 4: Abolishing GPCR signaling of Lphn2 by mutation of its 3rd cytoplasmic loop 371 blocks rescue of perforant-path synaptic connectivity in Lphn2-deficient CA1 region 372 neurons in vivo. Data are from patch-clamp whole-cell recordings in acute hippocampal slice from 373 Lphn2 conditional KO mice at P21-25. The CA1 region of the mice was injected at P0 with low titers 374 of lentiviruses expressing either Cre alone, or Cre together with the indicated rescue constructs to 375 induce sparse infection of CA1 pyramidal neurons (Sando et al., 2019). Control recordings were from 376 uninfected cells in slices in the opposite uninfected hemisphere. A-C. Sparse conditional KO of 377 Lphn2 in CA1-region neurons has no effect on the synaptic responses mediated by Schaffer- 378 collateral inputs, and overexpression of wild-type or T4L-mutant Lphn2 does not alter these 379 responses (A, sample traces; B, input-output curves; C, summary graph of the slopes of the input- 380 output curves). D-F. Sparse conditional KO of Lphn2 in CA1-region neurons impairs synaptic 381 responses mediated by entorhinal cortex inputs in a manner that can be rescued by wild-type but 382 not T4L-mutant Lphn2 (D, sample traces; E, input-output curves; F, summary graph of the slopes of 383 the input-output curves). Data are means ±SEM (numbers of cells/experiments are indicated in bars). 384 Statistical significance was assessed by one-way or two-way ANOVA with post hoc Tukey tests for 385 multiple comparisons (** denotes p<0.01; * denotes p<0.05). 386

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387 388 Figure 5: Abolishing Lphn3 GPCR activity by mutation of its 3rd cytoplasmic loop 389 blocks rescue of Schaffer collateral synaptic connectivity in CA1 region neurons in 390 vivo. Data are from patch-clamp whole-cell recordings in acute hippocampal slice from Lphn3 391 conditional KO mice at P21-25. The CA1 region of the mice was injected at P0 with low titers of 392 lentiviruses expressing either Cre alone, or Cre together with the indicated rescue constructs to 393 induce sparse infection of CA1 pyramidal neurons (Sando et al., 2019). Control recordings were from 394 uninfected cells in slices in the opposite uninfected hemisphere. A-C. Sparse conditional KO of 395 Lphn3 in CA1-region neurons impairs synaptic responses mediated by Schaffer-collateral inputs in 396 a manner that can be rescued by wild-type but not T4L-mutant Lphn3 (A, sample traces; B, input- 397 output curves; C, summary graph of the slopes of the input-output curves). D-F. Sparse conditional 398 KO of Lphn3 in CA1-region neurons has no effect on the synaptic responses mediated by perforant 399 pathway inputs, and overexpression of wild-type or T4L-mutant Lphn3 does not alter these 400 responses (D, sample traces; E, input-output curves; F, summary graph of the slopes of the input- 401 output curves). Data are means ±SEM (numbers of cells/experiments are indicated in bars). 402 Statistical significance was assessed by one-way or two-way ANOVA with post hoc Tukey tests for 403 multiple comparisons (** denotes p<0.01; * denotes p<0.05).

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404 405 Figure 6: Retrograde mono-synaptic rabies virus tracing confirms that Lphn3 GPCR 406 activity is essential for Schaffer-collateral synaptic connectivity in vivo. A. Schematic 407 of the experimental approach. The CA1 region of the hippocampus of Lphn3 conditional KO or

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408 control mice was injected at P0 with lentiviruses encoding Cre without or with the indicated rescue 409 constructs, at P21 with AAVs encoding Cre-dependent AAVs of rabies-complementing proteins, and 410 at P35 with pseudotyped rabies virus. Mice were analyzed by imaging at P40. B. Representative 411 images of monosynaptic rabies-virus tracing experiments from the four conditions analyzed. C. 412 Synaptic connectivity quantifications for the indicated synaptic inputs to CA1 region pyramidal 413 neurons starter cells in the hippocampal CA1 region. Rabies virus tracing demonstrates that the 414 impairment of Schaffer-collateral synaptic connectivity induced by deletion of Lphn3 is rescued by 415 wild-type Lphn3 but not by T4L-mutant Lphn3. Data are means ± SEM (numbers of mice are 416 indicated in bars). Statistical significance was assessed by one-way ANOVA with post hoc Tukey 417 tests for multiple comparisons (** denotes p<0.01; * denotes p<0.05). 418

