C1ql1-Bai3 signaling is necessary for climbing fiber synapse formation in mature Purkinje cells in coordination with neuronal activity

Takahiro Aimi; Keiko Matsuda; Michisuke Yuzaki

doi:10.1186/s13041-023-01048-4

C1ql1-Bai3 signaling is necessary for climbing fiber synapse formation in mature Purkinje cells in coordination with neuronal activity

Aimi, Takahiro; Matsuda, Keiko; Yuzaki, Michisuke 2023-07-24 00:00:00 Changes in neural activity induced by learning and novel environments have been reported to lead to the formation of new synapses in the adult brain. However, the underlying molecular mechanism is not well understood. Here, we show that Purkinje cells (PCs), which have established adult-type monosynaptic innervation by climbing fibers (CFs) after elimination of weak CFs during development, can be reinnervated by multiple CFs by increased expression of the synaptic organizer C1ql1 in CFs or Bai3, a receptor for C1ql1, in PCs. In the adult cerebellum, CFs are known to have transverse branches that run in a mediolateral direction without 2+ forming synapses with PCs. Electrophysiological, Ca -imaging and immunohistochemical studies showed that overexpression of C1ql1 or Bai3 caused these CF transverse branches to elongate and synapse on the distal dendrites of mature PCs. Mature PCs were also reinnervated by multiple CFs when the glutamate receptor GluD2, which is essential for the maintenance of synapses between granule cells and PCs, was deleted. Interestingly, the effect of GluD2 knockout was not observed in Bai3 knockout PCs. In addition, C1ql1 levels were significantly upregulated in CFs of GluD2 knockout mice, suggesting that endogenous, not overexpressed, C1ql1-Bai3 signaling could regulate the reinnervation of mature PCs by CFs. Furthermore, the effects of C1ql1 and Bai3 overexpression required neuronal activity in the PC and CF, respectively. C1ql1 immunoreactivity at CF-PC synapses was reduced when the neuronal activity of CFs was suppressed. These results suggest that C1ql1-Bai3 signaling may mediate CF synaptogenesis in mature PCs, potentially in concert with neuronal activity. Keywords Cerebellum, Purkinje cell, Climbing fiber, Synapse, Electrophysiology, C1ql1, Bai3 Introduction pathophysiology of various neurological and neuropsy- Neural circuits are formed and refined in response to chiatric disorders [5, 6]. However, the molecular mech- changes in neural activity associated with learning and anisms underlying structural plasticity in adult brains new environments, not only during development but remain largely unknown, partly due to the complexity of throughout life [1–4]. The increasing evidence suggests neuronal circuits consisting of many heterogenous syn- that such structural plasticity plays a key role in the apses. Activity-dependent structural plasticity during development has been extensively studied in the cerebel- lar cortex, which contains simple and well-defined neu - ronal circuits. Purkinje cells (PCs), which send the only *Correspondence: Michisuke Yuzaki outputs from the cerebellar cortex, receive two excitatory myuzaki@keio.jp inputs, parallel fibers (PFs) from granule cells and climb - Department of Physiology, Keio University School of Medicine, ing fibers (CFs) from the inferior olive neurons (IONs) at Tokyo 160-8582, Japan © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Aimi et al. Molecular Brain (2023) 16:61 Page 2 of 17 proximal and distal dendrites, respectively. Several hun- in CFs and PCs, respectively, transverse branches of CFs dred thousand PFs make synapses on spines located on elongated and formed new synapses with distal dendrites, the distal dendrites of a single PC. In contrast, although allowing PCs to be re-innervated by multiple CFs. This multiple CFs initially innervate a single immature PC, a process required neural activity of both CFs and PCs, single CF becomes dominant in an activity-dependent suggesting that C1ql1-Bai3 signaling may be involved in manner, forming synapses on the proximal dendrites the activity-dependent CF synapse modification in the while the rest of the CFs are eliminated by the end of the mature PCs. 3rd postnatal week in mice [7, 8]. Interestingly, the cer- ebellar cortex undergoes remarkable changes in its archi- Methods tecture after damage or alteration of neural activity, even Animals in adulthood. For example, PC spine density is increased All procedures relating to the care and treatment of by motor-skill learning [9] or exposure to an enriched mice were performed in accordance with the guide- environment [10]. Conversely, inhibition of neuronal lines approved by the animal resource committee of activities by application of the Na channel blocker tetro- Keio University. Mice of the following strains were used: dotoxin or the AMPA-receptor antagonist NBQX to the Htr5B-tTA and tetO-YC mice [25] (a gift from Prof. Kenji adult cerebellar cortex replaced CF synapses with PF syn- Tanaka, Keio Univ Sch Med), conditional Bai3 knockout f/f f/f apses on proximal dendrites [11–13]. u Th s, the cerebel - mice (Bai3 ) [18], conditional GluD2 (Grid2 ) mice [20], lar cortical circuits provide a valuable model to elucidate GluD2 knockout mice [17], C57B6/N mice (Japan SLC, molecular mechanisms underlying structural plasticity in Inc.). The constitutive Bai3 knockout mouse was gener- f/f the adult brain. ated by crossing Bai3 with telencephalin-Cre transgenic Many synaptic organizers regulate synapse forma- mice as previously reported [30, 37]. Mice were housed tion, maturation, or elimination during development with a 12:12 h light-dark cycle with food and water [14]. Among them, the C1q family is crucial in organiz- available ad libitum. The sex for the virus injections, ing excitatory inputs to PCs during development [15]. immunohistochemistry, and electrophysiology was not Cbln1, a member of the C1q family secreted from PFs, distinguished. regulates PF-PC synapses by binding to the GluD2 glu- tamate receptor expressed on distal dendrites of PCs [16, Cell lines 17]. In contrast, C1ql1, provided by CFs, mediates CF-PC HEK293 cells (tSA line; gift from Dr. R. Horn, Thomas synapse formation on the proximal dendrites by binding Jefferson Univ., PA) and AAV-293 cells (Cell Biolab) to the adhesion G-protein-coupled receptor Bai3 [18, 19]. were cultured with Dulbecco’s modified Eagle medium Interestingly, both Cbln1-GluD2 and C1ql1-Bai3 pro- (DMEM, D5796, Sigma-Aldrich) with 10% Fetal Bovine tein pairs remain expressed in the adult cerebellum after Serum (#004–00025, Japan Bioserum), 50 U/mL penicil- PF- and CF-PC synapses are established. When C1ql1 or lin/streptomycin (15140-122, Thermo Fisher Scientific), Bai3 is deleted in the IONs or PCs, respectively, CF syn- and 2 mM L-Glutamine. Cells were incubated at 37 °C in apses are lost from the proximal dendrites of PCs in the the 10% CO incubator. adult cerebellum [18]. Similarly, when GluD2 is deleted in the mature PCs, PF synapses are gradually lost from cDNA constructs the distal dendrites [20]. Furthermore, although PCs are Some constructs of Bai3, C1ql1 and plasmids for len- normally innervated by single CFs on proximal dendrites, tivirus preparation were previously reported [18]. Bai3 the loss of GluD2 in the adult cerebellum causes multiple mutants were generated by two-step overlapping PCRs. CFs to innervate distal PC dendrites where PFs normally To visually detect the cells expressing genes of inter- make synapses [20]. Intriguingly, similar changes in the est, the fluorescent proteins (GFP, YFP or mCherry) are innervation pattern of PCs by PFs and CFs are observed linked by a self-cleaving 2 A peptide (P2A, F2A) from when neuronal activity is inhibited in the adult cerebel- the foot-and-mouth-disease virus were co-expressed lum [11–13]. Furthermore, Cbln1 is secreted from PFs in [18]. cDNAs encoding ChR2-eYFP (#110,339), tdTomato an activity-dependent manner [21], while Cbln1 mRNA (#104,112) and Cre (#107,738) were obtained from Add- expression is repressed by prolonged neuronal activities gene. The minimal L7 promoter [ 39] was synthesized [22, 23]. These results suggest that C1q family proteins (Eurofin genomics). The sequence of ESKir2.1 and pAAV- may play a role in activity-dependent structural plasticity DJ was kindly provided by Dr. Kazuya Togashi (Grad in the cerebellum after PF- and CF synapses are matured. School of Science, Univ. Tokyo). pAAV-TRE, pAAV-L7 In the present study, we investigated whether and how and pAAV-Syn-DIO vectors were purchased from Add- C1ql1-Bai3 signaling is involved in CF-PC structural gene (#104,112, #58,867 and #50,459). pHelper were pur- plasticity in mature cerebellar circuits. We found that chased from Cell Biolab. when the expression of C1ql1 and Bai3 was upregulated Aimi et al. Molecular Brain (2023) 16:61 Page 3 of 17 Virus preparation KCl, 2 CaCl , 1 MgCl , 26 NaH PO , 10 Glucose and 0.1 2 2 2 4 Lentiviruses were used to express Bai3 or Cre in PCs. picrotoxin, bubbled with a mixture of 95%O and 5%CO . 2 2 Lentivirus was prepared as previously reported [18] with The transferred slices were incubated with ACSF at room small modifications. The cDNAs were cloned into the temperature for more than 1 h to recover. pCL20c vector and expressed under the MSCV promoter. Whole-cell patch-clamp recordings were made from Lentiviruses were produced by transfecting the pCL20c visually identified PCs using an x60 water-immersion vector and three helper plasmids (pCAG-KGRIR, pCAG- objective attached to an upright microscope (BX51WI, RTR2 and pCAG-VSVG) into HEK293tSA cells using a Olympus) at room temperature. Intracellular solutions calcium phosphate method. 36–40 h after the transfec- were composed of (in mM): 150 Cs-gluconate, 10 HEPES, tion, the culture media were collected and centrifuged at 4 MgCl , 4 Na ATP, 1 Na GTP, 0.4 EGTA and 5 lidocaine 2 2 2 24,000 rpm for 2 h at 2 ℃ to concentrate the virus. After N-ethyl bromide (QX-314) (pH 7.25, 290–300 mOsm/kg) the centrifugation, the pellets were dissolved with a small for the CF- and PF-EPSC recordings. The patch pipette volume of cold medium and stored frozen at -80 ℃. The resistance was 1–2 MΩ. 8–9 lentivirus was at a 10 titer unit. To evoke CF-EPSCs, a glass pipette filled with ACSF AAV-DJ vectors were used to express C1ql1 in CFs was placed on the granular layer near (20–100 μm) the and ESKir2.1 and Cre in PCs. AAVs were produced by PC voltage-clamped at -10 mV and square paired pulses transfecting the pAAV vector, pHelper and pAAV-DJ (20 µs duration, 0–300 µA and 50 ms inter-stimulus into 293AAV (Cell Biolab, lnc) using a calcium phosphate interval) were applied. Selective stimulation of CFs was method. 36–40 h after transfection, cells were collected confirmed by the paired-pulse depression of EPSC and AAVs were purified by using an AAV purification kit amplitudes. The number of functional CF synapses on (Takara #6666) following the manufacturer’s protocols. single PCs was estimated by varying the stimulus inten- As for the pAAV vectors, SmaI digestion was performed sity because a single CF input has a single threshold for to confirm that the two SmaI sites within the ITR were excitation. maintained. The titer of AAVs was determined by using To estimate the location of stimulation for evoking the AAVpro Titration Kit (Takara #6233) and calculated main or surplus CF-EPSC, the XY coordinates of the 10–11 as 10 vector genomes/mL. stimulation location were defined with the center of the PC soma as the origin. The stimulus electrode was moved Stereotaxic injection systematically every 10 μm in the XY direction on the Mice (3–4 weeks) were anesthetized with a mixture of surface of the granular layer. Square paired pulses (20 µs ketamine (80 mg/kg body weight) and xylazine (20 mg/ duration, 0–300 µA and 50 ms inter-stimulus interval) kg body weight) by intraperitoneal injection. To express were applied at about 40 stimulation points, respectively. the genes into PC, glass pipettes filled with the AAV and/ Nearest distances for evoking main or surplus CF-EPSC or lentivirus solution (2–4 µL) were inserted into the cer- were calculated as the mean of 3 stimulation points clos- ebellar vermis at the depth of 0.5 mm. The solution was est to the origin, at which main or surplus CF-EPSC were injected at a speed of 0.25 µL/min. evoked. To express genes in the CFs, glass pipettes were To ensure that EPSCs were evoked by CFs trans- inserted into the ION according to the reported method duced by AAV, we optogenetically stimulated CFs by with small modifications [ 40]. The AAV solution (1.0– co-expressing ChR2 in IO neurons in Figs. 2 and 7. Light 1.5 µL) was injected at a speed of 0.2 µL/min. After the stimulation (wavelengths: ~470 nm, 4–10 ms dura- injections, the incised skin was closed by using adhesive tion, 100 ms inter-stimulus interval) was applied from and the mice transiently stayed in the recovery cages for a mercury lamp (Olympus) combined with a mechani- 12–16 h. Then mice were returned to their home cages. cal shutter. The number of functional CF synapses on single PCs was estimated by varying the light intensi- Electrophysiology ties (0–4 mW/mm ) and durations. Some PCs (15–20%) Mice were anesthetized with isoflurane and the brains did not respond to light stimulation, most likely because were rapidly removed and immersed in an ice-cold cho- not all CFs expressed ChR2. Due to the exclusion of line-based cutting solution containing (in mM): 120 Cho- non-responding PCs, the number of functional CFs line Cl, 3 KCl, 1.25 NaH PO , 28 NaHCO , 8 MgCl , 22 determined by optogenetic CF stimulation is likely an 2 4 3 2 Glucose and 0.5 Ascorbate, bubbled with a mixture of underestimation. Indeed, unlike electric stimulation of 95%O and 5%CO . Parasagittal cerebellar slices (200- CFs [18], optogenetic stimulation failed to detect mul- 2 2 µm thick) were prepared by using a micro-slicer (Pro7N, tiple CF inputs on single PCs in Bai3 knockout mice Dosaka EM) in the ice-cold cutting solution. The pre - (Fig. 2H). pared slices were transferred into the artificial cerebro - To evoke PF-EPSCs, square paired pulses (20 µs dura- spinal fluid (ACSF) containing (in mM) 125 NaCl, 2.5 tion, 0–200 µA and 50 ms inter-stimulus interval) were Aimi et al. Molecular Brain (2023) 16:61 Page 4 of 17 applied through a glass pipette placed on the molecu- taken by using confocal microscopy (FV1000, Olympus). lar layer. PCs were voltage-clamped at -80 mV. Selective To observe and trace transverse CF branches, z-stack stimulation of PFs was confirmed by the paired-pulse images (1 μm step for 30 μm) were taken. Images were potentiation of EPSC amplitudes. analyzed by using Fiji software. Regions of interest (ROI), For recording mIPSCs, intracellular solutions were such as cell soma and vGluT2 puncta, were selected man- composed of (in mM): 120 Cs-Chloride, 20 HEPES, 1 ually, and signal intensities were measured in each ROI. MgCl , 4 Na ATP, 10 sucrose (pH 7.25, 290 mOsm/kg). Transverse CF branches were manually traced, and the 2 2 mIPSC were recorded from PCs in ACSF containing length of each branch was measured (µM): 10 NBQX, 20 D-AP5 and 1 TTX, in which picro- 2+ toxin was not added. PCs were voltage-clamped at -70 Ca imaging 2+ mV and mIPSCs were recorded as inward currents. Ca imaging was performed in PCs under the current Current responses were recorded with an Axopatch clamp condition using confocal laser-scanning micros- 200B amplifier (Molecular Devices) and pClamp software copy (FV-1200, Olympus). Intracellular solutions were (version 10, Molecular Devices) was used for data acqui- composed of (in mM): 130 K-Gluconate, 10 KCl, 10 sition and analysis. Signals were filtered at 1 kHz and dig - HEPES, 1 MgCl , 4 Na ATP, 1 Na GTP, 15 sucrose and 2 2 2 itized at 4 kHz. 0.1 Oregon Green 488 BAPTA-1 (OGB-1) (pH 7.25, 315 mOsm/kg). We waited at least 20 min after establish- Immunohistochemistry ing the whole-cell mode to fill PC dendrites with OGB-1 Mice were anesthetized by intraperitoneal injection of 2% and identified the stimulation site for evoking main and avertin and fixed by perfusion with 4%PFA/0.1 M sodium surplus CF inputs by CF-EPSC recording. Fluorescence phosphate buffer (PB). Fixed brains were submerged in images were acquired at 5–10 Hz while recording CF- 4%PFA/0.1 M PB at 4℃ for 12–16 h and