The Essential Role of Latrophilin-1 Adhesion GPCR Nanoclusters in Inhibitory Synapses

Latrophilin-1 (Lphn1, a.k.a. CIRL1 and CL1; gene symbol Adgrl1) is an Adhesion GPCR that has been implicated in excitatory synaptic transmission as a candidate receptor for α-latrotoxin. Here we analyzed conditional knockin/knockout mice for Lphn1 that contain an extracellular myc-epitope tag. Surprisingly, we found that Lphn1 is localized in cultured neurons to synaptic nanoclusters that are present in both excitatory and inhibitory synapses. Conditional deletion of Lphn1 in cultured neurons failed to elicit a detectable impairment in excitatory synapses but produced a decrease in inhibitory synapse numbers and synaptic transmission that was most pronounced for synapses close to the neuronal soma. No changes in axonal or dendritic outgrowth or branching were observed. Our data indicate that Lphn1 is among the few postsynaptic adhesion molecules that are present in both excitatory and inhibitory synapses and that Lphn1 by itself is not essential for excitatory synaptic transmission but contributes to inhibitory synaptic connections.


INTRODUCTION
Synapses are the fundamental unit of neuronal networks that mediate sensation, cognition and behavior.An enormous body of work described many of the fundamental processes that underlie synapse function, such as postsynaptic neurotransmitter reception, presynaptic vesicle release, or various forms of synaptic plasticity (reviewed in Kandel 2001, Südhof 2013, Basu and Siegelbaum 2015, Bailey et al. 2015, Chen and Gouaux 2019, Scott and Aricescu 2019).However, the mechanisms and molecules that enable synapses to be formed in the first place are poorly understood.Recent insights into synapse formation came from the study of synaptic adhesion molecules, such as neurexins, neuroligins, or adhesion GPCRs (aGPCRs) (reviewed in Südhof 2017, Cao and Tabuchi 2017, Südhof 2018, Sanes and Zipursky 2020, Gomez et al. 2021, Fuccillo and Pak 2021, Boxer and Aoto 2022, Cortés et al. 2023).Adhesion GPCRs are characterized by a combination of a large extracellular N-terminal region containing a variety of adhesion domains, a so-called GAIN-domain that mediates autoproteolysis and is invariably found in Adhesion GPCRs, a canonical 7 transmembrane domain typical for all GPCRs, and a rather long cytoplasmic region (reviewed in Hamann et al. 2015, Vizurraga et al. 2020, Rosa et al. 2021, Einspahr and Tilley 2022, Liebscher et al. 2022, Seufert et al. 2023).This domain combination places aGPCRs at the intersection of cell-cell or cell-matrix adhesion and signal transduction, although the precise functions of most aGPCRs are unclear.Multiple aGPCRs, including brain angiogenesis inhibitors (BAI's), latrophilins, and CELSR's, are thought to contribute to synapse formation (Najarro et al. 2012, Sigoillot et al. 2015, Wang et al. 2021, Wang et al. 2020, Bolliger et al. 2011, Shiu et al. 2022, Tu et al. 2018, Zhu et al. 2015, Stephenson et al. 2013, Aimi et al. 2023, Martinelli et al. 2016, Kakegawa et al. 2015, Freitas et al. 2023, Li et al. 2022, Zhou et al. 2021, Thakar et al. 2017, Anderson et al. 2017, Sando et al. 2019), although limited information is available about their mechanisms of action and scope of functions.