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437 SUPPLEMENTARY FIGURES and FIGURE LEGENDS

438 439 Figure S1: Representative images of aggregation assays for wild-type and mutant 440 Lphn3 constructs show that the mutations do not alter surface expression and ligand 441 binding. Wild-type and mutant Lphn3 proteins used in rescue experiments are robustly expressed 442 on the cell surface of transfected HEK293 cells and efficiently mediate trans-cellular adhesion by 443 binding to FLRT3 and Ten2 ligands. Representative images show trans-cellular aggregation assays 444 using the indicated Lphn3 proteins and either FLRT3 or Ten2 as ligands; for summary graph, see 445 Fig. 1B. 446 447

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448 449 Figure S2: Representative images of cAMP measurements using Pink Flamindo (A) 450 and of PKA activity measurements using a new reporter protein (B) in HEK293T cells 451 expressing wild-type and mutant Lphn2 and Lphn3 constructs. A. Further representative 452 images for the green GFP used as an internal control and the red pink Flamindo signal during the 453 cAMP measurements shown in Fig. 2A. B. Further representative images for the green phospho- 454 peptide and the red tdTomato signal used as an internal control during the PKA measurements 455 shown in Fig. 2D.

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456 457 Figure S3: Immunoblotting analyses of neurons expressing Lphn2 (A) and Lphn3 458 constructs (B), and demonstration that expression of various Lphn3 constructs does 459 not grossly alter synapse morphology (C). A & B. Immunoblotting for indicated FLAG-tagged 460 Lphn2 (A) and Lphn3 proteins (B) in primary hippocampal cultures. β-Actin was used as a loading 461 control. Wild-type and mutant Lphn2 and Lphn3 proteins used in rescue experiments are expressed 462 at the expected molecular weight. C. Representative high-magnification images of synapse 463 morphology from Lphn3 rescue experiments shown in Figure 3. 464

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465 466 Figure S4: Intrinsic electrical properties of CA1 neurons are not significantly altered by the 467 Lphn2 and Lphn3 deletions and by wild-type and T4L-mutant Lphn2 and Lphn3 rescues in 468 the CA1 region of the hippocampus in vivo. A. Capacitance (left) and input resistance 469 measurements (right) of neurons analyzed in acute slices for the Lphn2 in vivo rescue experiments 470 shown in Figure 4. B. Same as A, except that the data are from neurons analyzed in the Lphn3 in 471 vivo rescue experiments shown in Figure 5. 472 473

474

475 METHODS

476 Key Resource Table

Reagent type Source or Additional (species) or Designation Identifiers reference information resource genetic reagent PMID: Lphn2 cKO JAX ID 023401 (Mus musculus) 28972101 genetic reagent PMID: HA-Lphn3 cKO JAX ID 026684 (Mus musculus) 30792275 Pure CD-1 background mice used to generate genetic reagent Charles River primary hippocampal CD-1 (Mus musculus) Laboratory cultures for surface expression experiments. cell line (homo HEK293T ATCC CRL-11268 sapiens) cell line (homo Thermo HEK293F R79007 sapiens) Fisher recombinant DNA Lentiviral Syn PMID: Also used in PMID:

reagent NLS-GFP-Δcre 21241895 30792275

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recombinant DNA Lentiviral Syn PMID: Also used in PMID:

reagent NLS-GFP-CRE 21241895 30792275 PMID: N-terminal IgK signal recombinant DNA pCMV WT 30792275, peptide followed by reagent hLphn3 26235030 FLAG N-terminal PMID: recombinant DNA preprotrypsin signal pCMV hFLRT3 30792275, reagent peptide followed by 26235030 MYC PMID: C-terminal FLAG; recombinant DNA pcDNA hTen2 30792275, extracellular HA prior to reagent 26235030 transmembrane region N-terminal PMID: preprotrypsin signal recombinant DNA pCAG WT 28972101, peptide followed by reagent rLphn2 30792275 FLAG; intracellular mVenus hLphn3 residues 20- 923 with an N-terminal recombinant DNA pCMV Lphn3- NCAM1 signal peptide this paper reagent ECD and a C-terminal NCAM1 GPI-anchoring sequence hLphn3 with a stop codon at residue 1117, recombinant DNA pCMV Lphn3- this paper causing deletion of the reagent ΔCt C-terminal tail (amino acids 1117-1447) hLphn3 with T4 recombinant DNA pCMV Lphn3- lysozyme peptide this paper reagent T4L inserted at Ile1039 in intracellular loop 3 rLphn2 with T4 recombinant DNA pCAG Lphn2- lysozyme peptide this paper reagent T4L inserted at Gly1045 in intracellular loop 3 Lentiviral shuttle recombinant DNA Lentiviral Syn PMID: plasmid containing reagent WT Lphn3 30792275 FLAG-tagged hLphn3 Lentiviral shuttle plasmid containing recombinant DNA Lentiviral Syn this paper Lphn3-ECD for rescue reagent Lphn3-ECD experiments in primary cultures Lentiviral shuttle plasmid containing recombinant DNA Lentiviral Syn this paper Lphn3-ΔCt for rescue reagent Lphn3-ΔCt experiments in primary cultures

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Lentiviral shuttle plasmid containing recombinant DNA Lentiviral Syn this paper Lphn3-T4L for rescue reagent Lphn3-T4L experiments in primary cultures Lentiviral shuttle Lentiviral Syn plasmid co-expressing recombinant DNA PMID: NLS-GFP-CRE NLS-GFP-Cre and WT reagent 30792275 p2a WT Lphn3 hLphn3 for in vivo rescue experiments Lentiviral shuttle Lentiviral Syn plasmid co-expressing recombinant DNA NLS-GFP-CRE this paper NLS-GFP-Cre and reagent p2a Lphn3-T4L hLphn3-T4L for in vivo rescue experiments Lentiviral shuttle Lentiviral Syn plasmid co-expressing recombinant DNA PMID: NLS-GFP-CRE NLS-GFP-Cre and WT reagent 30792275 p2a WT Lphn2 rLphn2 for in vivo rescue experiments Lentiviral shuttle Lentiviral Syn plasmid co-expressing recombinant DNA NLS-GFP-CRE this paper NLS-GFP-Cre and reagent p2a Lphn2-T4L rLphn2-T4L for in vivo rescue experiments A PKA phosphopeptide substrate (MRKVSKGEGGRRVS N) was fused to the N- Lentiviral EF1a terminus of tdTomato recombinant DNA phospho-PKA this paper separated by a linker reagent tdTomato (GGSGGGSGG) and was detected with the PKA phosphopeptide substrate antibody (sc- 56941) PMID: recombinant DNA pink Flamindo 28779099; Addgene reagent cAMP reporter Addgene #102356 AAV CAG PMID: recombinant DNA Rabies complementing FLEX TVA- 26232228; Addgene reagent AAV shuttle plasmid mCherry Addgene #48332 PMID: recombinant DNA AAV CAG Rabies complementing 26232228; Addgene reagent FLEX rG AAV shuttle plasmid Addgene #48333 1:500 IHC; 1:1,000 antibody anti-HA mouse Covance #MMS101R ICC; 1:2,000 IB Cell Signaling 1:2,000 ICC; 1:1,000 antibody anti-HA rabbit Technologies #3724 IHC