solutions were evoked voltage changes. In some cases, the extracel- 2+ replaced with PBS containing 0.1% sodium azide. Brains lular Ca concentration was increased from 2 mM to 2+ were embedded in a 2% agarose gel just before section- 5 mM to enhance Ca changes induced by surplus CF ing. The cerebellar cortex (50 μm thickness, sagittal or stimulations. coronal) and the brainstem, including the IONs (50 μm, The acquired fluorescence images were analyzed 2+ coronal), were cut by a micro-slicer (DTK-1000; Dosaka by using Fiji software. CF-evoked Ca changes were EM). expressed as increases in the fluorescence value (ΔF) Immunostaining was performed in glass tubes. After divided by the averaged fluorescence value before CF sections were treated with 10% donkey serum, primary stimulations (F ). The area that showed a large Ca eleva - antibodies (Guinea pig anti-GFP [Frontier Institute], tion (ΔF/F within 30% of the peak value) was analyzed. Goat anti-GFP [Frontier Science], Chicken anti-GFP Voltage responses were recorded with an Axopatch 200B [Millipore], Rat anti-mCherry [Thermo Fisher Scientific], amplifier (Molecular Devices) and pClamp software (ver - Rabbit anti-vGluT2 [Frontier Institute], Guinea pig anti- sion 10, Molecular Devices) was used for data acquisi- vGluT2 [Frontier Institute], Goat anti-vGluT2 [Frontier tion. Signals were filtered at 1 kHz and digitized at 4 kHz Institute], Guinea pig anti-C1ql1 [a gift from Masahiko for the evoked voltage changes. Watanabe], Goat anti-calbindin[Frontier Institute], Rab- bit anti-Bai3 [a gift from Masahiko Watanabe], Rabbit Statistical analyses anti-HA [Cell Signaling Technology], Rabbit anti-c-Fos Electrophysiological data were analyzed offline using [Merck]) were applied and incubated at room tempera- Clampfit 10 (Molecular Devices). Immunohistochemical 2+ ture for 12–16 h. The specificity of the antibodies against and Ca imaging data were analyzed by using Fiji (Image C1ql1 and Bai3 was previously confirmed by the lack J) software. All bar graphs indicate mean ± standard of immunoreactivity in C1ql1 and Bai3 knockout mice, error of the mean. Statistical analyses were performed respectively [18]. Sections were washed with PBS 3 times using Mirosoft Excel (Microsoft) and BellCurve for Excel and incubated with secondary antibodies, which were (Social Survey Research Information Co., Ltd.). To com- conjugated with fluorescence dye such as Dylight 405, pare the number of CFs, which has discrete variables, we Alexa 488, 594, 647 and Cy3 (Molecular probes or Jack- used Mann-Whitney U test for two groups (Figs. 2D and son ImmunoResearch Laboratories) against the respec- H, 3D and 7D) and Kruskal-Wallis test followed by Steel tive primary antibody, together with DAPI at room test for multiple groups (Figs. 4D, 5D and 6D). For other temperature for 2 h. Sections were washed with PBS and continuous variables, we used Welch’s t-test for com- mounted on slide glasses with fluoromount G (Invitro - parison of two groups (Figs. 1C, 2C, E, G and I, 3E and gen). For staining HA-C1ql1, pepsin treatment (37℃ for F and 7C) and two-way ANOVA followed by Dunnett 5 min) was performed to expose antigens before treat- test (Fig. 5C) or Tukey test (for two parameters; Fig. 6C). ment with 10% donkey serum. Fluorescent images were Since the number of mice was small (n = 4), we also used Aimi et al. Molecular Brain (2023) 16:61 Page 5 of 17 Mann-Whitney U test for Fig. 1C and confirmed the C1ql1 expression in the soma of IONs was doubled by same statistical significance (*p = 0.0433) as Welch’s t-test. AAV-based expression (Fig. 1C). In the cerebellar cortex, The sample size, p-value, and the statistical test used in high levels of YFP signal were detected in the molecular each figure are also provided in figure legends. layer, with no evidence of misexpression in mossy fibers with rosette-like structures in the granular layer (Fig. 1D). Results Immunohistochemical staining for calbindin, a PC Selective gene expression in IONs and CFs using AAV and marker, and vesicular glutamate transporter 2 (vGluT2), a knock-in mice presynaptic marker for CFs, showed that HA-C1ql1 was To test the role of C1ql1-Bai3 signaling in structural localized to CF terminals along PC dendrites (Fig. 1E). changes in mature CF PC synapses, we first examined These results indicate that AAV-based delivery of C1ql1 the effect of increased expression of C1ql1 on CF syn - to Htr5b-tTA knock-in mice specifically and moderately apses. To avoid possible indirect effects of misexpres - increased the amount of C1ql1 in CF terminals. sion of C1ql1 in mossy fibers [ 24], we used Htr5b-tTA knock-in mice in which IONs, the origin of CFs, specifi - Increased C1ql1 levels in CFs induce re-innervation of cally express tTA [25]. We delivered adeno-associated mature PCs by multiple CFs virus (AAV) encoding the tetracycline response element Next, we examined the effect of increased C1ql1 expres - (TRE) followed by a channel rhodopsin-2 yellow fluo - sion on the function of CF-PC synapses using whole-cell rescent fusion protein (ChR2-YFP) and human influenza patch-clamp recordings from PCs in acute slices. AAV- hemagglutinin (HA)-tagged C1ql1. ChR2 was intro- TRE-ChR2-YFP (control) or AAV-TRE-ChR2-YFP-P2A- duced to directly assess the function of CFs in the later C1ql1 was injected into the ION of Htr5b-tTA knock-in experiments (Fig. 1A). Three weeks after injection into mice at 3–4 weeks of age (Fig. 2A). Application of two 3–4-week-old mice, HA-C1ql1 and YFP were detected light stimuli with an interval of 100 ms evoked paired- in IONs (Fig. 1B). C1ql1 immunostaining indicates that pulse depression of excitatory postsynaptic currents Fig. 1 CF selective gene delivery using Htr5B-tTA mice and AAV A Experimental scheme. AAVs encoding ChR2-YFP with or without C1ql1 were injected into the ION. The right panel shows YFP signals in the ION. Scale bar, 500 μm. B Expression levels of C1ql1 in the ION. Immunohistochemical staining shows total C1ql1, exogenous HA-C1ql1 and YFP in the ION infected with AAV-CTRL and AAV-C1ql1. DAPI staining shows the nucleus. Scale bar, 20 μm. C Quantification of C1ql1 immunoreactivity in the soma of IONs ( B ). p = 0.0389, Two-tailed Welch’s t-test; n = 4 mice each. D Selective expression of YFP and C1ql1 in the CFs. No YFP signals were detected in the mossy fibers in the granular layer. ML, molecular layer; PCL, Purkinje cell layer; GL, granular layer. Scale bar, 40 μm. E Immunohistochemical staining of vGluT2 (magenta), YFP or HA-C1ql1 (green) and calbindin (blue) indicates accumulation of HA-C1ql1 at CF synapses. Scale bar, 10 μm. Bars represent mean ± SEM. *p < 0.05 Aimi et al. Molecular Brain (2023) 16:61 Page 6 of 17 Fig. 2 Increased C1ql1 levels in CFs allow adult PCs to be innervated by multiple CFs A Experimental scheme for recording CF-EPSCs. B Representative CF-EPSC traces from wild-type PCs. The blue bar indicates the timing of light stimu- lation. Increasing the light intensity elicited a single EPSC in control slices (B ) in an all-or-none manner, but multiple EPSCs with a slower rise time in slices overexpressing C1ql1 (B ). C Total CF-EPSC amplitude. The graph shows the sum of the peak amplitudes of single CF-EPSCs or multiple CF-EPSCs. p = 0.0372, two-tailed Welch’s t-test; n = 28 cells from 4 mice (CTRL); n = 29 cells from 5 mice (+ C1ql1). D The percentage of the number of CFs innervating single PCs. The number of EPSCs evoked by distinct CF activation thresholds (number of steps) is shown. p = 0.0234, Mann–Whitney U test; n = 51 cells from 4 mice (CTRL); n = 49 cells from 5 mice (+ C1ql1). E Average of the 10–90% rise time of CF-EPSCs. p = 0.0028, two-tailed Welch’s t-test; n = 28 responses from 4 mice (CTRL); n = 46 responses from 5 mice (+ C1ql1). F Representative CF-EPSC traces from Bai3 knockout PCs (left: CTRL, right: +C1ql1). G Total CF-EPSC amplitude. The graph shows the sum of peak amplitudes of single CF-EPSCs or multiple CF-EPSCs. p = 0.8534, two-tailed Welch’s t-test; n = 31 cells from 3 mice (CTRL); n = 42 cells from 4 mice (+ C1ql1). H The percentage of the number of CFs innervating single PCs. p = 0.6159, Mann–Whitney U test; n = 38 cells from 3 mice (CTRL); n = 50 cells from 4 mice (+ C1ql1). I Average of the 10–90% rise time of CF-EPSCs. p = 0.0609, two-tailed Welch’s t-test; n = 33 responses from 3 mice (CTRL); n = 46 responses from 4 mice (+ C1ql1). Bars represent mean ± SEM. **p < 0.01; *p < 0.05; ns, not significant (EPSCs), a result consistent with a high release probabil- effect of C1ql1 overexpression on PC development, we ity of CF terminals. Furthermore, increasing light inten- examined PC dendritic arborization, which could affect sity elicited EPSCs in an all-or-none manner in control CF synapse formation. Immunohistochemical staining slices (Fig. 2B ), indicating that PCs are innervated by a of PCs with calbindin revealed no gross differences in single CF input with a single excitation threshold. In con- the dendritic arborization between PCs innervated by trast, overexpression of C1ql1 in CFs not only increased control and C1ql1-overexpressing CFs (Supplementary the amplitude of EPSCs, but also led to the appearance Fig. 1A). In addition, C1ql1 overexpression in CFs did not of CFs with two to three activation thresholds (Fig. 2B , affect the membrane capacitance of PCs, an electrophysi - C, D). In addition, EPSCs evoked by different activation ological estimate of the total surface area (Supplemen- thresholds had a slower rise time than EPSCs evoked tary Fig. 1B). Furthermore, AAV-based overexpression by most CFs (Fig. 2B , E). These results suggest that of C1ql1 in 6-week-old mice increased the percentage of increased expression of C1ql1 in CFs not only enhanced PCs innervated by multiple CFs in the same manner as the functions of existing CF-PC synapses, but also in 3-week-old mice (Supplementary Fig. 1C, D). These induced new CF synapses with distinct properties. results indicate that the effect of C1ql1 overexpression PCs establish a mature innervation pattern with a sin- was not confounded by the developmental stage of the gle CF by postnatal day 20 in rodents [7]. To rule out the PCs. Aimi et al. Molecular Brain (2023) 16:61 Page 7 of 17 C1ql1 regulates CF-PC synapse formation by binding To determine whether the effect of C1ql1 on CF trans - to the CUB domain of Bai3 during development [18, 19]. verse branches required Bai3, we next traced CFs in To determine whether the effect of C1ql1 requires Bai3, coronal cerebellar sections from Bai3 knockout mice we overexpressed C1ql1 in Bai3 knockout mice at 3–4 (Supplementary Fig. 2A) to which AAV-TRE-GFP (con- weeks of age. CF-evoked EPSC amplitudes were much trol) or AAV-TRE-GFP-P2A-C1ql1 was injected. In Bai3 smaller in Bai3 knockout than in wild-type mice (Fig. 2C knockout mice, the overexpression of C1ql1 in CFs did vs. 2G), a result consistent with previous reports [18, not elongate the transverse CF branch or increase the 19]. In contrast to conditional knockout mice in which percentage of vGluT2-positive terminals (Supplemen- the Bai3 gene was postnatally deleted [18], PCs in con- tary Fig. 2B-F). These results indicate that overexpression stitutional Bai3 knockout mice did not show a multiple of C1ql1, likely via interaction with Bai3, could induce innervation pattern by CFs (Fig. 2H). Importantly, over- the growth and synapse formation by of transverse CF expression of C1ql1 in CFs did not result in an increase branches, resulting in an increased number of PCs re- in the amplitude of CF-evoked EPSCs in Bai3 knockout innervated by multiple CFs after CF-PC synapses mature. PCs (Fig. 2G). In addition, overexpression of C1ql1 did not affect the percentage of PCs innervated by CFs with Bai3 overexpression in PCs induces re-innervation by multiple excitation thresholds (Fig. 2F2, H). Similarly, we surplus CFs through C1ql1 binding did not detect CF-evoked EPSCs with a slower rise time While new CF-PC synapses were induced by overexpres- (Fig. 2F2, I). Taken together, these results suggest that sion of C1ql1 in CFs, it was unclear whether PCs over- C1ql1 overexpression, likely via binding to Bai3, could expressing Bai3 could form new synapses with CFs with induce the formation of new CF synapses and increase normal levels of C1ql1. To address this question, we used the proportion of PCs innervated by multiple CFs in lentivirus with the murine stem cell virus (MSCV) pro- mature PCs. moter [28] to preferentially express EGFP and Bai3 in PCs of mice at 3–4 weeks of age (Fig. 4A). Bai3 expres- C1ql1-Bai3 signaling induces synapse formation by sion levels were estimated to be increased by approxi- transverse CF branches mately 1.8-fold (Supplementary Fig. 3A, B). We recorded How can a PC in which excess CFs have already been CF-evoked EPSCs from whole-cell patch-clamped PCs pruned except for a dominant single CF be innervated by placing the stimulating electrode in the granular layer again by other CFs? A previous in vivo time-lapse imag- near the PC soma. CF-EPSCs, which were confirmed by ing study showed that while ascending branches of CFs the paired-pulse depression, were elicited in an all-or- formed stable synapses with proximal dendrites of PCs, none manner in PCs expressing EGFP only, confirming the thin transverse branches were highly dynamic and that approximately 90% of wild-type PCs are innervated did not make synapses in adult wild-type mice [26]. u Th s, by a single CF input (Fig. 4 C, D). In contrast, EPSCs were to clarify the contribution by transverse CF branches, we evoked by two or three thresholds of stimulation in PCs traced GFP-positive CFs in coronal cerebellar sections overexpressing wild-type Bai3 (Fig. 4 C, D), suggesting from wild-type mice to which AAV-TRE-GFP (control) that Bai3 overexpression in PCs induces re-innervation or AAV-TRE-GFP-P2A-C1ql1 was injected at 3–4 weeks by surplus CFs. of age (Fig. 3A, B ). Co-immunostaining of GFP and To rule out an effect of Bai3 overexpression on PC vGluT2 revealed that the transverse CF branches were development, we examined the membrane capacitance of observed at various locations along the PC dendrites, but PCs. As in the case of C1ql1 overexpression in CFs, over- they mostly lacked vGluT2 in control sections (Fig. 3B , expression of Bai3 in PCs did not affect the membrane C, D), indicating their inability to form functional syn- capacitance of PCs (Supplementary Fig. 4A), suggest- apses as reported previously [20, 26, 27]. Interest- ing no gross differences in the total surface area of PCs. ingly, when C1ql1 was overexpressed in CFs, transverse Furthermore, lentivirus-based overexpression of Bai3 in branches elongated and often became positive for vGluT2 6-week-old mice increased the percentage of PCs inner- (Fig. 3B , C, D). The elongation of the transverse branch vated by multiple CFs in the same manner as injection occurred mostly in distal dendrites (80–160 μm from into 3-week-old mice (Supplementary Fig. 4B, C). These the soma) (Fig. 3 C, E). Transverse branches that were results indicate that the effect of Bai3 overexpression was positive for vGluT2 were longer than those negative for not confounded by the developmental stage of the PCs. vGluT2 (Fig. 3F). Since EPSCs at synapses farther elec- Bai3 belongs to the adhesion G protein-coupled recep- trotonic distance from the recording site show a slower tor family, which mediates intracellular signaling through rise time, these results suggest that an increased propor- distinct functional domains [29–32]. To gain insight into tion of PCs innervated by multiple CFs is at least partly the signaling mechanism mediated by Bai3, we expressed caused by the transverse CF branches forming synapses Bai3 with mutations in these functional domains on distal dendrites. (Fig. 4B). Expression of Bai3-AAA, disabling the ELMO Aimi et al. Molecular Brain (2023) 16:61 Page 8 of 17 Fig. 3 C1ql1-Bai3 signaling induces synapse formation by transverse CF branches A Experimental scheme. B Coronal cerebellar sections. GFP (CTRL) or GFP plus C1ql1 (+ C1ql1) was overexpressed in CFs. Maximum intensity z-projection images are shown. Dotted lines, upper and lower boundaries of the molecular layer (B ). Enlarged views of representative CF branches (B ). Arrowheads 1 2 indicate vGluT2-negative and positive branches in CTRL and + C1ql1, respectively. Scale bars, 20 μm. C Height and length of CF transverse branches in the molecular layer. Branch height was measured from the apical pole of PC somata. Transverse branches negative (-) and positive (+) for vGluT2 are indi- cated by white and red circles, respectively. n = 152 branches (CTRL), n = 132 branches (+ C1ql1). D Percentage of vGluT2-positive CF transverse branches − 5 in C TRL or + C1ql1. p = 1.352 × 10 , n = 152 (C TRL); n = 132 (+ C1ql1). Mann–Whitney U test. E Histogram showing the mean length of CF transverse branches as a function of their height in the molecular layer. Black and orange bars represent the cerebellum in CTRL and + C1ql1, respectively. 