Latrophilins are postsynaptic receptors (Anderson et al. 2017, Sando et al. 2019) that contain an N-terminal lectin-like and olfactomedin-like domains that bind to presynaptic teneurin (Silva et al. 2011, Boucard et al. 2014) and presynaptic Flrt adhesion molecules (O'Sullivan et al. 2012), respectively.Intracellularly, latrophilins contain long cytoplasmic sequences that are expressed in multiple alternatively spliced variants, of which the most prevalent variant interacts with intracellular SHANK proteins (Kreienkamp et al. 2000, Tobaben et al. 2000), which are a component of postsynaptic density-protein scaffolds (Naisbitt et al. 1999).Super-resolution microscopy of the latrophilin ligand teneurin-3 revealed that it is not ubiquitously distributed within a synapse but assembled into nanoclusters (Zhang et al. 2022).Interestingly, latrophilin-mediated synapse formation requires G-protein coupling (Sando and Südhof 2021), suggesting that synaptic adhesion complexes constitute sites of active signaling.In the hippocampus, Lphn2 and Lphn3 mediate excitatory synapse formation onto different subcellular regions of CA1 pyramidal neurons independent of one another (Sando et al. 2019), whereas in the cerebellum Lphn2/3 act redundantly in parallel-fiber synapse formation (Zhang et al. 2020).Such "molecular codes" are also employed by other synaptic adhesion molecules, such as neurexins or neuroligins (summarized in Südhof 2017, Cao and Tabuchi 2017, Südhof 2018;Sanes and Zipursky 2020, Gomez et al. 2021, Fuccillo and Pak 2021, Boxer and Aoto 2022, Cortés et al. 2023).It is essential to investigate how each synaptic adhesion molecule contributes to synapse formation in order to generate a comprehensive view of the intricate complexity of neuronal connections in the brain.
Previous studies on latrophilins have been focused on Lphn2 and Lphn3, while Lphn1 has not been studied as intensely.Progress in studying Lphn1 was hindered by the lack of specific antibodies and behavioral abnormalities of Lphn1 constitutive KO mice that impeded breeding (Tobaben et al. 2002, Vitobello et al. 2022).A recent study revealed that human loss-of-function mutations of the Lphn1 ortholog (ADGRL1) cause major neurodevelopmental impairments and that a mouse mutant with a constitutive loss of Lphn1 exhibits major synaptic impairments (Vitobello et al. 2022).To further investigate the role of Lphn1 in brain, we here generated conditional knockout (cKO) mice that also allow localization of the endogenous protein by virtue of a knocked-in myc epitope tag.We show using STED microscopy that Lphn1 forms nanoclusters in both excitatory and inhibitory synapses of hippocampal neurons.However, in contrast to the other two isoforms, Lphn1 specifically mediates inhibitory synapse formation onto the cell soma.
Neuronal morphology was unchanged after Lphn1 deletion.These findings expand our knowledge of how latrophilins define different forms of synaptic connections, be it in excitatory versus inhibitory synapses or in the spatial restriction of synapses to subcellular regions of target neurons.

Generation and validation of Lphn1 conditional knockin/knockout (cKO) mice
Earlier studies of Lphn2 and Lphn3 were facilitated by the availability of conditional knockin/knockout mice in which endogenous Lphn2 or Lphn3 was tagged with an epitope for immunocyto-/immunohistochemical localization but could be deleted by expression of Cre recombinase (Anderson et al. 2017, Sando et al. 2019).Although constitutive Lphn1 KO mice were described (Tobaben et al. 2002, Vitobello et al. 2022), breeding Lphn1 KO mice is challenging because of a major behavioral phenotype (Tobaben et al. 2002, Vitobello et al. 2022).
Moreover, no highly specific Lphn1 antibodies are available.To overcome these challenges, we generated new Lphn1 conditional knockin/knockout (cKO) mice in which endogenous Lphn1 is tagged at the N-terminus with a myc epitope but can be deleted with Cre recombinase (Figure 1A).Lphn1 cKO mice were viable and fertile with no apparent behavioral abnormality.To validate the Lphn1 myc-tagging and conditional Lphn1 deletion, we cultured primary hippocampal cells from newborn mice and infected them with lentiviruses expressing inactive mutant ∆Cre recombinase (as a control) or active Cre recombinase (test) under control of the neuronal synapsin-1 promoter.
Both active Cre and mutant inactive ∆Cre recombinase were expressed as tdTomato fusion proteins that contain a nuclear localization signal which translocates the Cre and ∆Cre proteins into the nucleus.Upon expression of Cre but not ∆Cre, the floxed exon in Lphn1 cKO neurons is recombined, leading to a frameshift in the mRNA that abolishes Lphn1 protein synthesis.To test the efficiency of Cre recombination, we analyzed total protein from infected cultures by quantitative immunoblotting (Fig 1B,Suppl. Figure 1A).Whereas ∆Cre virus-infected cultures exhibited a ~115 kDa band corresponding to the Lphn1 N-terminal fragment that results from physiological autocleavage by Lphn1's GAIN domain (Araç et al. 2012), cultures infected with Cre viruses showed a >97% reduction of Lphn1 protein expression (Figure 1B, C).Thus, Cre expression abolished Lphn1 expression in the cultured cells.