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anti-FLAG antibody Sigma 1:2,000 IB mouse #F3165 anti-beta Actin antibody Sigma 1:10,000 IB mouse #A1978 anti-MAP2 antibody Encor 1:2,000 ICC chicken #CPCA MAP2 anti-PSD95 Synaptic antibody 1:2,000 ICC mouse Systems #124011 anti-vGLUT1 antibody Millipore 1:2,000 ICC guinea pig #AB5905 anti-PKA phosphopeptid Santa Cruz antibody 1:1,000 ICC e substrate Biotechnology mouse #sc-56941 chemical Papain Worthington compound, drug #LS003127 chemical Matrigel Corning compound, drug #356235 chemical B-27 Gibco compound, drug supplement #17504044 Cytosine chemical arabinofuranosi Sigma compound, drug de #C6645 chemical DAPI Roche compound, drug #10236276001 chemical QX-314 Tocris compound, drug #1014 chemical Picrotoxin Tocris compound, drug #1128 chemical Tetrodotoxin Tocris compound, drug #1069 chemical Freestyle MAX Life compound, drug reagent Technologies #16447100 software, SnapGene GSL Biotech algorithm previously existing software, Molecular pClamp10 algorithm Devices previously existing software, Molecular Clampfit10 algorithm Devices previously existing software, NIS-Elements Nikon algorithm AR previously existing National software, ImageJ Institutes of algorithm Health previously existing software, Adobe Adobe algorithm Photoshop previously existing software, Adobe Adobe algorithm Illustrator previously existing software, Graphpad Graphpad algorithm Prism 8.0 software previously existing

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477 Mice. HA-Lphn3 cKO and Lphn2 cKO mice were previously described12,13. Overexpression studies 478 were conducted in primary hippocampal cultures from CD1 mice. Mice were weaned at 18-21 days 479 of age and housed in groups of 2 to 5 on a 12 h light/dark cycle with food and water ad libidum. 480 Stanford Animal Housing Facility: All procedures conformed to National Institutes of Health 481 Guidelines for the Care and Use of Laboratory Mice and were approved by the Stanford University 482 Administrative Panel on Laboratory Animal Care.

483 Plasmids. Schematic designs of all constructs utilized in this study are shown in Extended Data 484 Figure 1. Lentiviruses expressing NLS-GFP-∆Cre or NLS-GFP-Cre driven by the synapsin-1 485 promoter were described previously40 and were used for all Lphn3 cKO experiments in cultured 486 neurons or in in vivo manipulations. Lphn2 expression plasmids were based on rat Lphn2 lacking 487 SSA, with an exogenous N-terminal signal peptide from preprotrypsin followed by a FLAG epitope 488 (sequence: MSALLILALVGAAVADYKDDDDKLFSRAALPFGLVRRE…; signal peptide bolded and 489 FLAG epitope underlined), while the Lphn3 expression plasmids were based on human Lphn3 490 containing an insert in SSA, with an exogenous N-terminal signal peptide from IgKappa followed by 491 a FLAG epitope (sequence: 492 METDTLLLWVLLLWVPGSTGDAGAQDYKDDDDKFSRAPIPMAVVRRE…).

493 All latrophilin expression experiments in HEK293T cells were conducted with pCMV5 or pcDNA3 494 vectors using the CMV or CAG promoter, and all experiments in neurons and in vivo with lentiviruses 495 in which latrophilin expression was driven by the rat synapsin-1 promoter. For Lphn2 and Lphn3 496 rescue experiments, the following mutations and truncations were generated:

497 Lphn3-ECD: Encodes the Lphn3 extracellular sequence (residues 20-923) with an N-terminal 498 NCAM1 signal peptide and the C-terminal NCAM1 GPI-anchoring sequence.

499 Lphn2-T4L and Lphn3-T4L: Encodes Lphn2 or Lphn3 with an insertion of the T4 lysozyme 500 sequence into the intracellular loop 3 at Gly1045 of Lphn2 or at Ile1039 of Lphn3 (T4 lysozyme 501 sequence:

502 LENIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNTNGVITKDAEKLFNQ 503 DVDAAVRGILRNAKLKPVYDSLDAVRRAALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNL 504 AKSRWYNQTPNRAKRVITTFRTGTWDAY).

505 Lphn3-ΔCt: Encodes Lphn3 with a stop codon at residue 1117, causing deletion of the C-terminal 506 tail (amino acids 1117-1447). VRKEYGKCLR*

507 For phosphodiesterase PDE7b expression experiments, human PDE7b cDNA was used, with 508 HEK293T cell expression mediated by pCMV5 or pcDNA3 vectors.

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509 Phospho-PKA tdTomato Reporter: A PKA phosphopeptide substrate (MRKVSKGEGGRRVSN) 510 was fused to the N-terminus of tdTomato, separated by a linker (GGSGGGSGG) and was detected 511 via ICC with the PKA Phosphopeptide Substrate Antibody (Santa Cruz Biotechnology, BDI251, Cat# 512 sc-56941; 1:1,000 ICC).