0–40 μm: − 5 p = 0.8328, n = 3 (C TRL), n = 18 (+ C1ql1); 40–80 μm: p = 0.1731, n = 34 (C TRL), n = 36 (+ C1ql1); 80–120 μm: p = 2.648 × 10 , n = 63 branches (C TRL), n = 58 − 5 (+ C1ql1); 120–160 μm: p = 7.370 × 10 , n = 49 (C TRL), n = 20 (+ C1ql1); >160 μm: p = 0.1321, n = 3 (C TRL), n = 9 (+ C1ql1). Two-tailed Welch’s t-test. F Histogram showing the mean length of CF transverse branches with the presence or absence of vGluT2. CTRL: p = 0.4338; vGluT2(-), n = 137; vGluT2(+), n = 15. +C1ql1: p = 0.003482; vGluT2(-), n = 93; vGluT2(+), n = 39. Two-tailed Welch’s t-test. All datasets are from 3 mice per group (CTRL and + C1ql1). Bars represent mean ± SEM. **p < 0.01; *p < 0.05; ns, not significant binding motif [29, 30], Bai3-ΔCT7, which lacked the PDZ CFs by binding to C1ql1, but independently of ELMO, binding motif [32] and Bai3-S832A, which disrupted PDZ proteins or proteolysis at the GAIN domain. the proteolysis sequence in the GPCR auto-proteolysis- inducing (GAIN) domain [31], had similar effects as Bai3 overexpression in PCs induces re-innervation by CFs wild-type Bai3 in inducing re-innervation of PCs by sur- at distal dendrites plus CFs (Fig. 4 C, D). In contrast, the expression of Bai3- The largest EPSCs observed in PCs overexpressing Bai3, ΔCUB, which lacked the binding site for C1ql1 [18], did which we termed “main CF-EPSC”, had similar kinet- not affect the pattern of CF innervation in mature PCs ics to EPSCs seen in control PCs, but the smaller EPSCs (Fig. 4 C, D). These results indicate that overexpression (surplus CF-EPSCs) elicited by distinct stimulus thresh- of Bai3 in mature PCs induced innervation by additional olds had slower rise times (Fig. 4E). Since overexpres- sion of C1ql1 in CFs also caused the appearance of small Aimi et al. Molecular Brain (2023) 16:61 Page 9 of 17 Fig. 4 Bai3 overexpression in PCs induces re-innervation by CFs at distal dendrites by binding to C1ql1 A Experimental scheme. B Diagram of the functional domains of Bai3 and its mutants. C Representative CF-EPSC traces recorded from adult wild-type PCs overexpressing the indicated constructs. Paired-pulse stimulation with 50-ms interstimulus interval was applied. D The percentages of the number of CFs innervating single PCs. The number of EPSCs evoked by distinct CF activation thresholds (step numbers) is shown. Bai3-WT: p = 0.0100, n = 69 cells from 11 mice; Bai3-AAA: p = 0.0278, n = 30 cells from 4 mice; Bai3-ΔCT7: p = 0.0007, n = 60 cells from 8 mice; Bai3-S832A: p = 0.0186, n = 22 cells from 3 mice; Bai3- ΔCUB: p = 0.9981, n = 63 cells from 8 mice. Kruskal-Wallis test followed by Steel test vs. CTRL: n = 57 cells from 7 mice. E Average of the 10–90% rise time of CF-EPSCs in PCs overexpressing Bai3-WT. p = 0.0018, Two-tailed Welch’s t-test, n = 24 traces (main), n = 14 traces (surplus) from 11 mice. F Time course of 2+ CF-evoked Ca changes associated with main and surplus EPSPs. Changes in the fluorescence (ΔF) were normalized by the averaged fluorescence (F ) 2+ 2+ before the CF stimulation (arrowhead). Inset, representative CF-EPSPs during Ca imaging. G Representative CF-evoked Ca changes associated with 2+ 2+ main and surplus EPSPs. S, PC soma. Scale bar, 20 μm. H The mean area of Ca elevation associated with main or surplus CF-EPSPs. The area of large Ca − 5 elevation (ΔF/F within 30% of the peak value) was measured. p = 2.763 × 10 , two-tailed Welch’s t-test, n = 7 responses (main), n = 7 (surplus) from 5 mice. 2+ I The closest distance between the site of large Ca elevation and the PC soma was measured (see Methods). p = 0.0075, two-tailed Welch’s t-test, n = 7 responses (main), n = 7 (surplus) from 5 mice. Bars represent mean ± SEM. **p < 0.01; *p < 0.05; ns, not significant CF-evoked EPSCs with slower kinetics (Fig. 2E) and IONs by injecting AAV-TRE-tdTomato into Htr5b-tTA synapse formation at distal dendrites by transverse CF knock-in mice (Supplemental Fig. 3C). We found a few branches (Fig. 3C), we hypothesized that Bai3 overex- PCs that expressed Bai3 and were selectively innervated pression in PCs similarly induced new CF synapse forma- by transverse CF branches expressing tdTomato without tion on distal dendrites. labeled main CF inputs (Supplemental Fig. 3D). However, To test this hypothesis morphologically, we expressed since the identification of surplus CF branches relies on EGFP and Bai3 in PCs by lentivirus and sparsely labeled the coincidental sparse labeling of PCs and CFs, it was Aimi et al. Molecular Brain (2023) 16:61 Page 10 of 17 difficult to quantify the effect of Bai3 on the formation PCs preferentially induces CF synapses without signifi - of surplus CF synapses by the immunohistochemical cantly altering the number of other synapses. method. To clarify the location of surplus CF synapses that gave Endogenous Bai3 and C1ql1 are involved in the rise to EPSCs with slow kinetics, we next used the elec- re-innervation of CFs in mature PCs trophysiological mapping method. We systematically Can CFs form new synapses in mature cerebellar cir- moved the stimulating electrode every 10 μm in the XY cuits that do not overexpress C1ql1 or Bai3? Loss of direction in the granular layer (Supplementary Fig. 5A). PF-PC synapses in conditional GluD2 knockout mice has We found that at some locations, main and surplus been reported to trigger re-innervation of PCs through EPSCs could be evoked by varying the stimulus inten- CF transverse branches without exogenous manipula- sity, while at other locations, only main or surplus EPSCs tion of C1ql1-Bai3 signaling [20]. Therefore, we investi - were selectively evoked. Overall, the location of the stim- gated the role of endogenous Bai3 in conditional GluD2 ulating electrode that evoked surplus EPSCs was farther knockout mice. Using lentivirus with an MSCV pro- from the PC soma than that elicited main EPSCs (Supple- moter, we sparsely expressed a Cre recombinase and mentary Fig. 5B). These results suggest that in PCs over - EGFP in PCs of 3–4-week-old wild-type and conditional f/f f/f expressing Bai3, surplus EPSCs are evoked by CFs that GluD2 (Grid2 ) and/or Bai3 (Bai3 ) knockout mice travel farther from the cell body than the main CF. (Fig. 5A, Supplementary Fig. 7A). Whole-cell patch- To directly visualize where surplus CFs formed func- clamp recordings from acute cerebellar slices prepared tional synapses with PCs overexpressing Bai3, we loaded from conditional GluD2 knockout mice two months 2+ PCs with a Ca indicator (Oregon green BAPTA-1) after Cre introduction revealed that PCs were inner- through a patch electrode. We first identified the sites vated by multiple CFs with distinct thresholds (Fig. 5B, where only main or surplus CF-EPSCs were selectively D), as previously reported [20]. In contrast, CF evoked evoked (Supplementary Fig. 5C) and then performed smaller EPSCs in an all-or-none manner in conditional 2+ Ca imaging under the current-clamp mode (Fig. 4F-I). Bai3 knockout mice (Fig. 5B-D), indicating that the pat- Stimulation of sites where main EPSPs were selectively tern of innervation of PCs by a single CF is unaffected by 2+ elicited caused greater increases in Ca concentrations knocking out Bai3 in adult mice as reported previously from a larger dendritic area than stimulation of sites [12]. Interestingly, in contrast to GluD2 knockout mice, where surplus EPSPs were selectively elicited (Fig. 4G, when both GluD2 and Bai3 were knocked out, CF-EPSCs 2+ H). Furthermore, Ca elevations associated with sur- became smaller (Fig. 5B, C), but many PCs remained plus EPSPs were observed in dendrites more distal to innervated by a single CF (Fig. 5D). Furthermore, C1ql1 the PC soma than those associated with the main EPSPs immunopositive puncta were significantly upregulated (Fig. 4G, I). These results further support the hypothesis in the upper molecular layer of GluD2 knockout mice at that overexpression of Bai3 in PCs causes the formation 2–3 months of age (Supplemental Fig. 7B, C, D), suggest- of new CF synapses on dendrites more distal to the main ing the involvement of C1ql1 in CF synapse formation in CFs, resulting in multiple CF innervation of mature PCs. GluD2 knockout mice. These results suggest that endog - During development, the inhibitory inputs from enous Bai3, probably together with endogenous C1ql1, molecular layer interneurons and CFs compete for syn- is required for re-innervation of mature PCs by CFs in apses on PC somata [33]. In GluD2 knockout mice, PFs GluD2 knockout mice. and CFs compete for synapses on distal dendrites of PCs [20]. Therefore, to explore the possibility that Bai3 Bai3-induced re-innervation of PCs by CFs requires PC may affect other types of PC synapses, we recorded PF- activity evoked EPSCs, which were confirmed by paired-pulse Since structural synaptic plasticity occurs in an activity- facilitation, in PCs overexpressing Bai3 (Supplementary dependent manner throughout life in the mammalian Fig. 6A). The amplitudes of PF-EPSCs in response to brain [34, 35], we next investigated whether increased increasing stimulus intensities were similar between PCs C1ql1-Bai3 levels could bypass neuronal activity to form expressing EGFP only (control) and EGFP plus Bai3 (Sup- new CF synapses in mature PCs. Using an AAV-based plementary Fig. 6B, C). Miniature inhibitory postsynap- Cre-DIO (double-floxed inverse open reading frame) sys - tic currents (mIPSCs) recorded from PCs overexpressing tem, we expressed EGFP and ESKir2.1, a non-rectifying Bai3 Supplementary Fig. 6D) and control showed similar variant of Kir2.1 potassium channel [36], to specifically amplitudes and frequencies (Supplementary Fig. 6E, F). suppress levels of intrinsic PC activity (Supplementary Although local competition may be missed because PF Fig. 8A). As a control, we used ESKir2.1 , a mutant AAA and inhibitory synapses outnumber CF synapses, these channel lacking channel activity [36]. Loose-patch results indicate that overexpression of Bai3 in mature recordings in acute cerebellar slices prepared from mice 2–3 weeks after the AAV injection at 3–4 weeks of age Aimi et al. Molecular Brain (2023) 16:61 Page 11 of 17 Fig. 5 Endogenous Bai3 is required for synapse formation of re-innervating CFs A Experimental scheme. B Representative CF-EPSC traces recorded from PCs of the indicated genotypes. C Total CF-EPSC amplitude. The graph shows the − 6 sum of peak amplitudes of single CF-EPSCs or multiple CF-EPSCs. CTRL (n = 17 cells from 2 mice) vs. Bai3 knockout (KO), p = 1.272 × 10 (n = 29 cells from − 6 5 mice); vs. GluD2 KO, p = 0.8311 (n = 25 cells from 5 mice); vs. Bai3 KO::GluD2 KO, p = 1.336 × 10 (n = 33 cells from 4 mice). One way ANOVA followed by Dunnett’s test. D The percentages of the number of CFs innervating single PCs. The number of EPSCs evoked by distinct CF activation thresholds (step − 5 numbers) is shown. CTRL (n = 29 cells from 2 mice) vs. Bai3 KO, p = 0.9859 (n = 49 cells from 5 mice); vs. GluD2 KO, p = 1.885 × 10 (n = 46 cells from 5 mice); vs. Bai3 KO::GluD2 KO, p = 0.3750 (n = 61 cells from 4 mice). Kruskal-Wallis test followed by Steel test. Bars represent mean ± SEM. **p < 0.01; ns, not significant confirmed the absence of spontaneous action potentials only Bai3, surrounded by silent PCs expressing only in PCs expressing ESKir2.1, but not ESKir2.1 (Supple- ESKir2.1 (Fig. 6A, right; Supplementary Fig. 8E, bottom). AAA mentary Fig. 8B, top traces). Whole-cell voltage-clamp The amplitude of CF-EPSCs in such non-silenced PCs recordings revealed that the amplitude of CF-evoked was similar to that in control PCs expressing ESKir2.1 AAA EPCSs was reduced in PCs expressing ESKir2.1, but the and Bai3 (Fig. 6C). Unexpectedly, however, CF-EPSCs number of stimulus thresholds (reflecting the number of were evoked by a single threshold in these non-silenced CF inputs) was similar to control PCs (Supplementary PCs (Fig. 6B, right traces; Fig. 6D). These results suggest Fig. 8B-D). Similarly, the application of tetrodotoxin or that multiple innervation by CFs requires neuronal activ- NBQX is reported to reduce the amplitude of CF-EPSCs ity not only in the Bai3-expressing PCs but also in the and CF synapses in adult PCs [11–13]. While the site of surrounding PCs. action was unclear in these pharmacological studies, our findings indicate that the intrinsic activity of PCs is CF activity is required for C1ql1 to induce the innervation required to maintain CF synapses in mature PCs. of adult PCs by CFs Next, we examined whether Bai3 overexpression could Finally, we investigated whether increased C1ql1 lev- induce CF re-innervation in PCs expressing ESKir2.1 by els in CFs could induce new CF synapses in the absence coinfecting L7-Cre mice with AAV-Syn-DIO-ESKir2.1- of CF activities. To suppress CF activities in vivo, we T2A-EGFP and Lenti-MSCV-mCherry-P2A-Bai3 injected a mixture of AAV-TRE-ChR2-YFP and AAV- (Fig. 6A). In PCs overexpressing Bai3 and ESKir2.1 , TRE-mCherry-P2A-ESKir2.1 into the ION of Htr5B- AAA we detected CF-EPSCs with multiple thresholds (Fig. 6B, tTA knock-in mice at 3–4 weeks of age (Supplementary left traces; Fig. 6D), as observed in the absence of Fig. 9A). With acute slice preparations where input fibers ESKir2.1 (Fig. 4 C, D). In contrast, CF stimulation are not preserved, it is difficult to determine how effec - AAA evoked smaller EPSCs with a single threshold in most tively ESKir2.1 could silence the electrical activity of PCs coexpressing ESKir2.1 and Bai3 (Fig. 6B, middle IONs in vivo. Instead, we injected harmaline intraperito- traces; Fig. 6D). These results indicate that intrinsic PC neally, which transiently increases the synchronous fir - activity is required for Bai3 to re-innervate mature PCs. ing of IONs [26], and performed immunohistochemical In these experiments, expression of ESKir2.1 (as staining for c-Fos, a marker of neuronal activity, 10 min detected by EGFP) was widespread in many PCs at the after the injection (Supplementary Fig. 10A). We found injection site, whereas Bai3 (as detected by mCherry) was that the number of c-Fos-positive IONs was signifi - detected in only a few PCs (Supplementary Fig. 8E). How- cantly reduced by the expression of ESKir2.1 compared ever, we occasionally found non-silenced PCs expressing to ESKir2.1 (Supplementary Fig. 10B, C). In addition, AAA Aimi et al. Molecular Brain (2023) 16:61 Page 12 of 17 Fig. 6 Bai3-induced re-innervation by CFs requires PC activity A Experimental scheme. The right panel shows three cases: in all cases recorded PCs overexpress Bai3, but all PCs are active (spiking PCs, left), all PCs are silenced by ESKir2.1 (silent PCs, middle) and recorded PCs are active but neighboring PCs are silenced (right). B Representative action potentials by loose-patch recordings (upper traces) and CF-EPSCs by whole-cell patch-clamp recordings (lower traces) from PCs expressing the indicated constructs. C Total CF-EPSC amplitude. The graph shows the sum of peak amplitudes of single CF-EPSCs or multiple CF-EPSCs. p = 0.0098, CTRL + Bai3 vs. ESKir2.1 + Bai3; p = 0.9500, CTRL + Bai3 vs. ESKir2.1 + Bai3, GFP (-); p = 0.0393, ESKir2.1 + Bai3 vs. ESKir2.1 + Bai3, GFP (-). One way ANOVA followed by Tukey’s test. n = 16 cells from 3 mice (CTRL + Bai3), n = 18 cells from 3 mice (ESKir2.1 + Bai3), n = 12 cells from 3 mice (ESKir2.1 + Bai3, GFP (-)). D The percentages of the num- ber of CFs innervating single PCs. The number of EPSCs evoked by distinct CF activation thresholds (step numbers) is shown. p = 0.0061, CTRL + Bai3 vs. ESKir2.1 + Bai3; p = 0.0182, CTRL + Bai3 vs. ESKir2.1 + Bai3, GFP (-); p = 0.9894, ESKir2.1 + Bai3 vs. ESKir2.1 + Bai3, GFP (-). Kruskal-Wallis