We next performed surface labeling of the myc-Lphn1 protein in the same hippocampal cultures (Figure 1D).We observed punctate myc signals along dendrites in the ∆Cre condition that were largely absent from neurons in the Cre condition (Figure 1D, E), consistent with the Lphn1 protein deletion.Furthermore, when we stained cryosections of the brains from adult mice for myc-Lphn1 protein, we found that myc-Lphn1 was uniformly distributed among the various strata of the hippocampus (Figure 1F, G), indicating that the Lphn1 distribution differs from that of its paralogs Lphn2 and Lphn3 that are highly enriched in the S. lacunosum-moleculare (Lphn2) or the S. oriens and S. radiatum (Lphn3) (Anderson et al. 2017, Sando et al. 2019).

Lphn1 forms nanoclusters in both excitatory and inhibitory synapses
Lphn2 and Lphn3 are essential for excitatory but not inhibitory synapse formation and are localized to excitatory synapses, although their possible presence in inhibitory synapses has not been investigated (Anderson et al. 2017, Sando et al. 2019).We therefore examined whether Lphn1 localizes to excitatory and/or inhibitory synapses.As an initial approach, we surface-labelled myctagged Lphn1 in primary hippocampal cultures from the Lphn1 knockin/cKO mice, followed by detergent permeabilization of the cells and staining for excitatory and inhibitory synaptic markers (Figure 2, 3).We observed prominent co-labeling of myc-Lphn1 with synaptic markers but found that, surprisingly, Lphn1 co-localized with both excitatory (Figure 2A) and inhibitory synaptic markers (Figure 3A) in the cultured neurons.
Since conventional confocal microscopy is diffraction-limited and poorly resolves individual synapses, we aimed to confirm the presence of Lphn1 in both excitatory and inhibitory synapses using STED microscopy.To ensure that the Lphn1 signal we observe is specific, we examined both ∆Cre (Lphn1-expressing) and Cre (Lphn1-deleted) hippocampal cultures and stained them as described above.Intriguingly, we found that Lphn1 forms nanoclusters in excitatory synapses (Figure 2B, C) as previously shown for other synaptic adhesion proteins, for example for the Lphn ligand Teneurin-3 (Zhang et al. 2022).The majority of synapses in the ∆Cre condition displayed one to three nanoclusters, while the nanoclusters were absent in ~90% of synapses in the Cre condition (Figure 2D, E).The nanoclusters had a mean diameter of ~90-100 nm (Figure 2F), similar to the previously published dimensions of teneurin nanoclusters (Zhang et al. 2022).The deletion of Lphn1 did not significantly influence the area or staining intensity of pre-or postsynaptic specializations (Figure 2G-J), consistent with largely intact excitatory synapses after the Lphn1 deletion.
We next investigated inhibitory synapses.As suggested by conventional confocal microscopy, Lphn1 nanoclusters were prominently observed in inhibitory synapses, both on dendrites (Figure 3B, C) and the cell soma (Figure 3B, K).As a negative control, almost no clusters were found in inhibitory synapses in Cre-expressing neurons lacking Lphn1 (Figure 3D, L).Interestingly, in the ∆Cre condition, the number of nanoclusters per synapse was on average higher in somatic (mean = ~2.4nanoclusters, Figure 3L, M) than in dendritic inhibitory synapses (mean = ~1.8nanoclusters, Figure 3D, E), which in turn was higher than the average number of nanoclusters in excitatory synapses (mean = ~1.3nanoclusters, Figure 2D, E).However, the nanocluster size of ~90-100 nm was similar in all synapse types (Figure 2F, 3F, 3N).Pre-and postsynaptic areas had a small, statistically insignificant trend to being smaller in the Cre condition while the synapse-marker staining intensities were unchanged between ∆Cre and Cre expressing neurons (Figure 3G-J and 3O-R).
These data suggest that Lphn1 is localized to the majority of both excitatory and inhibitory synapses in cultured hippocampal neurons and that it assembles into similar nanoclusters in these synapses, although it is on average more abundant in inhibitory synapses.