513 Pink Flamindo cAMP Reporter: This plasmid was a gift from Tetsuya Kitaguchi (Addgene plasmid 514 #102356).

515 Rabies Complementing AAVs: AAV CAG FLEX TVA-mCherry and AAV CAG FLEX RG were gifts 516 from Liqun Luo (Addgene #48332 and #48333).

517 Antibodies. The following antibodies were used at the indicated concentrations (IHC- 518 immunohistochemistry; ICC-immunocytochemistry; IB-immunoblot): anti-HA mouse (Covance Cat# 519 MMS101R; 1:1,000 ICC, 1:2,000 IB), anti-HA rabbit (Cell Signaling Technologies Cat# 3724; 1:2,000 520 ICC), anti-FLAG mouse (Sigma Cat#F3165; 1:2,000 IB), anti-βactin mouse (Sigma Cat#A1978; 521 1:10,000 IB), anti-Map2 chicken (Encor Cat# CPCA MAP2; 1:2,000 ICC), anti-PSD95 mouse 522 (Synaptic Systems Cat# 124011; 1:2,000 ICC), anti-vGLUT1 guinea pig (Millipore Cat# AB5905; 523 1:2,000 ICC), PKA Phosphopeptide Substrate Antibody (Santa Cruz Biotechnology, BDI251, Cat# 524 sc-56941; 1:1,000 ICC), fluorescently-conjugated goat secondary antibodies from Life Technologies.

525 Cultured hippocampal neurons. Hippocampi were dissected from P0 Lphn3fl/fl or CD-1 mice, and 526 cells were dissociated by papain (Worthington, Cat# LS003127) digestion for 20 mins at 37ºC, 527 filtered through a 70 m cell strainer (Falcon Cat# 352350), and plated on Matrigel (Corning Cat# 528 356235)-coated 0 thickness glass coverslips (Assistent Cat# 01105209) in 24-well plates. Plating 529 media contained 5% fetal bovine serum (Atlanta), B27 (Gibco Cat# 17504044), 0.4% glucose 530 (Sigma), 2 mM glutamine (Gibco Cat# 25030164), in 1x MEM (Gibco Cat# 51200038). Culture media 531 was exchanged to Growth media 24 hrs later (1 DIV), which contained 5% fetal bovine serum 532 (Atlanta), B27 (Gibco), 2 mM glutamine (Gibco) in Neurobasal A (Gibco Cat# 10888022). Cytosine 533 β-D-arabinofuranoside hydrochloride (Sigma Cat#C6645) was added at a final concentration of 4 534 M on 3 DIV in a 50% growth media exchange, and neurons were analyzed 14-16 DIV.

535 Virus Production. For production of lentiviruses, the lentiviral expression shuttle vector and three 536 helper plasmids (pRSV-REV, pMDLg/pRRE and vesicular stomatitis virus G protein (VSVG)) were 537 co-transfected into HEK293T cells (ATCC), at 5 μg of each plasmid per 25 cm2 culture area, 538 respectively. Control conditions aside from GFP-∆Cre or GFP-infected controls were lentivirus 539 produced with empty shuttle vector. Transfections were performed using the calcium-phosphate 540 method. Media with viruses was collected at 48 hours after transfection, centrifuged at 5,000 x g for

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541 5 min to pellet cellular debris, filtered (0.45 μm pore size), and added directly to primary cultures for 542 in vitro experiments. For in vivo injections, virus was produced at the Janelia Farm Virus Core Facility 543 where infectious titer was determined, and equal numbers of viral particles were injected for each 544 experiment.