test followed by Steel test n = 44 cells from 5 mice (CTRL + Bai3), n = 49 cells from 9 mice (ESKir2.1 + Bai3), n = 37 cells from 6 mice (ESKir2.1 + Bai3, GFP (-)). Bars represent mean ± SEM. **p < 0.01; *p < 0.05; ns, not significant the amplitude of CF-EPSCs was decreased by the expres- co-expression of C1ql1 and ESKir2.1 (Fig. 7D). These sion of ESKir2.1 in IONs (Supplementary Fig. 9B, C) results indicate that CF activity is required for C1ql1 to while the innervation pattern of CF was unchanged (Sup- induce additional CF innervation onto adult PCs. plementary Fig. 9B, D), indicating that the intrinsic activ- To gain insight into why CF activity enhances synap- ity of IONs, which is required to maintain CF synapses in togenesis through C1ql1, we performed immunohis- adult PCs, was suppressed by ESKir2.1. tochemical staining of HA-C1ql1 in mice expressing To examine the effect of CF activities on C1ql1- HA-C1ql1 and either ESKir2.1 or ESKir2.1 in the AAA induced CF synapse formation, we next injected a IONs (Fig. 7E). In the cerebellar cortex, HA-Clql1 immu- mixture of AAV-TRE-ChR2-YFP-P2A-C1ql1 and AAV- noreactivity on vGluT2-positive CF terminals was signifi - TRE-mCherry-P2A-ESKir2.1 into the ION of Htr5B- cantly reduced in CFs expressing ESKir2.1 as compared tTA knock-in mice (Fig. 7A). Whole-cell patch-clamp to those expressing ESKir2.1 (Fig. 7F, H). In contrast, AAA recordings revealed that in mice expressing ChR2, C1ql1 no difference was observed in HA-C1ql1 immunoreac - and ESKir2.1 were expressed in the IONs, additional tivity in the cell bodies of IONs expressing ESKir2.1 ver- AAA EPSCs with a slow time course were elicited in response sus ESKir2.1 (Fig. 7G, I). While the neuronal activity AAA to an increasing light stimulus (Fig. 7B ), as observed in of IONs could affect CF synaptogenesis through various the absence of ESKir2.1 (Fig. 2B ). In contrast, when pathways (discussed in detail below), our findings sug - AAA 2 C1ql1 was overexpressed with ESKir2.1 in the IONs, gest that one possibility is its involvement in the secre- the amplitude of CF-EPSCs was significantly reduced tion from CF terminals since C1ql1 immunoreactivity in (Fig. 7B , C). Furthermore, the percentage of PCs that the adult cerebellar cortex is detected in the synaptic cleft were innervated by the surplus CFs was reduced by [18]. Aimi et al. Molecular Brain (2023) 16:61 Page 13 of 17 Fig. 7 CF activity is required for C1ql1 to induce CF innervation on adult PCs A Experimental scheme for silencing of CFs and recording of CF-EPSC. B Representative light-evoked CF-EPSC traces (blue lines). Multiple EPSCs with a slower rise time were recorded in slices overexpressing C1ql1 in CFs (B ). The boxed region is enlarged on the right to show the slow CF-EPSC. Single − 7 EPSC was evoked in an all-or-none manner in slices overexpressing C1ql1 and ESKir2.1 in CFs (B ). C Total CF-EPSC amplitude. p = 2.665 × 10 , two-sided Welch’s t-test. n = 27 cells from 5 mice (CTRL), n = 31 cells from 5 mice (ESKir2.1). D The percentage of the number of CFs innervating single PCs. The num- ber of EPSCs evoked by distinct CF activation thresholds (number of steps) is shown. p = 0.0221, Mann–Whitney U test. n = 34 cells from 5 mice (CTRL), n = 34 cells from 5 mice (ESKir2.1). E Experimental scheme to study the effect of CF neural activity on C1ql1 immunoreactivity. F Immunohistochemical analysis of HA-C1ql1 at CF synapses. Expression of ESKir2.1 (GFP, bottom panels) in IOs reduced C1ql1 (HA) immunoreactivity at CF synapses (vGluT2, arrowheads) compared to ESKir2.1 (GFP, top panels). Scale bar, 10 μm. G Immunohistochemical analysis of HA-C1ql1 in IONs. Scale bar, 20 μm. H AAA Quantification of HA-C1ql1 levels at CF synapses. HA-C1ql1 immunoreactivity was normalized by the GFP fluorescence in vGluT2-positive CF synapses. − 4 p = 2.353 × 10 , two-tailed Welch’s t-test. n = 103 areas from 3 mice (CTRL), n = 169 areas from 3 mice (ESKir2.1). I Quantification of HA-C1ql1 levels in IONs. HA-C1ql1 immunoreactivity was normalized by the GFP fluorescence in the soma of IONs. p = 0.5970, two-tailed Welch’s t-test. n = 149 cells from 3 mice (CTRL), n = 161 cells from 2 mice (ESKir2.1). Bars represent mean ± SEM. **p < 0.01; *p < 0.05; ns, not significant PCs (Fig. 2H, Supplementary Fig. 2 C, D). In addition, Discussion the effect of overexpression of Bai3 in PCs required the In the present study, we showed that mature PCs, which CUB domain, a binding site of Bai3 for C1ql1 (Fig. 4D). achieved innervation by a single strong CFs after prun- Although C1ql1 and Bai3 have additional binding part- ing weak CFs during development, became re-innervated ners, such as kainate receptors [37] and RTN4 [38], by surplus CFs when the expression of C1ql1 or Bai3 was respectively, these results indicate that CF-derived C1ql1 upregulated in CFs or PCs, respectively. Immunohisto- binds to Bai3 in PCs to induce the formation of new CF chemical (Fig. 3E, Supplementary Fig. 3D), electrophysi- synapses. Interestingly, endogenous Bai3 was required 2+ ological (Figs. 2E and 4E), and Ca imaging (Fig. 4G, I) for the re-innervation of mature PCs by CFs in GluD2 studies indicated that transverse CF branches most likely knockout mice (Fig. 5D). Furthermore, the effect of contributed to the formation of surplus CF synapses C1ql1-Bai3 signaling on CF innervation required neu- at distal dendrites of mature PCs. The effect of C1ql1 ronal activity of both PCs (Fig. 6D) and CFs (Fig. 7D). overexpression in CFs required normal levels of Bai3 in Together, we propose a model in which C1ql1-Bai3 Aimi et al. Molecular Brain (2023) 16:61 Page 14 of 17 signaling mediates CF structural plasticity in mature PCs activation of matrix metalloproteinase 9, an enzyme in a manner dependent on neuronal activity (Fig. 8). involved in ECM remodeling, leading to activity-depen- dent spine growth in hippocampal neurons [45]. Since Activity-dependent effect of C1ql1 and Bai3 on innervation by surplus CFs required neural activity not re-innervation of mature PCs by CFs only in the Bai3-expressing PCs but also in the surround- Why do C1ql1 and Bai3 require neuronal activity in ing PCs, activity-dependent modification of the ECM both PCs and IONs to induce CF synapses in mature may provide a permissive environment for new synapse 2+ PCs? P/Q-type Ca channels and aCaMKII regulate the formation in mature PCs. elimination of surplus CFs during development since PCs genetically lacking these molecules remain innervated by C1ql1-Bai3 exerts synaptogenic function through surplus CFs in adulthood [41–43]. However, the selective uncharacterized domains strengthening of the winner CFs is also impaired in PCs Bai3 is reported to inhibit dendritogenesis of PCs dur- 2+ 2+ lacking Ca influx through P/Q Ca channels [44], indi- ing development by regulating the activity of the small 2+ cating that Ca influx is not only required to eliminate GTPase Rac1 through the interaction with ELMO1 weak CFs, but also to selectively strengthen strong CFs and DOCK180 [29]. Similarly, Bai3 has been shown to 2+ during development. Thus, activity-induced Ca influx mediate the fusion of myoblasts by binding to ELMO1/ in PCs may play a role in maintaining newly formed CF DOCK1 during development [30]. Bai1, a close relative synapses in mature PCs. of Bai3, is reported to promote synaptogenesis through Since C1ql1 immunoreactivity at CF-PC synapses the PDZ binding motif by associating with Nlgn1 [46], was reduced when the activity of IONs was suppressed recruiting the Rac1-GEF complex Par3/Tiam1 [47] and (Fig. 7H), C1ql1 may be released from CFs in an activ- stabilizing PSD-95 [48]. An engineered truncation in Bai1 ity-dependent manner. Similarly, the C1q family pro- and Bai3, mimicking autocleavage at the GAIN domain, tein Cbln1 is released from PFs in an activity-dependent led to activation of Ga [32] and G [30], respectively. 12/13 ai1 manner [21]. It has been reported that the motility of However, in the present study, mutations disrupting the transverse CF branches lacking vGluT2-positive presyn- ELMO1 binding motif, the PDZ binding motif and the aptic sites was reduced 3 h after the application of har- GAIN domain did not affect the ability of Bai3 to induce 2+ maline [26]. Thus, the activity-induced Ca increase in CF synapses in adult mice (Fig. 4D). Since mutant Bai3 CF transverse branches may slow down their motility to was expressed in wild-type mice, the mutant Bai3 may facilitate the accumulation of vesicles containing synapse have associated with endogenous Bai3 to compensate for organizers, such as C1ql1, to facilitate synapse forma- the function of the mutated site. However, the inability of tion with mature PCs. In addition, changes in the extra- Bai3-ΔCUB to induce surplus CF innervation indicates cellular matrix (ECM) associated with CF activity may that at least the CUB domain defect cannot be compen- allow C1ql1 to be stabilized at synapses. Indeed, activity- sated for by endogenous Bai3. Thus, considering that dependent release of cathepsin B, a lysosomal enzyme Bai1 can mediate at least five downstream signaling path - co-released with Cbln1, allows presynaptic morphologi- ways by differentially coupling to ELMO, MDM2, Par3/ cal changes associated with PF-PC synapse formation Tiam1, Ga and Bcr, depending on the cellular context 12/13 [21]. Back-propagating action potentials also trigger the [49], we postulate that C1ql1 binding to Bai3 likely exerts exocytosis of cathepsin B from dendrites and subsequent Fig. 8 Proposed model for re-innervation of mature PCs by excess CFs through C1ql1-Bai3 signaling Increased C1ql1-Bai3 signaling induces re-innervation of mature PCs by excess CFs. These new CF inputs, derived from transverse CF branches of neigh- boring PCs, synapse onto the distal dendrites of PCs (left). Neuronal activity of both CFs and PCs is required for C1ql1-Bai3 signaling to induce surplus CFs (right) Aimi et al. Molecular Brain (2023) 16:61 Page 15 of 17 its synaptogenic function through distinct domains that lesions [58] or in GluD2 knockout mice [56] can partially interact with uncharacterized signaling pathways. cross the zebrin II boundary, leading to the connection of some PCs belonging to different microzones. Thus, the Functional implication of added CF synapses by transverse physiological role of newly formed synapses by CF trans- branches versal branches could be complicated depending on the It is difficult to chronically increase the activities of IONs extent to which CF synapses are formed across the zebrin in vivo to investigate whether the expression of endog- II boundary. Since C1ql3, a closely related family member enous C1ql1 or Bai3 changes and induces re-innervation of C1ql1, and Bai3 are expressed in other neural circuits, by CFs. For example, although harmaline administration such as the basolateral amygdala-medial prefrontal cor- rapidly induces tremor-like movements in rodents asso- tex [60] and the anterior olfactory neuron-olfactory bulb ciated with increased activity of IONs, the effect is tran - [61], further studies are warranted to clarify whether and sient and lasts only a few days [50]. However, knockout of how C1ql-Bai3 signaling mediates activity-dependent GluD2 caused re-innervation of mature PCs by multiple structural plasticity in these brain regions in adulthood. CFs without overexpression of C1ql1 or Bai3, but only in Abbreviations the presence of endogenous Bai3 in PCs (Fig. 5D). Impor- AAV Adeno-associated virus tantly, C1ql1 immunopositive puncta were upregulated AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Bai3 Brain specific angiogenesis inhibitor 3 in adult GluD2 knockout mice (Supplemental Fig. 7B, C, C1ql1 C1q-like protein 1 D). These findings suggest that endogenous C1ql1-Bai3 Cbln1 Cerebellin-1 signaling is involved in the re-innervation of PCs by CFs CF Climbing fiber ChR2 Channel rhodopsin-2 under certain pathological conditions. DIO Double-floxed inverse open reading frame The main branches of CFs are distributed parasagit - EPSC Excitatory postsynaptic currents tally in microzones [51], which likely contain ~ 100 PCs in GluD2 Delta-type glutamate receptor 2 HA Human influenza hemagglutinin mice [52]. In contrast, CFs mediolaterally extend trans- ION Inferior olive neurons verse branches for 5–300 μm without forming synapses mIPSC Miniature inhibitory postsynaptic currents in adult mice [26, 53]. Many PCs belonging to the same MSCV Murine stem cell virus NBQX 2,3-Dioxo-6-nitro-1,2,3,4- tetrahydrobenzo[f] microzone show synchronous activity due to electrical quinoxaline-7-sulfonamide coupling between IONs. A computer simulation study PC Purkinje cell indicates that the higher level of electrical coupling of PF Parallel fiber TRE Tetracycline response element IONs accelerates the crude learning at the initial stage tTA Tetracycline transactivator by facilitating the synchronized firing of PCs, while the vGluT2 Vesicular glutamate transporter 2 reduced electrical coupling at the later stage of learning allows more sophisticated and complicated learning [54]. Alternatively, synchronized firing by enhanced electrical Supplementary Information The online version contains supplementary material available at https://doi. coupling of IONs may represent a state change underly- org/10.1186/s13041-023-01048-4. ing skilled movements [55]. Interestingly, PCs in GluD2 knockout mice are reported to show enhanced synchro- Supplementary Material 1 nous firing in the mediolateral direction, which was not mediated by electrical coupling of IONs, but by surplus Acknowledgements transverse CF branches making synapses onto distal PC We would like to acknowledge Drs. Wataru Kakegawa, Itaru Arai and Ayako W. Ishikawa for technical support in the electrophysiological analysis, Dr. Eriko dendrites in vivo [56]. Similarly, after partial lesion of Miura for technical support in immunohistochemistry, Drs. Keiko Matsuda. IONs, surviving CFs are reported to sprout new collater- Tokiwa Yamasaki and Ms. Shihomi Kuwano for assisting virus preparation and als in the mediolateral direction and innervate PCs [57]. sharing cDNA constructs. A live imaging study showed that these newly sprouted Authors’ contributions transverse CF branches preferentially synapse on PCs T.A.: Conceptualization, Methodology, Investigation, Writing–Original draft near the target of the original PCs [58]. Thus, although preparation. K. M.: Investigation. M.Y: Conceptualization, Funding acquisition, 2+ Supervision, Writing–Review & Editing. the EPSCs and Ca transients elicited by newly formed transverse CF synapses are generally small (e.g., Fig. 2B , Funding 4 F, G), they can cause synchronous firing of adjacent PCs This work was supported by the JST CREST (JPMJCR1854 to M.Y.), MEXT KAKENHI (20H05628 to M.Y.), Grant-in-Aid for JSPS Research Fellow (19J10096 in the mediolateral direction, thereby affecting cerebellar to T.A.) and Ushioda Memorial Scholarship (to T.A). learning or recovery after injury. Cerebellar microzones show differential immunore - Data Availability The datasets and plasmids used in this study are available from the activity to zebrin II, largely corresponding to a distinct corresponding author on reasonable request. ensemble of PCs with similar functional properties [59]. However, transverse CF branches induced by partial ION Aimi et al. Molecular Brain (2023) 16:61 Page 16 of 17 connectivity of excitatory inputs converging on cerebellar Purkinje cells. Cell Declarations Rep. 2015 Feb;10(5):820–32. 20. Miyazaki T, Yamasaki M, Takeuchi T, Sakimura K, Mishina M, Watanabe M. Abla- Ethics approval and consent to participate tion of glutamate receptor GluRdelta2 in adult Purkinje cells causes multiple All procedures relating to the care and treatment of mice were performed in innervation of climbing fibers by inducing aberrant invasion to parallel fiber accordance with the guidelines approved by the animal resource committee innervation territory. 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Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2023
eISSN: 1756-6606
DOI: 10.1186/s13041-023-01048-4
Publisher site: See Article on Publisher Site

Abstract

