Network Activity Shapes Inhibitory Synaptic Development in the Mouse Hippocampus

Erin M. Johnson-Venkatesh; Hisashi Umemori

doi:10.1523/JNEUROSCI.1182-24.2025

Abstract

The proper development of excitatory/inhibitory (E/I) balance is critical for brain function, as any imbalance has been associated with myriad neuropsychiatric disorders. How this balance evolves during synaptic development remains unclear. To address this question, we examine how manipulations of signal-regulatory protein α (SIRPα), a cell adhesion molecule that organizes excitatory synaptogenesis in the hippocampus, affect inhibitory synaptogenesis to maintain E/I balance, using mice of either sex. SIRPα primarily localizes to excitatory synapses. Overexpression or inactivation of SIRPα in a single neuron in hippocampal cultures affects excitatory, but not inhibitory, synapses formed onto the SIRPα-manipulated neuron, indicating that SIRPα is an excitatory, but not inhibitory, synapse organizer. Despite this, bath application of SIRPα's ectodomain increases inhibitory synapses in culture, and global inactivation of SIRPα during critical periods functionally decreases both excitatory and inhibitory synapses in the hippocampus. By using various conditional knock-out mice, we found that SIRPα from pyramidal neurons, but not from interneurons, astrocytes, or microglia, is necessary for proper inhibitory synapse development. Interestingly, inactivation of SIRPα from most pyramidal neurons is necessary to impact inhibitory synaptic development, suggesting that inhibitory synaptogenesis in the hippocampus is driven by the strength of excitation in the pyramidal–neuron network, and not by a change in excitatory input to a single cell. Consistently, the effect of SIRPα's ectodomain on inhibitory, but not excitatory, synaptogenesis is blocked by global neural activity inhibition. We propose that the development of inhibitory synapses in the hippocampus is regulated by network-level excitatory activity to evolve E/I balance.

Significance Statement

How excitatory/inhibitory (E/I) balance evolves during development is still unknown. We manipulated an excitatory synapse organizing cell adhesion molecule, signal-regulatory protein α (SIRPα), in the hippocampus and examined how inhibitory synaptogenesis is affected to maintain E/I balance. Global inactivation of SIRPα during a critical period functionally decreases both excitatory and inhibitory synapses. Using many mouse mutants and manipulations, we identified that inactivation of SIRPα from most pyramidal neurons is necessary to impact inhibitory synaptogenesis and that the effect of SIRPα on inhibitory synaptogenesis is blocked by global neural activity inhibition. We propose that inhibitory synaptogenesis is regulated by the excitatory drive at the network level and not at the single-cell level. Our work reveals fundamental mechanisms that develop E/I balance.

Introduction

Healthy brain development and function require a balanced network. This balance is plastic and multifaceted, changing across developmental time, brain region, and experience-dependent learning. One level of balance critical for neural network function is the correct ratio of excitatory (glutamatergic) and inhibitory (GABAergic) synapses, also known as excitatory/inhibitory (E/I) balance. Alterations to E/I balance are evident in many neurodevelopmental disorders, including epilepsy, autism spectrum disorders, schizophrenia, and attention deficit/hyperactivity disorder (Duman et al., 2019; Lopatina et al., 2019; Liu et al., 2021; Nomura, 2021). Various factors can affect E/I balance, including the development of interneurons (Sohal and Rubenstein, 2019). However, despite its importance, how E/I balance develops during development is largely unknown.

In the developing rodent hippocampus, development for both glutamatergic and GABAergic synapses begins during the first postnatal week (Steward and Falk, 1991; Danglot et al., 2006). During the initial stages of synapse formation, GABAergic synapses are excitatory and then switch to their typical inhibitory function around P10 (Rivera et al., 1999; Ben-Ari, 2001). The growth of these GABAergic synapses then continues through ∼P30. On the other hand, glutamatergic synapses in the hippocampus develop in two main stages: (1) synaptic differentiation from ∼P0 to ∼P14 when the number of glutamatergic synapses increases and (2) synaptic maturation from ∼P15 to ∼P30 when synapses mature in an activity-dependent manner (Hsia et al., 1998; Yasuda et al., 2011; Lohmann and Kessels, 2014). The coordination between the development of GABAergic and glutamatergic systems is not fully understood. In this study, we wanted to investigate how alterations in glutamatergic excitatory synapse development affect GABAergic inhibitory synapse development.

Previously, we have identified signal-regulatory protein α (SIRPα) as a critical promoter of glutamatergic excitatory synaptic development during the synaptic maturation stage (Umemori and Sanes, 2008; Toth et al., 2013). SIRPα is a transmembrane cell adhesion molecule that is abundant in the immune and nervous systems (Matozaki et al., 2009; Barclay and van den Berg, 2014). In the hippocampus, SIRPα is expressed by many different cell types including glutamatergic pyramidal neurons, GABAergic interneurons, astrocytes, and microglia (Zhang et al., 2014). In neurons, SIRPα is predominantly localized at glutamatergic postsynaptic terminals (Toth et al., 2013; Nagappan-Chettiar et al., 2018). We have shown that neuronal SIRPα promotes glutamatergic synaptic maturation in an activity-dependent manner: neural activity leads to the cleavage of the extracellular portion (ectodomain) of neuronal SIRPα, and, after cleavage, the SIRPα ectodomain crosses the synaptic cleft to promote presynaptic maturation of glutamatergic synapses (Toth et al., 2013; Nagappan-Chettiar et al., 2018). When SIRPα was globally inactivated during the period of glutamatergic synaptic maturation in mice, the mice had deficits in excitatory synaptic density, synaptic vesicle clustering, synaptic function, and plasticity (Toth et al., 2013).