545 Immunocytochemistry. All solutions were made fresh and filtered via a 0.2 m filter prior to starting 546 experiments. Cells were washed briefly 1 x with PBS, fixed with 4% PFA (Electron Microscopy 547 Science Cat# 15714)/4% sucrose/PBS for 20 min at 4ºC, washed 3 x 5 min in PBS, and 548 permeabilized in 0.2% Triton x100/PBS for 5 min at room temperature. Cells were subsequently 549 placed in blocking buffer (4% BSA (Sigma Cat# 10735086001)/3% goat serum(Sigma Cat# 550 G9023)/PBS) 1 h, incubated with diluted primary antibodies in blocking buffer overnight at 4ºC, 551 washed 3 x 5 min in PBS, incubated with diluted fluorescently-conjugated secondary antibodies in 552 blocking buffer for 1 h, counterstained with DAPI in PBS for 15 mins at room temperature (Sigma 553 Cat# 10236276001), washed 3 x in PBS, and mounted on UltraClear microscope slides (Denville 554 Scientific Cat# M1021) using Fluoromount-G (Southern Biotech Cat#0100-01). For surface labeling 555 of FLAG-tagged Lphn constructs, cultures were immunolabeled for surface FLAG by staining without 556 permeabilization, and subsequently permeabilized in 0.2% Triton X-100/PBS and immunolabeled for 557 MAP2.

558 Immunoblotting. Samples were collected in sample buffer containing 312.5 mM Tris-HCl pH 6.8, 559 10% SDS, 50% glycerol, 12.5% 2-mercaptoethanol, bromophenol blue, and protease inhibitors 560 (Sigma Cat#5056489001) and run on SDS-PAGE gels (8% PAGE gels for FLAG-Lphn immunoblots 561 and 12% PAGE gels β-actin immunoblots) at 30 mA/gel constant current. The Precision Plus Protein 562 Dual Color Protein Standard (BioRad Cat# 1610374) was used as a protein molecular weight ladder. 563 Protein was transferred onto nitrocellulose transfer membrane in transfer buffer (25.1 mM Tris, 192 564 mM glycine, 20% methanol) at 200 mA constant current for 2 hrs at 4°C. Membranes were blocked 565 in 5% non-fat dry milk/TBST (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween-20) for 1 hr at 566 room temperature, incubated in primary antibodies diluted into 5% milk/TBST overnight at 4°C, 567 washed 3 x 5 mins in TBST, incubated in corresponding secondary antibodies (Licor IRDye 800CW 568 donkey anti-mouse Cat#92632212; IRDye 800CW Donkey anti-rabbit Cat#92632213) diluted into 569 TBST, washed 5 x 5 mins in TBST, and imaged on a Licor Odyssey system.

570 Electrophysiology of cultured neurons. For whole-cell patch clamp physiology experiments, the 571 patch pipettes were pulled from borosilicate glass capillary tubes (World Precision Instruments, Cat# 572 TW150-4) using a PC-10 pipette puller (Narishige). The resistance of pipettes filled with intracellular 573 solution varied between 2-4 MOhm. Synaptic currents were monitored with a Multiclamp 700B

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574 amplifier (Molecular Devices). The frequency, duration, and magnitude of the extracellular 575 stimulation were controlled with a Model 2100 Isolated Pulse Stimulator (A-M Systems, Inc.) 576 synchronized with Clampex 10 data acquisition software (Molecular Devices). For excitatory voltage- 577 clamp recordings, a whole-cell pipette solution was used containing (in mM) 135 Cs-

578 Methanesulfonate, 8 CsCl, 10 HEPES, 0.25 EGTA, 0.3 Na2GTP, 2 MgATP, 7 phosphocreatine, and 579 10 QX-314 (Tocris Cat#1014) (pH 7.3, adjusted with CsOH and 303 Osm). The external bath solution

580 contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 0.8 MgCl2, 10 HEPES, 10 glucose (pH 7.4, adjusted 581 with NaOH). AMPAR- and NMDAR-EPSC postsynaptic currents were pharmacologically isolated by

582 adding the GABAA receptor blocker picrotoxin (50 μM) (Tocris Cat#1128) to the extracellular bath 583 solution. AMPAR-EPSCs and mEPSC recordings were performed while holding the cell at -70 mV, 584 and NMDAR-EPSCs at +40 mV. Spontaneous miniature excitatory postsynaptic currents (mEPSCs) 585 were monitored in the presence of tetrodotoxin (1 µM) (Tocris Cat#1069) to block action potentials, 586 at -70 mV holding potential. Synaptic currents were sampled at 10 kHz and analyzed offline using 587 Clampfit 10 (Molecular Devices) software. Miniature events were analyzed using the template 588 matching search and a minimal threshold of 5 pA and each event was visually inspected for inclusion 589 or rejection by an experimenter blind to the recording condition.