Changes in neural activity induced by learning and novel environments have been reported to lead to the formation of new synapses in the adult brain. However, the underlying molecular mechanism is not well understood. Here, we show that Purkinje cells (PCs), which have established adult-type monosynaptic innervation by climbing fibers (CFs) after elimination of weak CFs during development, can be reinnervated by multiple CFs by increased expression of the synaptic organizer C1ql1 in CFs or Bai3, a receptor for C1ql1, in PCs. In the adult cerebellum, CFs are known to have transverse branches that run in a mediolateral direction without 2+ forming synapses with PCs. Electrophysiological, Ca -imaging and immunohistochemical studies showed that overexpression of C1ql1 or Bai3 caused these CF transverse branches to elongate and synapse on the distal dendrites of mature PCs. Mature PCs were also reinnervated by multiple CFs when the glutamate receptor GluD2, which is essential for the maintenance of synapses between granule cells and PCs, was deleted. Interestingly, the effect of GluD2 knockout was not observed in Bai3 knockout PCs. In addition, C1ql1 levels were significantly upregulated in CFs of GluD2 knockout mice, suggesting that endogenous, not overexpressed, C1ql1-Bai3 signaling could regulate the reinnervation of mature PCs by CFs. Furthermore, the effects of C1ql1 and Bai3 overexpression required neuronal activity in the PC and CF, respectively. C1ql1 immunoreactivity at CF-PC synapses was reduced when the neuronal activity of CFs was suppressed. These results suggest that C1ql1-Bai3 signaling may mediate CF synaptogenesis in mature PCs, potentially in concert with neuronal activity. Keywords Cerebellum, Purkinje cell, Climbing fiber, Synapse, Electrophysiology, C1ql1, Bai3 Introduction pathophysiology of various neurological and neuropsy- Neural circuits are formed and refined in response to chiatric disorders [5, 6]. However, the molecular mech- changes in neural activity associated with learning and anisms underlying structural plasticity in adult brains new environments, not only during development but remain largely unknown, partly due to the complexity of throughout life [1–4]. The increasing evidence suggests neuronal circuits consisting of many heterogenous syn- that such structural plasticity plays a key role in the apses. Activity-dependent structural plasticity during development has been extensively studied in the cerebel- lar cortex, which contains simple and well-defined neu - ronal circuits. Purkinje cells (PCs), which send the only *Correspondence: Michisuke Yuzaki outputs from the cerebellar cortex, receive two excitatory myuzaki@keio.jp inputs, parallel fibers (PFs) from granule cells and climb - Department of Physiology, Keio University School of Medicine, ing fibers (CFs) from the inferior olive neurons (IONs) at Tokyo 160-8582, Japan © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Aimi et al. Molecular Brain (2023) 16:61 Page 2 of 17 proximal and distal dendrites, respectively. Several hun- in CFs and PCs, respectively, transverse branches of CFs dred thousand PFs make synapses on spines located on elongated and formed new synapses with distal dendrites, the distal dendrites of a single PC. In contrast, although allowing PCs to be re-innervated by multiple CFs. This multiple CFs initially innervate a single immature PC, a process required neural activity of both CFs and PCs, single CF becomes dominant in an activity-dependent suggesting that C1ql1-Bai3 signaling may be involved in manner, forming synapses on the proximal dendrites the activity-dependent CF synapse modification in the while the rest of the CFs are eliminated by the end of the mature PCs. 3rd postnatal week in mice [7, 8]. Interestingly, the cer- ebellar cortex undergoes remarkable changes in its archi- Methods tecture after damage or alteration of neural activity, even Animals in adulthood. For example, PC spine density is increased All procedures relating to the care and treatment of by motor-skill learning [9] or exposure to an enriched mice were performed in accordance with the guide- environment [10]. Conversely, inhibition of neuronal lines approved by the animal resource committee of activities by application of the Na channel blocker tetro- Keio University. Mice of the following strains were used: dotoxin or the AMPA-receptor antagonist NBQX to the Htr5B-tTA and tetO-YC mice [25] (a gift from Prof. Kenji adult cerebellar cortex replaced CF synapses with PF syn- Tanaka, Keio Univ Sch Med), conditional Bai3 knockout f/f f/f apses on proximal dendrites [11–13]. u Th s, the cerebel - mice (Bai3 ) [18], conditional GluD2 (Grid2 ) mice [20], lar cortical circuits provide a valuable model to elucidate GluD2 knockout mice [17], C57B6/N mice (Japan SLC, molecular mechanisms underlying structural plasticity in Inc.). The constitutive Bai3 knockout mouse was gener- f/f the adult brain. ated by crossing Bai3 with telencephalin-Cre transgenic Many synaptic organizers regulate synapse forma- mice as previously reported [30, 37]. Mice were housed tion, maturation, or elimination during development with a 12:12 h light-dark cycle with food and water [14]. Among them, the C1q family is crucial in organiz- available ad libitum. The sex for the virus injections, ing excitatory inputs to PCs during development [15]. immunohistochemistry, and electrophysiology was not Cbln1, a member of the C1q family secreted from PFs, distinguished. regulates PF-PC synapses by binding to the GluD2 glu- tamate receptor expressed on distal dendrites of PCs [16, Cell lines 17]. In contrast, C1ql1, provided by CFs, mediates CF-PC HEK293 cells (tSA line; gift from Dr. R. Horn, Thomas synapse formation on the proximal dendrites by binding Jefferson Univ., PA) and AAV-293 cells (Cell Biolab) to the adhesion G-protein-coupled receptor Bai3 [18, 19]. were cultured with Dulbecco’s modified Eagle medium Interestingly, both Cbln1-GluD2 and C1ql1-Bai3 pro- (DMEM, D5796, Sigma-Aldrich) with 10% Fetal Bovine tein pairs remain expressed in the adult cerebellum after Serum (#004–00025, Japan Bioserum), 50 U/mL penicil- PF- and CF-PC synapses are established. When C1ql1 or lin/streptomycin (15140-122, Thermo Fisher Scientific), Bai3 is deleted in the IONs or PCs, respectively, CF syn- and 2 mM L-Glutamine. Cells were incubated at 37 °C in apses are lost from the proximal dendrites of PCs in the the 10% CO incubator. adult cerebellum [18]. Similarly, when GluD2 is deleted in the mature PCs, PF synapses are gradually lost from cDNA constructs the distal dendrites [20]. Furthermore, although PCs are Some constructs of Bai3, C1ql1 and plasmids for len- normally innervated by single CFs on proximal dendrites, tivirus preparation were previously reported [18]. Bai3 the loss of GluD2 in the adult cerebellum causes multiple mutants were generated by two-step overlapping PCRs. CFs to innervate distal PC dendrites where PFs normally To visually detect the cells expressing genes of inter- make synapses [20]. Intriguingly, similar changes in the est, the fluorescent proteins (GFP, YFP or mCherry) are innervation pattern of PCs by PFs and CFs are observed linked by a self-cleaving 2 A peptide (P2A, F2A) from when neuronal activity is inhibited in the adult cerebel- the foot-and-mouth-disease virus were co-expressed lum [11–13]. Furthermore, Cbln1 is secreted from PFs in [18]. cDNAs encoding ChR2-eYFP (#110,339), tdTomato an activity-dependent manner [21], while Cbln1 mRNA (#104,112) and Cre (#107,738) were obtained from Add- expression is repressed by prolonged neuronal activities gene. The minimal L7 promoter [ 39] was synthesized [22, 23]. These results suggest that C1q family proteins (Eurofin genomics). The sequence of ESKir2.1 and pAAV- may play a role in activity-dependent structural plasticity DJ was kindly provided by Dr. Kazuya Togashi (Grad in the cerebellum after PF- and CF synapses are matured. School of Science, Univ. Tokyo). pAAV-TRE, pAAV-L7 In the present study, we investigated whether and how and pAAV-Syn-DIO vectors were purchased from Add- C1ql1-Bai3 signaling is involved in CF-PC structural gene (#104,112, #58,867 and #50,459). pHelper were pur- plasticity in mature cerebellar circuits. We found that chased from Cell Biolab. when the expression of C1ql1 and Bai3 was upregulated Aimi et al. Molecular Brain (2023) 16:61 Page 3 of 17 Virus preparation KCl, 2 CaCl , 1 MgCl , 26 NaH PO , 10 Glucose and 0.1 2 2 2 4 Lentiviruses were used to express Bai3 or Cre in PCs. picrotoxin, bubbled with a mixture of 95%O and 5%CO . 2 2 Lentivirus was prepared as previously reported [18] with The transferred slices were incubated with ACSF at room small modifications. The cDNAs were cloned into the temperature for more than 1 h to recover. pCL20c vector and expressed under the MSCV promoter. Whole-cell patch-clamp recordings were made from Lentiviruses were produced by transfecting the pCL20c visually identified PCs using an x60 water-immersion vector and three helper plasmids (pCAG-KGRIR, pCAG- objective attached to an upright microscope (BX51WI, RTR2 and pCAG-VSVG) into HEK293tSA cells using a Olympus) at room temperature. Intracellular solutions calcium phosphate method. 36–40 h after the transfec- were composed of (in mM): 150 Cs-gluconate, 10 HEPES, tion, the culture media were collected and centrifuged at 4 MgCl , 4 Na ATP, 1 Na GTP, 0.4 EGTA and 5 lidocaine 2 2 2 24,000 rpm for 2 h at 2 ℃ to concentrate the virus. After N-ethyl bromide (QX-314) (pH 7.25, 290–300 mOsm/kg) the centrifugation, the pellets were dissolved with a small for the CF- and PF-EPSC recordings. The patch pipette volume of cold medium and stored frozen at -80 ℃. The resistance was 1–2 MΩ. 8–9 lentivirus was at a 10 titer unit. To evoke CF-EPSCs, a glass pipette filled with ACSF AAV-DJ vectors were used to express C1ql1 in CFs was placed on the granular layer near (20–100 μm) the and ESKir2.1 and Cre in PCs. AAVs were produced by PC voltage-clamped at -10 mV and square paired pulses transfecting the pAAV vector, pHelper and pAAV-DJ (20 µs duration, 0–300 µA and 50 ms inter-stimulus into 293AAV (Cell Biolab, lnc) using a calcium phosphate interval) were applied. Selective stimulation of CFs was method. 36–40 h after transfection, cells were collected confirmed by the paired-pulse depression of EPSC and AAVs were purified by using an AAV purification kit amplitudes. The number of functional CF synapses on (Takara #6666) following the manufacturer’s protocols. single PCs was estimated by varying the stimulus inten- As for the pAAV vectors, SmaI digestion was performed sity because a single CF input has a single threshold for to confirm that the two SmaI sites within the ITR were excitation. maintained. The titer of AAVs was determined by using To estimate the location of stimulation for evoking the AAVpro Titration Kit (Takara #6233) and calculated main or surplus CF-EPSC, the XY coordinates of the 10–11 as 10 vector genomes/mL. stimulation location were defined with the center of the PC soma as the origin. The stimulus electrode was moved Stereotaxic injection systematically every 10 μm in the XY direction on the Mice (3–4 weeks) were anesthetized with a mixture of surface of the granular layer. Square paired pulses (20 µs ketamine (80 mg/kg body weight) and xylazine (20 mg/ duration, 0–300 µA and 50 ms inter-stimulus interval) kg body weight) by intraperitoneal injection. To express were applied at about 40 stimulation points, respectively. the genes into PC, glass pipettes filled with the AAV and/ Nearest distances for evoking main or surplus CF-EPSC or lentivirus solution (2–4 µL) were inserted into the cer- were calculated as the mean of 3 stimulation points clos- ebellar vermis at the depth of 0.5 mm. The solution was est to the origin, at which main or surplus CF-EPSC were injected at a speed of 0.25 µL/min. evoked. To express genes in the CFs, glass pipettes were To ensure that EPSCs were evoked by CFs trans- inserted into the ION according to the reported method duced by AAV, we optogenetically stimulated CFs by with small modifications [ 40]. The AAV solution (1.0– co-expressing ChR2 in IO neurons in Figs. 2 and 7. Light 1.5 µL) was injected at a speed of 0.2 µL/min. After the stimulation (wavelengths: ~470 nm, 4–10 ms dura- injections, the incised skin was closed by using adhesive tion, 100 ms inter-stimulus interval) was applied from and the mice transiently stayed in the recovery cages for a mercury lamp (Olympus) combined with a mechani- 12–16 h. Then mice were returned to their home cages. cal shutter. The number of functional CF synapses on single PCs was estimated by varying the light intensi- Electrophysiology ties (0–4 mW/mm ) and durations. Some PCs (15–20%) Mice were anesthetized with isoflurane and the brains did not respond to light stimulation, most likely because were rapidly removed and immersed in an ice-cold cho- not all CFs expressed ChR2. Due to the exclusion of line-based cutting solution containing (in mM): 120 Cho- non-responding PCs, the number of functional CFs line Cl, 3 KCl, 1.25 NaH PO , 28 NaHCO , 8 MgCl , 22 determined by optogenetic CF stimulation is likely an 2 4 3 2 Glucose and 0.5 Ascorbate, bubbled with a mixture of underestimation. Indeed, unlike electric stimulation of 95%O and 5%CO . Parasagittal cerebellar slices (200- CFs [18], optogenetic stimulation failed to detect mul- 2 2 µm thick) were prepared by using a micro-slicer (Pro7N, tiple CF inputs on single PCs in Bai3 knockout mice Dosaka EM) in the ice-cold cutting solution. The pre - (Fig. 2H). pared slices were transferred into the artificial cerebro - To evoke PF-EPSCs, square paired pulses (20 µs dura- spinal fluid (ACSF) containing (in mM) 125 NaCl, 2.5 tion, 0–200 µA and 50 ms inter-stimulus interval) were Aimi et al. Molecular Brain (2023) 16:61 Page 4 of 17 applied through a glass pipette placed on the molecu- taken by using confocal microscopy (FV1000, Olympus). lar layer. PCs were voltage-clamped at -80 mV. Selective To observe and trace transverse CF branches, z-stack stimulation of PFs was confirmed by the paired-pulse images (1 μm step for 30 μm) were taken. Images were potentiation of EPSC amplitudes. analyzed by using Fiji software. Regions of interest (ROI), For recording mIPSCs, intracellular solutions were such as cell soma and vGluT2 puncta, were selected man- composed of (in mM): 120 Cs-Chloride, 20 HEPES, 1 ually, and signal intensities were measured in each ROI. MgCl , 4 Na ATP, 10 sucrose (pH 7.25, 290 mOsm/kg). Transverse CF branches were manually traced, and the 2 2 mIPSC were recorded from PCs in ACSF containing length of each branch was measured (µM): 10 NBQX, 20 D-AP5 and 1 TTX, in which picro- 2+ toxin was not added. PCs were voltage-clamped at -70 Ca imaging 2+ mV and mIPSCs were recorded as inward currents. Ca imaging was performed in PCs under the current Current responses were recorded with an Axopatch clamp condition using confocal laser-scanning micros- 200B amplifier (Molecular Devices) and pClamp software copy (FV-1200, Olympus). Intracellular solutions were (version 10, Molecular Devices) was used for data acqui- composed of (in mM): 130 K-Gluconate, 10 KCl, 10 sition and analysis. Signals were filtered at 1 kHz and dig - HEPES, 1 MgCl , 4 Na ATP, 1 Na GTP, 15 sucrose and 2 2 2 itized at 4 kHz. 0.1 Oregon Green 488 BAPTA-1 (OGB-1) (pH 7.25, 315 mOsm/kg). We waited at least 20 min after establish- Immunohistochemistry ing the whole-cell mode to fill PC dendrites with OGB-1 Mice were anesthetized by intraperitoneal injection of 2% and identified the stimulation site for evoking main and avertin and fixed by perfusion with 4%PFA/0.1 M sodium surplus CF inputs by CF-EPSC recording. Fluorescence phosphate buffer (PB). Fixed brains were submerged in images were acquired at 5–10 Hz while recording CF- 4%PFA/0.1 M PB at 4℃ for 12–16 h and solutions were evoked voltage changes. In some cases, the extracel- 2+ replaced with PBS containing 0.1% sodium azide. Brains lular Ca concentration was increased from 2 mM to 2+ were embedded in a 2% agarose gel just before section- 5 mM to enhance Ca changes induced by surplus CF ing. The cerebellar cortex (50 μm thickness, sagittal or stimulations. coronal) and the brainstem, including the IONs (50 μm, The acquired fluorescence images were analyzed 2+ coronal), were cut by a micro-slicer (DTK-1000; Dosaka by using Fiji software. CF-evoked Ca changes were