Lphn1 is not required for neuronal morphogenesis
Earlier analyses of constitutive Lphn1 KO neurons suggested that Lphn1 interactions with teneurins promotes axonal outgrowth (Vysokov et al. 2018), whereas Lphn3 was proposed to mediate an axon-repellant signal (Pederick et al. 2021).Since functions in dendritic or axonal outgrowth could confound analysis of a role for Lphn1 in synapse assembly, we examined neuronal morphology in developing hippocampal neurons cultured from Lphn1 cKO mice.We lentivirally expressed Cre or ∆Cre in the neurons and additionally sparsely transfected them with an EGFP-expression plasmid, which allowed us to quantify the size and shape of axons and dendrites as a function of the Lphn1 deletion (Figure 4A-G).However, we detected no significant morphological changes caused by the Lphn1 deletion.In particular, the total axon length (Figure 4B), number of axon branch points (Figure 4C), total dendrite length (Figure 4D), number of dendrite branch points (Figure 4E) and soma size (Figure 4F) were not altered in Lphn1-deficient neurons compared to control neurons analyzed in parallel.The GFP fluorescence intensity as a measure of transfection efficiency was also unchanged between groups (Figure 4G).Thus, in culture, the Lphn1 deletion had no effect on axonal or dendritic development.

Lphn1 is also not required for excitatory synapse assembly
Previously, deletions of Lphn2 and Lphn3 were shown to decrease spine density and excitatory synapse numbers in hippocampal neurons both in culture and in vivo (Anderson et al. 2017, Sando et al. 2019).We therefore imaged EGFP-transfected cultured hippocampal neurons expressing or lacking Lphn1 at high resolution to monitor single spines (Figure 5A).Quantifications revealed only a minor trend towards a decreased spine density in Lphn1-deficient neurons, suggesting that different from the Lphn2 and Lphn3 deletions, the Lphn1 deletion does not significantly lower spine numbers (Figure 5B).
The lack of a change in spine numbers in Lphn1-deficient neurons suggests that, surprisingly, the Lphn1 deletion may not affect excitatory synapse formation, different from the Lphn2 and Lphn3 deletions (Anderson et al. 2017, Sando et al. 2019).Contrary to this suggestion, however, a previous study showed that cultured hippocampal Lphn1-deficient neurons exhibited a reduction in the staining intensities for presynaptic markers, suggesting a loss of synapses, although no measurements of synapse numbers were performed (Vitobello et al. 2022).To test this question directly, we stained Lphn1-deficient and control neurons by immunocytochemistry for vGLUT1 and SHANK, which are markers for pre-and postsynaptic excitatory synapses (Figure 5C), and measured the synapse density.Consistent with the lack of a change in spine density, the density of synaptic puncta was identical between control and Lphn1-deficient neurons (Figure 5D).The area and staining intensities of pre-and postsynaptic regions were also unchanged (Figure 5E-H).
The morphological analyses suggest that the Lphn1 deletion does not affect excitatory synapse numbers in pyramidal neurons.To verify these findings, we performed whole-cell patch-clamp recordings in control (∆Cre) and Lphn1-deficient (Cre) neurons in the presence of tetrodotoxin (TTX), and measured spontaneous miniature excitatory postsynaptic currents (mEPSCs) (Figure 5I).The mEPSC frequency and amplitude of these events were slightly, but not significantly elevated by the Lphn1 deletion (Figure 5J-M), while their kinetics were unchanged (Figure 5N, O).
Recording parameters and passive cell characteristics were similar in both conditions throughout all electrophysiological experiments (Suppl.Figure 2).The absence of an excitatory synapse phenotype in Lphn1-deficient pyramidal neurons is surprising in view of the robust Lphn1 expression in these synapses, the fact that an excitatory synapse phenotype was reported for a constitutive Lphn1 KO mouse (Vitobello et al. 2022), and the large decreases observed in excitatory synapse and spine numbers in Lphn2-and Lphn3deficient pyramidal neurons (Anderson et al. 2017, Sando et al. 2019).To independently confirm this observation, we generated a second Lphn1 cKO mouse using ES cells obtained from the EUCOMM consortium (Figure 6A).The initial mice generated in this approach express LacZ from the endogenous Lphn1 gene (Adgrl1), enabling an independent assessment of Lphn1 expression.