590 Slice Electrophysiology. For acute slice electrophysiology, lentiviruses were injected into P0 mice, 591 and infected CA1 pyramidal neurons were analyzed at P21-25. Transverse hippocampal slices 592 (300 μm) were prepared by cutting in ice-cold solution containing (in mM): 228 Sucrose, 2.5 KCl, 1

593 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 7 MgSO4, 11 D-Glucose saturated with 95% O2/5% CO2. Slices 594 were transferred to a holding chamber containing artificial cerebrospinal fluid (ACSF, in mM): 119

595 NaCl, 2.5 KCl, 1 NaH2PO4, 26 NaHCO3, 2.5 CaCl2, 1.3 MgSO4, 11 D-Glucose, ∼290 mOsm. Slices 596 recovered at 32°C for 30 min, followed by holding at room temperature for 1 hr. Acute slices were 597 transferred to a recording chamber continuously superfused with oxygenated ACSF (1.5 ml/min) 598 maintained at 32°C. Neurons were clamped at -70 mV, and two-pathways of extracellular-evoked 599 EPSCs in hippocampal slices were monitored. AMPAR-EPSCs were evoked by electrical stimulation 600 using tungsten electrodes (Matrix electrode, 2x1, FHC Cat# MX21AEW(RT2)) positioned at the S. 601 radiatum 150 µm proximal to CA3, and the S. lacunosum-moleculare proximal to the molecular layer 602 of the dentate gyrus.

603 Sparse Lentiviral Infections in vivo. P0 Lphn2 fl/fl orLphn3 fl/fl pups were anesthetized on ice for 604 5 minutes and subsequently placed in a homemade clay mold. Lentivirus was loaded and injected 605 with a glass pipette connected to an infusion pump (Harvard Apparatus) completely continuous with 606 mineral oil. A stereotactic injection rig (Kopf) was used to target injections. Lentiviruses were diluted

32 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.10.430631; this version posted February 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

607 to a 1x107 IU/mL titer and 0.3 µL were injected at the following stereotactic coordinates from the 608 lambda at a 1 µL/min rate: A-P 1.0, M-L 0.9, D-V three subsequent injections at 1.4, 1.2, 1.0. Pups 609 then recovered in a clean cage placed on a heating pad, and were transferred back to their home 610 cage after completely recovering.

611 Monosynaptic retrograde rabies tracing. Lentiviral injections were performed at P0 as described 612 above. Complementing AAVs containing CAG-FLEX-TCB-mCherry and CAG-FLEX-RG were 613 generated at the Janelia Farm Viral Core Facility in capsid 2/5 and 2/8, respectively, and injected in 614 P21 mice. Adult mice were stereotactically injected mice by anesthetizing with an intraperitoneal 615 injection of tribromoethanol (Avertin) (250 mg/kg). Mouse heads were shaved, the shaved area was 616 cleaned with Betadine, lubricant was placed on eyes (Puralube Vet Ointment), and heads were 617 secured in a stereotactic injection rig (Kopf) and a small incision was made through the scalp with 618 sterilized tools. Viral solution was injected with a glass pipette at a flow rate of 0.15 µL/min and 0.5 619 µL volume per injection. Coordinates used for unilateral CA1 injections were AP −1.80 mm, ML 620 +/−1.35 mm, DV −1.25 mm. The injection pipette was left at the injection site for 5 mins after the 621 injection to prevent the spread of virus into neighboring brain regions. Incisions were sutured and 622 sealed with Vetbond tissue adhesive (1469SB), and mice were subsequently removed from the 623 stereotactic injection rig. Mice were monitored in a warmed recovery cage until full recovery. RbV- 624 CVS-N2c-deltaG-GFP (EnvA) was produced at the Janelia Farm Viral core facility and injected two 625 weeks after AAV injections at an infectious titer of 1x108 IU/mL and 0.5 µL volume as described 626 above. Mice were subsequently perfused and analyzed five days later. Brains were post-fixed 627 overnight in 4% PFA/PBS and sliced on a vibratome in 50 µm sections. Sections were labeled with 628 DAPI, washed with PBS, mounted and imaged using a Nikon A1 confocal microscope with a 20x 629 objective.