EM). expressed as increases in the fluorescence value (ΔF) Immunostaining was performed in glass tubes. After divided by the averaged fluorescence value before CF sections were treated with 10% donkey serum, primary stimulations (F ). The area that showed a large Ca eleva - antibodies (Guinea pig anti-GFP [Frontier Institute], tion (ΔF/F within 30% of the peak value) was analyzed. Goat anti-GFP [Frontier Science], Chicken anti-GFP Voltage responses were recorded with an Axopatch 200B [Millipore], Rat anti-mCherry [Thermo Fisher Scientific], amplifier (Molecular Devices) and pClamp software (ver - Rabbit anti-vGluT2 [Frontier Institute], Guinea pig anti- sion 10, Molecular Devices) was used for data acquisi- vGluT2 [Frontier Institute], Goat anti-vGluT2 [Frontier tion. Signals were filtered at 1 kHz and digitized at 4 kHz Institute], Guinea pig anti-C1ql1 [a gift from Masahiko for the evoked voltage changes. Watanabe], Goat anti-calbindin[Frontier Institute], Rab- bit anti-Bai3 [a gift from Masahiko Watanabe], Rabbit Statistical analyses anti-HA [Cell Signaling Technology], Rabbit anti-c-Fos Electrophysiological data were analyzed offline using [Merck]) were applied and incubated at room tempera- Clampfit 10 (Molecular Devices). Immunohistochemical 2+ ture for 12–16 h. The specificity of the antibodies against and Ca imaging data were analyzed by using Fiji (Image C1ql1 and Bai3 was previously confirmed by the lack J) software. All bar graphs indicate mean ± standard of immunoreactivity in C1ql1 and Bai3 knockout mice, error of the mean. Statistical analyses were performed respectively [18]. Sections were washed with PBS 3 times using Mirosoft Excel (Microsoft) and BellCurve for Excel and incubated with secondary antibodies, which were (Social Survey Research Information Co., Ltd.). To com- conjugated with fluorescence dye such as Dylight 405, pare the number of CFs, which has discrete variables, we Alexa 488, 594, 647 and Cy3 (Molecular probes or Jack- used Mann-Whitney U test for two groups (Figs. 2D and son ImmunoResearch Laboratories) against the respec- H, 3D and 7D) and Kruskal-Wallis test followed by Steel tive primary antibody, together with DAPI at room test for multiple groups (Figs. 4D, 5D and 6D). For other temperature for 2 h. Sections were washed with PBS and continuous variables, we used Welch’s t-test for com- mounted on slide glasses with fluoromount G (Invitro - parison of two groups (Figs. 1C, 2C, E, G and I, 3E and gen). For staining HA-C1ql1, pepsin treatment (37℃ for F and 7C) and two-way ANOVA followed by Dunnett 5 min) was performed to expose antigens before treat- test (Fig. 5C) or Tukey test (for two parameters; Fig. 6C). ment with 10% donkey serum. Fluorescent images were Since the number of mice was small (n = 4), we also used Aimi et al. Molecular Brain (2023) 16:61 Page 5 of 17 Mann-Whitney U test for Fig. 1C and confirmed the C1ql1 expression in the soma of IONs was doubled by same statistical significance (*p = 0.0433) as Welch’s t-test. AAV-based expression (Fig. 1C). In the cerebellar cortex, The sample size, p-value, and the statistical test used in high levels of YFP signal were detected in the molecular each figure are also provided in figure legends. layer, with no evidence of misexpression in mossy fibers with rosette-like structures in the granular layer (Fig. 1D). Results Immunohistochemical staining for calbindin, a PC Selective gene expression in IONs and CFs using AAV and marker, and vesicular glutamate transporter 2 (vGluT2), a knock-in mice presynaptic marker for CFs, showed that HA-C1ql1 was To test the role of C1ql1-Bai3 signaling in structural localized to CF terminals along PC dendrites (Fig. 1E). changes in mature CF PC synapses, we first examined These results indicate that AAV-based delivery of C1ql1 the effect of increased expression of C1ql1 on CF syn - to Htr5b-tTA knock-in mice specifically and moderately apses. To avoid possible indirect effects of misexpres - increased the amount of C1ql1 in CF terminals. sion of C1ql1 in mossy fibers [ 24], we used Htr5b-tTA knock-in mice in which IONs, the origin of CFs, specifi - Increased C1ql1 levels in CFs induce re-innervation of cally express tTA [25]. We delivered adeno-associated mature PCs by multiple CFs virus (AAV) encoding the tetracycline response element Next, we examined the effect of increased C1ql1 expres - (TRE) followed by a channel rhodopsin-2 yellow fluo - sion on the function of CF-PC synapses using whole-cell rescent fusion protein (ChR2-YFP) and human influenza patch-clamp recordings from PCs in acute slices. AAV- hemagglutinin (HA)-tagged C1ql1. ChR2 was intro- TRE-ChR2-YFP (control) or AAV-TRE-ChR2-YFP-P2A- duced to directly assess the function of CFs in the later C1ql1 was injected into the ION of Htr5b-tTA knock-in experiments (Fig. 1A). Three weeks after injection into mice at 3–4 weeks of age (Fig. 2A). Application of two 3–4-week-old mice, HA-C1ql1 and YFP were detected light stimuli with an interval of 100 ms evoked paired- in IONs (Fig. 1B). C1ql1 immunostaining indicates that pulse depression of excitatory postsynaptic currents Fig. 1 CF selective gene delivery using Htr5B-tTA mice and AAV A Experimental scheme. AAVs encoding ChR2-YFP with or without C1ql1 were injected into the ION. The right panel shows YFP signals in the ION. Scale bar, 500 μm. B Expression levels of C1ql1 in the ION. Immunohistochemical staining shows total C1ql1, exogenous HA-C1ql1 and YFP in the ION infected with AAV-CTRL and AAV-C1ql1. DAPI staining shows the nucleus. Scale bar, 20 μm. C Quantification of C1ql1 immunoreactivity in the soma of IONs ( B ). p = 0.0389, Two-tailed Welch’s t-test; n = 4 mice each. D Selective expression of YFP and C1ql1 in the CFs. No YFP signals were detected in the mossy fibers in the granular layer. ML, molecular layer; PCL, Purkinje cell layer; GL, granular layer. Scale bar, 40 μm. E Immunohistochemical staining of vGluT2 (magenta), YFP or HA-C1ql1 (green) and calbindin (blue) indicates accumulation of HA-C1ql1 at CF synapses. Scale bar, 10 μm. Bars represent mean ± SEM. *p < 0.05 Aimi et al. Molecular Brain (2023) 16:61 Page 6 of 17 Fig. 2 Increased C1ql1 levels in CFs allow adult PCs to be innervated by multiple CFs A Experimental scheme for recording CF-EPSCs. B Representative CF-EPSC traces from wild-type PCs. The blue bar indicates the timing of light stimu- lation. Increasing the light intensity elicited a single EPSC in control slices (B ) in an all-or-none manner, but multiple EPSCs with a slower rise time in slices overexpressing C1ql1 (B ). C Total CF-EPSC amplitude. The graph shows the sum of the peak amplitudes of single CF-EPSCs or multiple CF-EPSCs. p = 0.0372, two-tailed Welch’s t-test; n = 28 cells from 4 mice (CTRL); n = 29 cells from 5 mice (+ C1ql1). D The percentage of the number of CFs innervating single PCs. The number of EPSCs evoked by distinct CF activation thresholds (number of steps) is shown. p = 0.0234, Mann–Whitney U test; n = 51 cells from 4 mice (CTRL); n = 49 cells from 5 mice (+ C1ql1). E Average of the 10–90% rise time of CF-EPSCs. p = 0.0028, two-tailed Welch’s t-test; n = 28 responses from 4 mice (CTRL); n = 46 responses from 5 mice (+ C1ql1). F Representative CF-EPSC traces from Bai3 knockout PCs (left: CTRL, right: +C1ql1). G Total CF-EPSC amplitude. The graph shows the sum of peak amplitudes of single CF-EPSCs or multiple CF-EPSCs. p = 0.8534, two-tailed Welch’s t-test; n = 31 cells from 3 mice (CTRL); n = 42 cells from 4 mice (+ C1ql1). H The percentage of the number of CFs innervating single PCs. p = 0.6159, Mann–Whitney U test; n = 38 cells from 3 mice (CTRL); n = 50 cells from 4 mice (+ C1ql1). I Average of the 10–90% rise time of CF-EPSCs. p = 0.0609, two-tailed Welch’s t-test; n = 33 responses from 3 mice (CTRL); n = 46 responses from 4 mice (+ C1ql1). Bars represent mean ± SEM. **p < 0.01; *p < 0.05; ns, not significant (EPSCs), a result consistent with a high release probabil- effect of C1ql1 overexpression on PC development, we ity of CF terminals. Furthermore, increasing light inten- examined PC dendritic arborization, which could affect sity elicited EPSCs in an all-or-none manner in control CF synapse formation. Immunohistochemical staining slices (Fig. 2B ), indicating that PCs are innervated by a of PCs with calbindin revealed no gross differences in single CF input with a single excitation threshold. In con- the dendritic arborization between PCs innervated by trast, overexpression of C1ql1 in CFs not only increased control and C1ql1-overexpressing CFs (Supplementary the amplitude of EPSCs, but also led to the appearance Fig. 1A). In addition, C1ql1 overexpression in CFs did not of CFs with two to three activation thresholds (Fig. 2B , affect the membrane capacitance of PCs, an electrophysi - C, D). In addition, EPSCs evoked by different activation ological estimate of the total surface area (Supplemen- thresholds had a slower rise time than EPSCs evoked tary Fig. 1B). Furthermore, AAV-based overexpression by most CFs (Fig. 2B , E). These results suggest that of C1ql1 in 6-week-old mice increased the percentage of increased expression of C1ql1 in CFs not only enhanced PCs innervated by multiple CFs in the same manner as the functions of existing CF-PC synapses, but also in 3-week-old mice (Supplementary Fig. 1C, D). These induced new CF synapses with distinct properties. results indicate that the effect of C1ql1 overexpression PCs establish a mature innervation pattern with a sin- was not confounded by the developmental stage of the gle CF by postnatal day 20 in rodents [7]. To rule out the PCs. Aimi et al. Molecular Brain (2023) 16:61 Page 7 of 17 C1ql1 regulates CF-PC synapse formation by binding To determine whether the effect of C1ql1 on CF trans - to the CUB domain of Bai3 during development [18, 19]. verse branches required Bai3, we next traced CFs in To determine whether the effect of C1ql1 requires Bai3, coronal cerebellar sections from Bai3 knockout mice we overexpressed C1ql1 in Bai3 knockout mice at 3–4 (Supplementary Fig. 2A) to which AAV-TRE-GFP (con- weeks of age. CF-evoked EPSC amplitudes were much trol) or AAV-TRE-GFP-P2A-C1ql1 was injected. In Bai3 smaller in Bai3 knockout than in wild-type mice (Fig. 2C knockout mice, the overexpression of C1ql1 in CFs did vs. 2G), a result consistent with previous reports [18, not elongate the transverse CF branch or increase the 19]. In contrast to conditional knockout mice in which percentage of vGluT2-positive terminals (Supplemen- the Bai3 gene was postnatally deleted [18], PCs in con- tary Fig. 2B-F). These results indicate that overexpression stitutional Bai3 knockout mice did not show a multiple of C1ql1, likely via interaction with Bai3, could induce innervation pattern by CFs (Fig. 2H). Importantly, over- the growth and synapse formation by of transverse CF expression of C1ql1 in CFs did not result in an increase branches, resulting in an increased number of PCs re- in the amplitude of CF-evoked EPSCs in Bai3 knockout innervated by multiple CFs after CF-PC synapses mature. PCs (Fig. 2G). In addition, overexpression of C1ql1 did not affect the percentage of PCs innervated by CFs with Bai3 overexpression in PCs induces re-innervation by multiple excitation thresholds (Fig. 2F2, H). Similarly, we surplus CFs through C1ql1 binding did not detect CF-evoked EPSCs with a slower rise time While new CF-PC synapses were induced by overexpres- (Fig. 2F2, I). Taken together, these results suggest that sion of C1ql1 in CFs, it was unclear whether PCs over- C1ql1 overexpression, likely via binding to Bai3, could expressing Bai3 could form new synapses with CFs with induce the formation of new CF synapses and increase normal levels of C1ql1. To address this question, we used the proportion of PCs innervated by multiple CFs in lentivirus with the murine stem cell virus (MSCV) pro- mature PCs. moter [28] to preferentially express EGFP and Bai3 in PCs of mice at 3–4 weeks of age (Fig. 4A). Bai3 expres- C1ql1-Bai3 signaling induces synapse formation by sion levels were estimated to be increased by approxi- transverse CF branches mately 1.8-fold (Supplementary Fig. 3A, B). We recorded How can a PC in which excess CFs have already been CF-evoked EPSCs from whole-cell patch-clamped PCs pruned except for a dominant single CF be innervated by placing the stimulating electrode in the granular layer again by other CFs? A previous in vivo time-lapse imag- near the PC soma. CF-EPSCs, which were confirmed by ing study showed that while ascending branches of CFs the paired-pulse depression, were elicited in an all-or- formed stable synapses with proximal dendrites of PCs, none manner in PCs expressing EGFP only, confirming the thin transverse branches were highly dynamic and that approximately 90% of wild-type PCs are innervated did not make synapses in adult wild-type mice [26]. u Th s, by a single CF input (Fig. 4 C, D). In contrast, EPSCs were to clarify the contribution by transverse CF branches, we evoked by two or three thresholds of stimulation in PCs traced GFP-positive CFs in coronal cerebellar sections overexpressing wild-type Bai3 (Fig. 4 C, D), suggesting from wild-type mice to which AAV-TRE-GFP (control) that Bai3 overexpression in PCs induces re-innervation or AAV-TRE-GFP-P2A-C1ql1 was injected at 3–4 weeks by surplus CFs. of age (Fig. 3A, B ). Co-immunostaining of GFP and To rule out an effect of Bai3 overexpression on PC vGluT2 revealed that the transverse CF branches were development, we examined the membrane capacitance of observed at various locations along the PC dendrites, but PCs. As in the case of C1ql1 overexpression in CFs, over- they mostly lacked vGluT2 in control sections (Fig. 3B , expression of Bai3 in PCs did not affect the membrane C, D), indicating their inability to form functional syn- capacitance of PCs (Supplementary Fig. 4A), suggest- apses as reported previously [20, 26, 27]. Interest- ing no gross differences in the total surface area of PCs. ingly, when C1ql1 was overexpressed in CFs, transverse Furthermore, lentivirus-based overexpression of Bai3 in branches elongated and often became positive for vGluT2 6-week-old mice increased the percentage of PCs inner- (Fig. 3B , C, D). The elongation of the transverse branch vated by multiple CFs in the same manner as injection occurred mostly in distal dendrites (80–160 μm from into 3-week-old mice (Supplementary Fig. 4B, C). These the soma) (Fig. 3 C, E). Transverse branches that were results indicate that the effect of Bai3 overexpression was positive for vGluT2 were longer than those negative for not confounded by the developmental stage of the PCs. vGluT2 (Fig. 3F). Since EPSCs at synapses farther elec- Bai3 belongs to the adhesion G protein-coupled recep- trotonic distance from the recording site show a slower tor family, which mediates intracellular signaling through rise time, these results suggest that an increased propor- distinct functional domains [29–32]. To gain insight into tion of PCs innervated by multiple CFs is at least partly the signaling mechanism mediated by Bai3, we expressed caused by the transverse CF branches forming synapses Bai3 with mutations in these functional domains on distal dendrites. (Fig. 4B). Expression of Bai3-AAA, disabling the ELMO Aimi et al. Molecular Brain (2023) 16:61 Page 8 of 17 Fig. 3 C1ql1-Bai3 signaling induces synapse formation by transverse CF branches A Experimental scheme. B Coronal cerebellar sections. GFP (CTRL) or GFP plus C1ql1 (+ C1ql1) was overexpressed in CFs. Maximum intensity z-projection images are shown. Dotted lines, upper and lower boundaries of the molecular layer (B ). Enlarged views of representative CF branches (B ). Arrowheads 1 2 indicate vGluT2-negative and positive branches in CTRL and + C1ql1, respectively. Scale bars, 20 μm. C Height and length of CF transverse branches in the molecular layer. Branch height was measured from the apical pole of PC somata. Transverse branches negative (-) and positive (+) for vGluT2 are indi- cated by white and red circles, respectively. n = 152 branches (CTRL), n = 132 branches (+ C1ql1). D Percentage of vGluT2-positive CF transverse branches − 5 in C TRL or + C1ql1. p = 1.352 × 10 , n = 152 (C TRL); n = 132 (+ C1ql1). Mann–Whitney U test. E Histogram showing the mean length of CF transverse branches as a function of their height in the molecular layer. Black and orange bars represent the cerebellum in CTRL and + C1ql1, respectively. 