LacZ staining confirmed that Lphn1 is broadly expressed throughout the brain (Figure 6B).We then crossed the EUCOMM mice with a germline Flp recombinase-expressing mouse to generate Lphn1 cKO mice that contain normal Lphn1 levels (Figure 6A).Infection of cultured cKO hippocampal neurons with ubiquitin promoter-driven Cre recombinase resulted in a > 97% deletion of the floxed exon compared to a ∆Cre infected control (Figure 6C).We additionally crossed the cKO mice with a Cre-dependent tdTomato reporter mouse line (Ai14), and analyzed the spine density in the Lphn1 cKO/Ai14 mice as a function of the Lphn1 deletion in vivo.Specifically, we stereotactically infected CA1 pyramidal neurons in neonatal Lphn1 cKO mice with Cre-expressing lentiviruses and filled infected Lphn1-deficient and uninfected control cells with biocytin via the patch pipette (Figure 6D, E).Subsequent analyses of the spine density as a proxy for synapse density again failed to detect any change in Lphn1-deficient neurons compared to control neurons in the S. oriens, radiatum, or lacunosum-moleculare (Figure 6F-I).These findings provide further support for the unexpected conclusion that Lphn1 is not required in hippocampal pyramidal neurons for synapse formation.

Lphn1 is essential for normal assembly of somatic inhibitory synapses
Since the expression of Lphn1 in inhibitory synapses seems to be higher than in excitatory synapses (Figure 2, 3), we next stained cultured Lphn1-deficient and control hippocampal neurons for the inhibitory synapse markers vGAT and Gephyrin (Figure 7A).Dendritic inhibitory synapses lacking Lphn1 were nearly unchanged in density, size or marker intensity (Figure 7B-F), with only a small decrease in size and intensity.When examining somatic inhibitory synapses (Figure 7G), however, we observed a dramatic decrease (~50-60%) in puncta density (Figure 7H) as well as a minor decrease in pre-and postsynaptic area and staining intensity (Figure 7I-L).
To ensure that the decrease in inhibitory innervation is not a secondary effect due to lower numbers of inhibitory neurons in cultures infected with Cre virus, we determined the percentage of GABAergic neurons using GAD67 staining (Suppl.Figure 3A).In both conditions we estimated an interneuron density of ~10% (Suppl.Figure 3B).Staining intensity of GAD67 was also not significantly altered (Suppl.Figure 3C).Thus, in contrast to Lphn2 and Lphn3, Lphn1 is not required for excitatory synapse formation but is essential for inhibitory synapse formation, in particular for somatic inhibitory synapses.To verify these findings, we performed patch-clamp whole-cell recordings of miniature inhibitory postsynaptic currents (mIPSCs) in control and Lphn1-deficient hippocampal neurons in the presence of TTX (Figure 8A).When we analyzed total mIPSCs, we observed a minor decrease in mIPSC frequency of ~20% and a similar decrease in amplitude (Figure 8B-E), while the mIPSC kinetics were largely unchanged (Figure 8F, G).Interestingly, large amplitude mIPSCs seemed to be affected more severely than small amplitude mIPSCs (Figure 8A).mIPSCs originating from the cell soma are thought to exhibit larger amplitudes and faster rise times than mIPSCs generated in more distant dendrites (Wierenga and Wadman 1999).We therefore stratified mIPSCs into highamplitude and low-amplitude events using an amplitude threshold, defined as the 50% percentile in the amplitude distribution of the ∆Cre condition (average ~32 pA).Intriguingly, the highamplitude event frequency was reduced by ~40-50% in Lphn1 KO neurons (Figure 8H, I), while low amplitude events were unchanged (Figure 8N, O).Amplitudes and kinetics of both subsets were not altered between the Cre and ∆Cre conditions (Figure 8J-M and 8P-S).
To further validate an inhibitory synapse phenotype induced by the Lphn1 deletion, we next analyzed evoked inhibitory postsynaptic currents (eIPSCs) (Figure 9A).We observed a ~50% decrease in eIPSC amplitude as well as a modest increase in the coefficient of variation (Figure 9B, C).These results indicate that the Lphn1 deletion strongly impaired inhibitory synaptic transmission.The effect of the Lphn1 deletion is probably more robust as measured by eIPSCs than as measured by mIPSCs because somatic inhibitory synapses are functionally more potent than dendritic inhibitory synapses since they are closer to the recording electrode.Rise and decay times of eIPSCs were not significantly altered between Cre and ∆Cre expressing neurons (Figure 9D, E).