630 Imaging. Images were acquired using a Nikon A1 Eclipse Ti confocal microscope with a 10x, 20x 631 and 60x objective, operated by NIS-Elements AR v4.5 acquisition software. Laser intensities and 632 acquisition settings were established for individual channels and applied to entire experiments. 633 Image analysis was conducted using Nikon Elements, ImageJ, and Adobe Photoshop.

634 In vitro Synapse Imaging. Primary hippocampal cultures infected with lentiviruses encoding GFP- 635 ΔCre or GFP-Cre with or without co-expression of indicated rescue cDNAs were immunolabeled for 636 vGluT1 (guinea pig, Millipore Cat# AB5905; 1:2,000 ICC; in 488 channel), PSD-95 (mouse, Synaptic 637 Systems Cat# 124011; 1:2,000 ICC; in 546 channel), and MAP2 (chicken, Encor Cat# CPCA MAP2; 638 1:2,000 ICC; in 647 channel). Images were collected as described above with a 60x objective at 0.1 639 µm/pixel resolution and 0.2 µm z-stack intervals. Overlapping vGLUT1/PSD-95 puncta were

33 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.10.430631; this version posted February 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

640 automatically quantified along measured distances of MAP2-labeled primary and secondary 641 dendrites using Nikon NIS-Elements AR software.

642 Cell Aggregation Assay. FreeStyle HEK293F cells (Life Technologies Cat# R79007) grown to a 643 density of 1x106 cells/mL in a 30 mL volume were co-transfected with 30 μg of either pCMV5-Emerald 644 or pCMV5-tdTomato and 30 μg of the indicated construct using a FreeStyle Max reagent (Life 645 Technologies Cat# 16447100). All cDNAs were encoded in the pCMV5 vector. FreeStyle HEK293F

646 cells were grown at 37°C/8%CO2 with shaking at 125 rpms. Transfected cells were mixed in non- 647 coated 12-well plates at a 1:1 ratio two-days post-transfection and subsequently incubated for an 648 additional 2 h. Live cells were imaged in 12 well plates using a Nikon A1 confocal microscope. 649 Aggregation index was calculated using ImageJ, measuring the percentage of signal/frame occupied 650 by cells forming complexes of two or more cells relative to the total signal of the frame.

651 Statistics. Experiments were performed in a blinded manner whenever possible by coding viral 652 solutions. All data are expressed as means ± SEM and represent the results of at least three 653 independent biological replicates. Statistical significance was determined using the two-tailed 654 Student’s t-test, one-way ANOVA, or two-way ANOVA with post hoc Tukey tests for multiple 655 comparisons, as indicated in the Figure Legends. Data analysis and statistics were performed with 656 Microsoft Excel and GraphPad Prism 8.0.

657

658 ACKNOWLEDGEMENTS

659 We thank Irina Huryeva for technical assistance, and Shoji Maeda (Kobilka laboratory, 660 Stanford University), Brian Kobilka (Stanford University) and Demet Arac (University of 661 Chicago) for extensive discussions and advice.

662

663 ADDITIONAL INFORMATION

664 Funding

665 This study was supported by a grant from the NIH (K99-MH117235) to RS.

666

667 Author Contributions

34 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.10.430631; this version posted February 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

668 R.S. and T.C.S. designed the experiments and analyzed the experimental results; R.S., 669 performed the experiments, and R.S. and T.C.S. wrote the manuscript.

670

671 Author ORCIDs

672 Richard Sando

673 Thomas C. Südhof

674

675 Ethics

676 Animal experimentation: All animal procedures conformed to the National Institute of Health 677 Guidelines for the Care and Use of Laboratory Animals and were approved by the Stanford 678 University Administrative Panel on Laboratory Animal Care (protocol #2822).

679

680

681 CONFLICT OF INTEREST

682 The authors declare no conflict of interest.

683

35 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.10.430631; this version posted February 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

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39 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.10.430631; this version posted February 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

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