0–40 μm: − 5 p = 0.8328, n = 3 (C TRL), n = 18 (+ C1ql1); 40–80 μm: p = 0.1731, n = 34 (C TRL), n = 36 (+ C1ql1); 80–120 μm: p = 2.648 × 10 , n = 63 branches (C TRL), n = 58 − 5 (+ C1ql1); 120–160 μm: p = 7.370 × 10 , n = 49 (C TRL), n = 20 (+ C1ql1); >160 μm: p = 0.1321, n = 3 (C TRL), n = 9 (+ C1ql1). Two-tailed Welch’s t-test. F Histogram showing the mean length of CF transverse branches with the presence or absence of vGluT2. CTRL: p = 0.4338; vGluT2(-), n = 137; vGluT2(+), n = 15. +C1ql1: p = 0.003482; vGluT2(-), n = 93; vGluT2(+), n = 39. Two-tailed Welch’s t-test. All datasets are from 3 mice per group (CTRL and + C1ql1). Bars represent mean ± SEM. **p < 0.01; *p < 0.05; ns, not significant binding motif [29, 30], Bai3-ΔCT7, which lacked the PDZ CFs by binding to C1ql1, but independently of ELMO, binding motif [32] and Bai3-S832A, which disrupted PDZ proteins or proteolysis at the GAIN domain. the proteolysis sequence in the GPCR auto-proteolysis- inducing (GAIN) domain [31], had similar effects as Bai3 overexpression in PCs induces re-innervation by CFs wild-type Bai3 in inducing re-innervation of PCs by sur- at distal dendrites plus CFs (Fig. 4 C, D). In contrast, the expression of Bai3- The largest EPSCs observed in PCs overexpressing Bai3, ΔCUB, which lacked the binding site for C1ql1 [18], did which we termed “main CF-EPSC”, had similar kinet- not affect the pattern of CF innervation in mature PCs ics to EPSCs seen in control PCs, but the smaller EPSCs (Fig. 4 C, D). These results indicate that overexpression (surplus CF-EPSCs) elicited by distinct stimulus thresh- of Bai3 in mature PCs induced innervation by additional olds had slower rise times (Fig. 4E). Since overexpres- sion of C1ql1 in CFs also caused the appearance of small Aimi et al. Molecular Brain (2023) 16:61 Page 9 of 17 Fig. 4 Bai3 overexpression in PCs induces re-innervation by CFs at distal dendrites by binding to C1ql1 A Experimental scheme. B Diagram of the functional domains of Bai3 and its mutants. C Representative CF-EPSC traces recorded from adult wild-type PCs overexpressing the indicated constructs. Paired-pulse stimulation with 50-ms interstimulus interval was applied. D The percentages of the number of CFs innervating single PCs. The number of EPSCs evoked by distinct CF activation thresholds (step numbers) is shown. Bai3-WT: p = 0.0100, n = 69 cells from 11 mice; Bai3-AAA: p = 0.0278, n = 30 cells from 4 mice; Bai3-ΔCT7: p = 0.0007, n = 60 cells from 8 mice; Bai3-S832A: p = 0.0186, n = 22 cells from 3 mice; Bai3- ΔCUB: p = 0.9981, n = 63 cells from 8 mice. Kruskal-Wallis test followed by Steel test vs. CTRL: n = 57 cells from 7 mice. E Average of the 10–90% rise time of CF-EPSCs in PCs overexpressing Bai3-WT. p = 0.0018, Two-tailed Welch’s t-test, n = 24 traces (main), n = 14 traces (surplus) from 11 mice. F Time course of 2+ CF-evoked Ca changes associated with main and surplus EPSPs. Changes in the fluorescence (ΔF) were normalized by the averaged fluorescence (F ) 2+ 2+ before the CF stimulation (arrowhead). Inset, representative CF-EPSPs during Ca imaging. G Representative CF-evoked Ca changes associated with 2+ 2+ main and surplus EPSPs. S, PC soma. Scale bar, 20 μm. H The mean area of Ca elevation associated with main or surplus CF-EPSPs. The area of large Ca − 5 elevation (ΔF/F within 30% of the peak value) was measured. p = 2.763 × 10 , two-tailed Welch’s t-test, n = 7 responses (main), n = 7 (surplus) from 5 mice. 2+ I The closest distance between the site of large Ca elevation and the PC soma was measured (see Methods). p = 0.0075, two-tailed Welch’s t-test, n = 7 responses (main), n = 7 (surplus) from 5 mice. Bars represent mean ± SEM. **p < 0.01; *p < 0.05; ns, not significant CF-evoked EPSCs with slower kinetics (Fig. 2E) and IONs by injecting AAV-TRE-tdTomato into Htr5b-tTA synapse formation at distal dendrites by transverse CF knock-in mice (Supplemental Fig. 3C). We found a few branches (Fig. 3C), we hypothesized that Bai3 overex- PCs that expressed Bai3 and were selectively innervated pression in PCs similarly induced new CF synapse forma- by transverse CF branches expressing tdTomato without tion on distal dendrites. labeled main CF inputs (Supplemental Fig. 3D). However, To test this hypothesis morphologically, we expressed since the identification of surplus CF branches relies on EGFP and Bai3 in PCs by lentivirus and sparsely labeled the coincidental sparse labeling of PCs and CFs, it was Aimi et al. Molecular Brain (2023) 16:61 Page 10 of 17 difficult to quantify the effect of Bai3 on the formation PCs preferentially induces CF synapses without signifi - of surplus CF synapses by the immunohistochemical cantly altering the number of other synapses. method. To clarify the location of surplus CF synapses that gave Endogenous Bai3 and C1ql1 are involved in the rise to EPSCs with slow kinetics, we next used the elec- re-innervation of CFs in mature PCs trophysiological mapping method. We systematically Can CFs form new synapses in mature cerebellar cir- moved the stimulating electrode every 10 μm in the XY cuits that do not overexpress C1ql1 or Bai3? Loss of direction in the granular layer (Supplementary Fig. 5A). PF-PC synapses in conditional GluD2 knockout mice has We found that at some locations, main and surplus been reported to trigger re-innervation of PCs through EPSCs could be evoked by varying the stimulus inten- CF transverse branches without exogenous manipula- sity, while at other locations, only main or surplus EPSCs tion of C1ql1-Bai3 signaling [20]. Therefore, we investi - were selectively evoked. Overall, the location of the stim- gated the role of endogenous Bai3 in conditional GluD2 ulating electrode that evoked surplus EPSCs was farther knockout mice. Using lentivirus with an MSCV pro- from the PC soma than that elicited main EPSCs (Supple- moter, we sparsely expressed a Cre recombinase and mentary Fig. 5B). These results suggest that in PCs over - EGFP in PCs of 3–4-week-old wild-type and conditional f/f f/f expressing Bai3, surplus EPSCs are evoked by CFs that GluD2 (Grid2 ) and/or Bai3 (Bai3 ) knockout mice travel farther from the cell body than the main CF. (Fig. 5A, Supplementary Fig. 7A). Whole-cell patch- To directly visualize where surplus CFs formed func- clamp recordings from acute cerebellar slices prepared tional synapses with PCs overexpressing Bai3, we loaded from conditional GluD2 knockout mice two months 2+ PCs with a Ca indicator (Oregon green BAPTA-1) after Cre introduction revealed that PCs were inner- through a patch electrode. We first identified the sites vated by multiple CFs with distinct thresholds (Fig. 5B, where only main or surplus CF-EPSCs were selectively D), as previously reported [20]. In contrast, CF evoked evoked (Supplementary Fig. 5C) and then performed smaller EPSCs in an all-or-none manner in conditional 2+ Ca imaging under the current-clamp mode (Fig. 4F-I). Bai3 knockout mice (Fig. 5B-D), indicating that the pat- Stimulation of sites where main EPSPs were selectively tern of innervation of PCs by a single CF is unaffected by 2+ elicited caused greater increases in Ca concentrations knocking out Bai3 in adult mice as reported previously from a larger dendritic area than stimulation of sites [12]. Interestingly, in contrast to GluD2 knockout mice, where surplus EPSPs were selectively elicited (Fig. 4G, when both GluD2 and Bai3 were knocked out, CF-EPSCs 2+ H). Furthermore, Ca elevations associated with sur- became smaller (Fig. 5B, C), but many PCs remained plus EPSPs were observed in dendrites more distal to innervated by a single CF (Fig. 5D). Furthermore, C1ql1 the PC soma than those associated with the main EPSPs immunopositive puncta were significantly upregulated (Fig. 4G, I). These results further support the hypothesis in the upper molecular layer of GluD2 knockout mice at that overexpression of Bai3 in PCs causes the formation 2–3 months of age (Supplemental Fig. 7B, C, D), suggest- of new CF synapses on dendrites more distal to the main ing the involvement of C1ql1 in CF synapse formation in CFs, resulting in multiple CF innervation of mature PCs. GluD2 knockout mice. These results suggest that endog - During development, the inhibitory inputs from enous Bai3, probably together with endogenous C1ql1, molecular layer interneurons and CFs compete for syn- is required for re-innervation of mature PCs by CFs in apses on PC somata [33]. In GluD2 knockout mice, PFs GluD2 knockout mice. and CFs compete for synapses on distal dendrites of PCs [20]. Therefore, to explore the possibility that Bai3 Bai3-induced re-innervation of PCs by CFs requires PC may affect other types of PC synapses, we recorded PF- activity evoked EPSCs, which were confirmed by paired-pulse Since structural synaptic plasticity occurs in an activity- facilitation, in PCs overexpressing Bai3 (Supplementary dependent manner throughout life in the mammalian Fig. 6A). The amplitudes of PF-EPSCs in response to brain [34, 35], we next investigated whether increased increasing stimulus intensities were similar between PCs C1ql1-Bai3 levels could bypass neuronal activity to form expressing EGFP only (control) and EGFP plus Bai3 (Sup- new CF synapses in mature PCs. Using an AAV-based plementary Fig. 6B, C). Miniature inhibitory postsynap- Cre-DIO (double-floxed inverse open reading frame) sys - tic currents (mIPSCs) recorded from PCs overexpressing tem, we expressed EGFP and ESKir2.1, a non-rectifying Bai3 Supplementary Fig. 6D) and control showed similar variant of Kir2.1 potassium channel [36], to specifically amplitudes and frequencies (Supplementary Fig. 6E, F). suppress levels of intrinsic PC activity (Supplementary Although local competition may be missed because PF Fig. 8A). As a control, we used ESKir2.1 , a mutant AAA and inhibitory synapses outnumber CF synapses, these channel lacking channel activity [36]. Loose-patch results indicate that overexpression of Bai3 in mature recordings in acute cerebellar slices prepared from mice 2–3 weeks after the AAV injection at 3–4 weeks of age Aimi et al. Molecular Brain (2023) 16:61 Page 11 of 17 Fig. 5 Endogenous Bai3 is required for synapse formation of re-innervating CFs A Experimental scheme. B Representative CF-EPSC traces recorded from PCs of the indicated genotypes. C Total CF-EPSC amplitude. The graph shows the − 6 sum of peak amplitudes of single CF-EPSCs or multiple CF-EPSCs. CTRL (n = 17 cells from 2 mice) vs. Bai3 knockout (KO), p = 1.272 × 10 (n = 29 cells from − 6 5 mice); vs. GluD2 KO, p = 0.8311 (n = 25 cells from 5 mice); vs. Bai3 KO::GluD2 KO, p = 1.336 × 10 (n = 33 cells from 4 mice). One way ANOVA followed by Dunnett’s test. D The percentages of the number of CFs innervating single PCs. The number of EPSCs evoked by distinct CF activation thresholds (step − 5 numbers) is shown. CTRL (n = 29 cells from 2 mice) vs. Bai3 KO, p = 0.9859 (n = 49 cells from 5 mice); vs. GluD2 KO, p = 1.885 × 10 (n = 46 cells from 5 mice); vs. Bai3 KO::GluD2 KO, p = 0.3750 (n = 61 cells from 4 mice). Kruskal-Wallis test followed by Steel test. Bars represent mean ± SEM. **p < 0.01; ns, not significant confirmed the absence of spontaneous action potentials only Bai3, surrounded by silent PCs expressing only in PCs expressing ESKir2.1, but not ESKir2.1 (Supple- ESKir2.1 (Fig. 6A, right; Supplementary Fig. 8E, bottom). AAA mentary Fig. 8B, top traces). Whole-cell voltage-clamp The amplitude of CF-EPSCs in such non-silenced PCs recordings revealed that the amplitude of CF-evoked was similar to that in control PCs expressing ESKir2.1 AAA EPCSs was reduced in PCs expressing ESKir2.1, but the and Bai3 (Fig. 6C). Unexpectedly, however, CF-EPSCs number of stimulus thresholds (reflecting the number of were evoked by a single threshold in these non-silenced CF inputs) was similar to control PCs (Supplementary PCs (Fig. 6B, right traces; Fig. 6D). These results suggest Fig. 8B-D). Similarly, the application of tetrodotoxin or that multiple innervation by CFs requires neuronal activ- NBQX is reported to reduce the amplitude of CF-EPSCs ity not only in the Bai3-expressing PCs but also in the and CF synapses in adult PCs [11–13]. While the site of surrounding PCs. action was unclear in these pharmacological studies, our findings indicate that the intrinsic activity of PCs is CF activity is required for C1ql1 to induce the innervation required to maintain CF synapses in mature PCs. of adult PCs by CFs Next, we examined whether Bai3 overexpression could Finally, we investigated whether increased C1ql1 lev- induce CF re-innervation in PCs expressing ESKir2.1 by els in CFs could induce new CF synapses in the absence coinfecting L7-Cre mice with AAV-Syn-DIO-ESKir2.1- of CF activities. To suppress CF activities in vivo, we T2A-EGFP and Lenti-MSCV-mCherry-P2A-Bai3 injected a mixture of AAV-TRE-ChR2-YFP and AAV- (Fig. 6A). In PCs overexpressing Bai3 and ESKir2.1 , TRE-mCherry-P2A-ESKir2.1 into the ION of Htr5B- AAA we detected CF-EPSCs with multiple thresholds (Fig. 6B, tTA knock-in mice at 3–4 weeks of age (Supplementary left traces; Fig. 6D), as observed in the absence of Fig. 9A). With acute slice preparations where input fibers ESKir2.1 (Fig. 4 C, D). In contrast, CF stimulation are not preserved, it is difficult to determine how effec - AAA evoked smaller EPSCs with a single threshold in most tively ESKir2.1 could silence the electrical activity of PCs coexpressing ESKir2.1 and Bai3 (Fig. 6B, middle IONs in vivo. Instead, we injected harmaline intraperito- traces; Fig. 6D). These results indicate that intrinsic PC neally, which transiently increases the synchronous fir - activity is required for Bai3 to re-innervate mature PCs. ing of IONs [26], and performed immunohistochemical In these experiments, expression of ESKir2.1 (as staining for c-Fos, a marker of neuronal activity, 10 min detected by EGFP) was widespread in many PCs at the after the injection (Supplementary Fig. 10A). We found injection site, whereas Bai3 (as detected by mCherry) was that the number of c-Fos-positive IONs was signifi - detected in only a few PCs (Supplementary Fig. 8E). How- cantly reduced by the expression of ESKir2.1 compared ever, we occasionally found non-silenced PCs expressing to ESKir2.1 (Supplementary Fig. 10B, C). In addition, AAA Aimi et al. Molecular Brain (2023) 16:61 Page 12 of 17 Fig. 6 Bai3-induced re-innervation by CFs requires PC activity A Experimental scheme. The right panel shows three cases: in all cases recorded PCs overexpress Bai3, but all PCs are active (spiking PCs, left), all PCs are silenced by ESKir2.1 (silent PCs, middle) and recorded PCs are active but neighboring PCs are silenced (right). B Representative action potentials by loose-patch recordings (upper traces) and CF-EPSCs by whole-cell patch-clamp recordings (lower traces) from PCs expressing the indicated constructs. C Total CF-EPSC amplitude. The graph shows the sum of peak amplitudes of single CF-EPSCs or multiple CF-EPSCs. p = 0.0098, CTRL + Bai3 vs. ESKir2.1 + Bai3; p = 0.9500, CTRL + Bai3 vs. ESKir2.1 + Bai3, GFP (-); p = 0.0393, ESKir2.1 + Bai3 vs. ESKir2.1 + Bai3, GFP (-). One way ANOVA followed by Tukey’s test. n = 16 cells from 3 mice (CTRL + Bai3), n = 18 cells from 3 mice (ESKir2.1 + Bai3), n = 12 cells from 3 mice (ESKir2.1 + Bai3, GFP (-)). D The percentages of the num- ber of CFs innervating single PCs. The number of EPSCs evoked by distinct CF activation thresholds (step numbers) is shown. p = 0.0061, CTRL + Bai3 vs. ESKir2.1 + Bai3; p = 0.0182, CTRL + Bai3 vs. ESKir2.1 + Bai3, GFP (-); p = 0.9894, ESKir2.1 + Bai3 vs. ESKir2.1 + Bai3, GFP (-). Kruskal-Wallis test followed by Steel test n = 44 cells from 5 mice (CTRL + Bai3), n = 49 cells from 9 mice (ESKir2.1 + Bai3), n = 37 cells from 6 mice (ESKir2.1 + Bai3, GFP (-)). Bars represent mean ± SEM. **p < 0.01; *p < 0.05; ns, not significant the amplitude of CF-EPSCs was decreased by the expres- co-expression of C1ql1 and ESKir2.1 (Fig. 7D). These sion of ESKir2.1 in IONs (Supplementary Fig. 9B, C) results indicate that CF activity is required for C1ql1 to while the innervation pattern of CF was unchanged (Sup- induce additional CF innervation onto adult PCs. plementary