DISCUSSION
In the current study we generated conditional Lphn1 knockout mice that carry a knocked-in Nterminal myc epitope tag to enable us to immunolocalize the Lphn1 protein (Figure 1A-E).Using immunocytochemistry, we show that in the hippocampal CA1 region, Lphn1 is uniformly present throughout the dendritic tree of pyramidal neurons (Figure 1F, G), different from Lphn2 and Lphn3 that are enriched in the S. lacunosum-moleculare or the S. oriens and S. radiatum, respectively (Anderson et al. 2017, Sando et al. 2019).Moreover, we find in cultured hippocampal neurons using STED super-resolution microscopy that, unexpectedly, Lphn1 forms nanoclusters in both excitatory and inhibitory synapses (Figure 2, 3).The majority of excitatory and inhibitory synapses have at least one nanocluster, with a third of excitatory synapses (Figure 2E) and approximately 44% of dendritic and 59% of somatic inhibitory synapses featuring more than one Lphn1 nanocluster (Figure 3E, M).Consistent with the enhanced presence of Lphn1 in somatic inhibitory synapses, we found that in cultured hippocampal neurons the deletion of Lphn1 caused a significant decrease in the number of somatic but not dendritic inhibitory synapses (Figure 7).In parallel, we observed an impairment in inhibitory synaptic strength that most likely was due to the decrease in inhibitory somatic synapses.Specifically, measurements of mIPSCs revealed that the frequency of high-amplitude mIPSCs but not of low-amplitude mIPSCs that probably reflect synaptic events close to or distant from the soma, respectively, was significantly decreased (Figure 8H, N).Furthermore, the amplitude of evoked IPSCs was lower in Lphn1-deficient than control neurons without a significant change in the coefficient of variation (Figure 9).However, we observed no other major changes in Lphn1-deficient neurons.Neither dendritic nor axonal arborizations were changed (Figure 4), spine numbers were not decreased, excitatory synapse numbers were not lowered (Figure 5), and mEPSCs were not significantly changed (Figure 5).
The lack of a change in spines was confirmed by in vivo deletion of Lphn1 using a second independent Lphn1 cKO mouse line, suggesting that it is not an artifact of one particular mouse line (Figure 6).
By demonstrating that Lphn1 is present in both excitatory and inhibitory synapses, Lphn1 emerges as one among few postsynaptic adhesion molecules that are in both classes of synapses, which otherwise share few postsynaptic components since their receptors and scaffolding molecules are different.With a diameter of approximately 90 nm, the sizes of the nanoclusters formed by Lphn1 in excitatory and inhibitory synapses are comparable (Figure 2F, 3F, 3N), suggesting a similar molecular organization.The Lphn1 nanoclusters we describe here using STED super-resolution microscopy closely resemble those previously observed using dSTORM for presynaptic teneurin-3 (Zhang et al., 2022), neurexins (Lloyd et al., 2023;Trotter et al., 2019), and neuroligins (Haas et al., 2018;Nozawa et al., 2022;Han et al., 2022), suggesting that such nanoclusters are a general feature of synaptic adhesion molecules.The detection of synaptic Lphn1 nanoclusters in both excitatory and inhibitory synapses also resolves the previously puzzling observation that teneurins, which are presynaptic latrophilin ligands, are broadly expressed in both excitatory and inhibitory neurons and could thus represent presynaptic interaction partners for postsynaptic Lphn1 in both types of synapses.
Our findings also raise important questions.First, are Lphn2 and Lphn3 also present in inhibitory synapses like Lphn1?This seems likely but has not yet been studied.Second and more puzzlingly, why doesn't the Lphn1 deletion in our experiments produce a major phenotype in excitatory synapses, given that the Lphn2 and Lphn3 deletions cause major impairments in excitatory synapses and that previous studies on constitutive Lphn1 KO mice characterized significant impairments in excitatory synapse function (Vitobello et al. 2022)?The most plausible explanation for this discrepancy is that the Lphn1 deletions were analyzed on very different genetic backgrounds in mice and that analyses were performed on conditional vs. constitutive deletions.