Fig. 9B, D), indicating that the intrinsic activ- To gain insight into why CF activity enhances synap- ity of IONs, which is required to maintain CF synapses in togenesis through C1ql1, we performed immunohis- adult PCs, was suppressed by ESKir2.1. tochemical staining of HA-C1ql1 in mice expressing To examine the effect of CF activities on C1ql1- HA-C1ql1 and either ESKir2.1 or ESKir2.1 in the AAA induced CF synapse formation, we next injected a IONs (Fig. 7E). In the cerebellar cortex, HA-Clql1 immu- mixture of AAV-TRE-ChR2-YFP-P2A-C1ql1 and AAV- noreactivity on vGluT2-positive CF terminals was signifi - TRE-mCherry-P2A-ESKir2.1 into the ION of Htr5B- cantly reduced in CFs expressing ESKir2.1 as compared tTA knock-in mice (Fig. 7A). Whole-cell patch-clamp to those expressing ESKir2.1 (Fig. 7F, H). In contrast, AAA recordings revealed that in mice expressing ChR2, C1ql1 no difference was observed in HA-C1ql1 immunoreac - and ESKir2.1 were expressed in the IONs, additional tivity in the cell bodies of IONs expressing ESKir2.1 ver- AAA EPSCs with a slow time course were elicited in response sus ESKir2.1 (Fig. 7G, I). While the neuronal activity AAA to an increasing light stimulus (Fig. 7B ), as observed in of IONs could affect CF synaptogenesis through various the absence of ESKir2.1 (Fig. 2B ). In contrast, when pathways (discussed in detail below), our findings sug - AAA 2 C1ql1 was overexpressed with ESKir2.1 in the IONs, gest that one possibility is its involvement in the secre- the amplitude of CF-EPSCs was significantly reduced tion from CF terminals since C1ql1 immunoreactivity in (Fig. 7B , C). Furthermore, the percentage of PCs that the adult cerebellar cortex is detected in the synaptic cleft were innervated by the surplus CFs was reduced by [18]. Aimi et al. Molecular Brain (2023) 16:61 Page 13 of 17 Fig. 7 CF activity is required for C1ql1 to induce CF innervation on adult PCs A Experimental scheme for silencing of CFs and recording of CF-EPSC. B Representative light-evoked CF-EPSC traces (blue lines). Multiple EPSCs with a slower rise time were recorded in slices overexpressing C1ql1 in CFs (B ). The boxed region is enlarged on the right to show the slow CF-EPSC. Single − 7 EPSC was evoked in an all-or-none manner in slices overexpressing C1ql1 and ESKir2.1 in CFs (B ). C Total CF-EPSC amplitude. p = 2.665 × 10 , two-sided Welch’s t-test. n = 27 cells from 5 mice (CTRL), n = 31 cells from 5 mice (ESKir2.1). D The percentage of the number of CFs innervating single PCs. The num- ber of EPSCs evoked by distinct CF activation thresholds (number of steps) is shown. p = 0.0221, Mann–Whitney U test. n = 34 cells from 5 mice (CTRL), n = 34 cells from 5 mice (ESKir2.1). E Experimental scheme to study the effect of CF neural activity on C1ql1 immunoreactivity. F Immunohistochemical analysis of HA-C1ql1 at CF synapses. Expression of ESKir2.1 (GFP, bottom panels) in IOs reduced C1ql1 (HA) immunoreactivity at CF synapses (vGluT2, arrowheads) compared to ESKir2.1 (GFP, top panels). Scale bar, 10 μm. G Immunohistochemical analysis of HA-C1ql1 in IONs. Scale bar, 20 μm. H AAA Quantification of HA-C1ql1 levels at CF synapses. HA-C1ql1 immunoreactivity was normalized by the GFP fluorescence in vGluT2-positive CF synapses. − 4 p = 2.353 × 10 , two-tailed Welch’s t-test. n = 103 areas from 3 mice (CTRL), n = 169 areas from 3 mice (ESKir2.1). I Quantification of HA-C1ql1 levels in IONs. HA-C1ql1 immunoreactivity was normalized by the GFP fluorescence in the soma of IONs. p = 0.5970, two-tailed Welch’s t-test. n = 149 cells from 3 mice (CTRL), n = 161 cells from 2 mice (ESKir2.1). Bars represent mean ± SEM. **p < 0.01; *p < 0.05; ns, not significant PCs (Fig. 2H, Supplementary Fig. 2 C, D). In addition, Discussion the effect of overexpression of Bai3 in PCs required the In the present study, we showed that mature PCs, which CUB domain, a binding site of Bai3 for C1ql1 (Fig. 4D). achieved innervation by a single strong CFs after prun- Although C1ql1 and Bai3 have additional binding part- ing weak CFs during development, became re-innervated ners, such as kainate receptors [37] and RTN4 [38], by surplus CFs when the expression of C1ql1 or Bai3 was respectively, these results indicate that CF-derived C1ql1 upregulated in CFs or PCs, respectively. Immunohisto- binds to Bai3 in PCs to induce the formation of new CF chemical (Fig. 3E, Supplementary Fig. 3D), electrophysi- synapses. Interestingly, endogenous Bai3 was required 2+ ological (Figs. 2E and 4E), and Ca imaging (Fig. 4G, I) for the re-innervation of mature PCs by CFs in GluD2 studies indicated that transverse CF branches most likely knockout mice (Fig. 5D). Furthermore, the effect of contributed to the formation of surplus CF synapses C1ql1-Bai3 signaling on CF innervation required neu- at distal dendrites of mature PCs. The effect of C1ql1 ronal activity of both PCs (Fig. 6D) and CFs (Fig. 7D). overexpression in CFs required normal levels of Bai3 in Together, we propose a model in which C1ql1-Bai3 Aimi et al. Molecular Brain (2023) 16:61 Page 14 of 17 signaling mediates CF structural plasticity in mature PCs activation of matrix metalloproteinase 9, an enzyme in a manner dependent on neuronal activity (Fig. 8). involved in ECM remodeling, leading to activity-depen- dent spine growth in hippocampal neurons [45]. Since Activity-dependent effect of C1ql1 and Bai3 on innervation by surplus CFs required neural activity not re-innervation of mature PCs by CFs only in the Bai3-expressing PCs but also in the surround- Why do C1ql1 and Bai3 require neuronal activity in ing PCs, activity-dependent modification of the ECM both PCs and IONs to induce CF synapses in mature may provide a permissive environment for new synapse 2+ PCs? P/Q-type Ca channels and aCaMKII regulate the formation in mature PCs. elimination of surplus CFs during development since PCs genetically lacking these molecules remain innervated by C1ql1-Bai3 exerts synaptogenic function through surplus CFs in adulthood [41–43]. However, the selective uncharacterized domains strengthening of the winner CFs is also impaired in PCs Bai3 is reported to inhibit dendritogenesis of PCs dur- 2+ 2+ lacking Ca influx through P/Q Ca channels [44], indi- ing development by regulating the activity of the small 2+ cating that Ca influx is not only required to eliminate GTPase Rac1 through the interaction with ELMO1 weak CFs, but also to selectively strengthen strong CFs and DOCK180 [29]. Similarly, Bai3 has been shown to 2+ during development. Thus, activity-induced Ca influx mediate the fusion of myoblasts by binding to ELMO1/ in PCs may play a role in maintaining newly formed CF DOCK1 during development [30]. Bai1, a close relative synapses in mature PCs. of Bai3, is reported to promote synaptogenesis through Since C1ql1 immunoreactivity at CF-PC synapses the PDZ binding motif by associating with Nlgn1 [46], was reduced when the activity of IONs was suppressed recruiting the Rac1-GEF complex Par3/Tiam1 [47] and (Fig. 7H), C1ql1 may be released from CFs in an activ- stabilizing PSD-95 [48]. An engineered truncation in Bai1 ity-dependent manner. Similarly, the C1q family pro- and Bai3, mimicking autocleavage at the GAIN domain, tein Cbln1 is released from PFs in an activity-dependent led to activation of Ga [32] and G [30], respectively. 12/13 ai1 manner [21]. It has been reported that the motility of However, in the present study, mutations disrupting the transverse CF branches lacking vGluT2-positive presyn- ELMO1 binding motif, the PDZ binding motif and the aptic sites was reduced 3 h after the application of har- GAIN domain did not affect the ability of Bai3 to induce 2+ maline [26]. Thus, the activity-induced Ca increase in CF synapses in adult mice (Fig. 4D). Since mutant Bai3 CF transverse branches may slow down their motility to was expressed in wild-type mice, the mutant Bai3 may facilitate the accumulation of vesicles containing synapse have associated with endogenous Bai3 to compensate for organizers, such as C1ql1, to facilitate synapse forma- the function of the mutated site. However, the inability of tion with mature PCs. In addition, changes in the extra- Bai3-ΔCUB to induce surplus CF innervation indicates cellular matrix (ECM) associated with CF activity may that at least the CUB domain defect cannot be compen- allow C1ql1 to be stabilized at synapses. Indeed, activity- sated for by endogenous Bai3. Thus, considering that dependent release of cathepsin B, a lysosomal enzyme Bai1 can mediate at least five downstream signaling path - co-released with Cbln1, allows presynaptic morphologi- ways by differentially coupling to ELMO, MDM2, Par3/ cal changes associated with PF-PC synapse formation Tiam1, Ga and Bcr, depending on the cellular context 12/13 [21]. Back-propagating action potentials also trigger the [49], we postulate that C1ql1 binding to Bai3 likely exerts exocytosis of cathepsin B from dendrites and subsequent Fig. 8 Proposed model for re-innervation of mature PCs by excess CFs through C1ql1-Bai3 signaling Increased C1ql1-Bai3 signaling induces re-innervation of mature PCs by excess CFs. These new CF inputs, derived from transverse CF branches of neigh- boring PCs, synapse onto the distal dendrites of PCs (left). Neuronal activity of both CFs and PCs is required for C1ql1-Bai3 signaling to induce surplus CFs (right) Aimi et al. Molecular Brain (2023) 16:61 Page 15 of 17 its synaptogenic function through distinct domains that lesions [58] or in GluD2 knockout mice [56] can partially interact with uncharacterized signaling pathways. cross the zebrin II boundary, leading to the connection of some PCs belonging to different microzones. Thus, the Functional implication of added CF synapses by transverse physiological role of newly formed synapses by CF trans- branches versal branches could be complicated depending on the It is difficult to chronically increase the activities of IONs extent to which CF synapses are formed across the zebrin in vivo to investigate whether the expression of endog- II boundary. Since C1ql3, a closely related family member enous C1ql1 or Bai3 changes and induces re-innervation of C1ql1, and Bai3 are expressed in other neural circuits, by CFs. For example, although harmaline administration such as the basolateral amygdala-medial prefrontal cor- rapidly induces tremor-like movements in rodents asso- tex [60] and the anterior olfactory neuron-olfactory bulb ciated with increased activity of IONs, the effect is tran - [61], further studies are warranted to clarify whether and sient and lasts only a few days [50]. However, knockout of how C1ql-Bai3 signaling mediates activity-dependent GluD2 caused re-innervation of mature PCs by multiple structural plasticity in these brain regions in adulthood. CFs without overexpression of C1ql1 or Bai3, but only in Abbreviations the presence of endogenous Bai3 in PCs (Fig. 5D). Impor- AAV Adeno-associated virus tantly, C1ql1 immunopositive puncta were upregulated AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Bai3 Brain specific angiogenesis inhibitor 3 in adult GluD2 knockout mice (Supplemental Fig. 7B, C, C1ql1 C1q-like protein 1 D). These findings suggest that endogenous C1ql1-Bai3 Cbln1 Cerebellin-1 signaling is involved in the re-innervation of PCs by CFs CF Climbing fiber ChR2 Channel rhodopsin-2 under certain pathological conditions. DIO Double-floxed inverse open reading frame The main branches of CFs are distributed parasagit - EPSC Excitatory postsynaptic currents tally in microzones [51], which likely contain ~ 100 PCs in GluD2 Delta-type glutamate receptor 2 HA Human influenza hemagglutinin mice [52]. In contrast, CFs mediolaterally extend trans- ION Inferior olive neurons verse branches for 5–300 μm without forming synapses mIPSC Miniature inhibitory postsynaptic currents in adult mice [26, 53]. Many PCs belonging to the same MSCV Murine stem cell virus NBQX 2,3-Dioxo-6-nitro-1,2,3,4- tetrahydrobenzo[f] microzone show synchronous activity due to electrical quinoxaline-7-sulfonamide coupling between IONs. A computer simulation study PC Purkinje cell indicates that the higher level of electrical coupling of PF Parallel fiber TRE Tetracycline response element IONs accelerates the crude learning at the initial stage tTA Tetracycline transactivator by facilitating the synchronized firing of PCs, while the vGluT2 Vesicular glutamate transporter 2 reduced electrical coupling at the later stage of learning allows more sophisticated and complicated learning [54]. Alternatively, synchronized firing by enhanced electrical Supplementary Information The online version contains supplementary material available at https://doi. coupling of IONs may represent a state change underly- org/10.1186/s13041-023-01048-4. ing skilled movements [55]. Interestingly, PCs in GluD2 knockout mice are reported to show enhanced synchro- Supplementary Material 1 nous firing in the mediolateral direction, which was not mediated by electrical coupling of IONs, but by surplus Acknowledgements transverse CF branches making synapses onto distal PC We would like to acknowledge Drs. Wataru Kakegawa, Itaru Arai and Ayako W. Ishikawa for technical support in the electrophysiological analysis, Dr. Eriko dendrites in vivo [56]. Similarly, after partial lesion of Miura for technical support in immunohistochemistry, Drs. Keiko Matsuda. IONs, surviving CFs are reported to sprout new collater- Tokiwa Yamasaki and Ms. Shihomi Kuwano for assisting virus preparation and als in the mediolateral direction and innervate PCs [57]. sharing cDNA constructs. A live imaging study showed that these newly sprouted Authors’ contributions transverse CF branches preferentially synapse on PCs T.A.: Conceptualization, Methodology, Investigation, Writing–Original draft near the target of the original PCs [58]. Thus, although preparation. K. M.: Investigation. M.Y: Conceptualization, Funding acquisition, 2+ Supervision, Writing–Review & Editing. the EPSCs and Ca transients elicited by newly formed transverse CF synapses are generally small (e.g., Fig. 2B , Funding 4 F, G), they can cause synchronous firing of adjacent PCs This work was supported by the JST CREST (JPMJCR1854 to M.Y.), MEXT KAKENHI (20H05628 to M.Y.), Grant-in-Aid for JSPS Research Fellow (19J10096 in the mediolateral direction, thereby affecting cerebellar to T.A.) and Ushioda Memorial Scholarship (to T.A). learning or recovery after injury. Cerebellar microzones show differential immunore - Data Availability The datasets and plasmids used in this study are available from the activity to zebrin II, largely corresponding to a distinct corresponding author on reasonable request. ensemble of PCs with similar functional properties [59]. However, transverse CF branches induced by partial ION Aimi et al. Molecular Brain (2023) 16:61 Page 16 of 17 connectivity of excitatory inputs converging on cerebellar Purkinje cells. Cell Declarations Rep. 2015 Feb;10(5):820–32. 20. Miyazaki T, Yamasaki M, Takeuchi T, Sakimura K, Mishina M, Watanabe M. Abla- Ethics approval and consent to participate tion of glutamate receptor GluRdelta2 in adult Purkinje cells causes multiple All procedures relating to the care and treatment of mice were performed in innervation of climbing fibers by inducing aberrant invasion to parallel fiber accordance with the guidelines approved by the animal resource committee innervation territory. 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Journal

Molecular Brain – Springer Journals

Published: Jul 24, 2023

Keywords: Cerebellum; Purkinje cell; Climbing fiber; Synapse; Electrophysiology; C1ql1; Bai3

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C1ql1-Bai3 signaling is necessary for climbing fiber synapse formation in mature Purkinje cells in coordination with neuronal activity

C1ql1-Bai3 signaling is necessary for climbing fiber synapse formation in mature Purkinje cells in coordination with neuronal activity

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C1ql1-Bai3 signaling is necessary for climbing fiber synapse formation in mature Purkinje cells in coordination with neuronal activity

C1ql1-Bai3 signaling is necessary for climbing fiber synapse formation in mature Purkinje cells in coordination with neuronal activity

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