It seems unlikely that the discrepancy is due to a technical issue in our study or that of Vitobello et al. (2022) since we have established that our deletion indeed abolishes Lphn1 protein expression as expected from the genetic strategy (Figure 1A-E) and the phenotypes we observed in inhibitory synapses (Figure 7-9) and Vitobello et al. (2022) described in excitatory/inhibitory synapses appear to be very robust.Note, however, that another previous paper on constitutive Lphn1 knockout mice using neurotransmitter release from synaptosomes as an assay also did not detect a major change in excitatory synapses, although a synaptic density phenotype would have been missed in that study (Tobaben et al., 2002).
Another difference between the present results and another previous analysis of Lphn1 constitutive KO mice is that we did not detect changes in axonal outgrowth (Figure 4), whereas the constitutive Lphn1 KO neurons were found to display axonal growth defects (Vysokov et al. 2018).Such defects are interesting since functions in neuronal morphogenesis were also reported for the C. elegans latrophilin homolog LAT-1 (Matúš et al. 2022).However, only fluorescence intensities of stained axons in fields of view were measured in the previous analysis and axon lengths were not tracked in cultured neurons (Vysokov et al. 2018).While these methods can give hints towards neuronal functions, they are easily confounded by variations in culture density or composition.Moreover, it is puzzling that some experiments assign an attractive function of the latrophilin-teneurin interaction in axonal outgrowth (Vysokov et al. 2018), whereas others propose a repellent function (Pederick et al. 2021).Moreover, latrophilins were proposed to have an essential role in neuronal migration during brain development (Del Toro et al. 2020), even though major developmental effects on cortical layers by the constitutive Lphn1 deletion were not apparent (Tobaben et al. 2002;Vysokov et al. 2018;Vitobello et al. 2022).At present these contradictions cannot be resolved althoughagaingenetic backgrounds may play a role, an issue that is challenging to resolve.
In summary, our results reveal the presence of similar Lphn1 nanoclusters in both excitatory and inhibitory synapses, with most synapses in hippocampal cultures featuring at least one nanocluster.Moreover, our findings uncover an essential function for Lphn1 in inhibitory synapses.
These results expand our view of the synaptic role of latrophilins, suggesting a general function in most synapses independent of their transmitter type.However, major questions arose that need to be addressed in future, such as that of the role of Lphn1 in excitatory synapses.Such a role might be occluded by redundancy, or Lphn1 might be without function in these synapses.Given the differences in results with distinct mouse lines and the possibility of major genetic background effects, a more reductionist approach may be needed to deconstruct the contribution of latrophilins in general and Lphn1 in particular to different types of synapses.For example, it is possible that latrophilins function in the same pathway as other aGPCRs, such as BAI's and CELSRs that also have synaptic functions (Najarro et al. 2012, Sigoillot et al. 2015, Wang et al. 2020, Wang et al. 2021, Bolliger et al. 2011, Shiu et al. 2022, Tu et al. 2018, Zhu et al. 2015, Stephenson et al. 2013, Aimi et al. 2023, Martinelli et al. 2016, Kakegawa et al. 2015, Freitas et al. 2023, Li et al. 2022, Zhou et al. 2021, Thakar et al. 2017), and that a more extensive deletion of multiple genes will be necessary to unravel the making of a synapse!(B) Immunoblotting validation of Lphn1 cKO mice.Three independent primary hippocampal cultures from Lphn1 cKO mice were infected with lentiviruses encoding active (Cre) or enzymatically inactive recombinases (ΔCre) that contained a nuclear localization signal and were fused to tdTomato.In addition, hippocampal cells from a single wild-type culture (wt) were analyzed.Blots were stained for the knocked-in myc-tag (top) or actin as a loading control (bottom).Note that owing to GAIN-domain mediated autocleavage, the myc-tag labels the N-

Figure 1 :
Figure 1: Generation of Latrophilin-1 (Lphn1) conditional knockout (cKO) mice in which endogenous Lphn1 carries an N-terminal myc-tag (A) Design of Lphn1 cKO mice.A 2xmyc tag sequence was introduced into the second exon of the Lphn1 gene (Adgrl1) and the exon was flanked by loxP sites using homologous recombination in ES cells.