Here we use manipulation of SIRPα as a way to alter excitation in the developing hippocampus. We show that unlike its role at excitatory synapses, SIRPα does not alter inhibitory synaptic development in a cell-autonomous manner. However, we also found that global manipulation of SIRPα expression in the hippocampus affects both excitation and inhibition to maintain E/I balance. Using various knock-out (KO) mice, we show that SIRPα from pyramidal neurons, but not from inhibitory interneurons, astrocytes, or microglia, is necessary for proper inhibitory synapse development. Furthermore, we show that SIRPα must be inactivated in most pyramidal neurons to affect inhibitory synaptogenesis. This is in contrast to excitatory synapses, where inactivation of SIRPα in single pyramidal neurons negatively impacts excitatory synaptogenesis. Finally, the effect of SIRPα's ectodomain on inhibitory synaptogenesis is blocked by global neural activity inhibition, while its effect on excitatory synaptogenesis is not. Our results suggest that development of inhibitory synapses is regulated by the excitatory drive at the network level, and not at the single cell level, and reveal a mechanism that regulates E/I balance during development.

Materials and Methods

Mice

All animal care and use were in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee at Boston Children's Hospital or the University Committee on Use and Care of Animals at the University of Michigan. Mice were maintained in standard housing conditions on a 12 h light/dark cycle with food and water provided ad libitum.

SIRPα^fl/fl (flox/flox) mice were previously described (Toth et al., 2013). These mice were crossed with one of the following Cre driver lines: Actin-Cre-ER [Tg(CAG-cre/Esr1*)1Lbe; Guo et al., 2002], Dlx5/6-Cre [Tg(dlx5a-Cre)1Mekk, Jackson Laboratory; Monory et al., 2006], Aldh1l1-Cre [B6;FVB-Tg(Aldh1l1-Cre)JD1884Htz/J, Jackson Laboratory; Tien et al., 2012], Vglut1-Cre [B6;129S-Slc17a7tm1.1(Cre)Hze/J, Jackson Laboratory; Harris et al., 2014], Cx3cr1-Cre-ER [B6.129P2(C)-Cx3cr1^{tm2.1(cre/ERT2)Jung}/J, Jackson Laboratory; Yona et al., 2013], and CAG-Cre-ER [B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J, Jackson Laboratory; Hayashi and McMahon, 2002 ]. In all cases, control mice were littermates, and their genotype was either Cre+::SIRPα^wt/wt or Cre-::SIRPα^fl/fl. No difference was detected between the control genotypes, and so they were combined.

Tamoxifen (100 μg to 1 mg) was injected at the age stated in each experiment to induce the Cre recombinase-mediated excision of the Sirpa gene. This tamoxifen delivery was only for mice crossed with Actin-Cre-ER (global KO), CAG-Cre-ER (global KO), or Cx3cr1-Cre-ER (microglial specific KO). Control mice were littermates that were either Actin-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice (so Cre negative) with tamoxifen; CAG-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice (Cre negative) with tamoxifen; and Cx3cr1-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice (Cre negative) with tamoxifen, per relative experiment. No difference was detected between the genotypes, so they were combined together in each experiment.

Both male and female mice were used in our experiments. We did not detect any significant differences between males and females.

Primary hippocampal cultures and treatment

For all hippocampal cultures used, hippocampi were dissected from Postnatal Day (P)0 to P1 mice and dissociated with 0.5% trypsin. For single-cell overexpression (Fig. 1E–H) and sSIRPα (soluble SIRPα; Fig. 2A–D) experiments, 50,000 cells were plated on poly-d-lysine–coated glass coverslips (12 mm) and grown in Neurobasal culture medium (Invitrogen) supplemented with B27 (Invitrogen), 2 mM l-glutamine, and 100 units/ml penicillin–streptomycin. For single-cell KO (Fig. 1I–L) experiments, 80,000 neurons were plated and grown in NeuralQ culture medium (Sigma-Aldrich) supplemented with GS21 (MTI-GlobalStem, Sigma-Aldrich), 2 mM l-glutamine, and 100 units/ml penicillin–streptomycin. For tamoxifen-treated (Fig. 2I–Q) and AAV-infected (Fig. 9A–I) cultures, 80,000–100,000 cells were plated on poly-d-lysine–coated glass coverslips (12 mm) and grown in Neurobasal plus culture medium (Invitrogen) supplemented with B27 plus (Invitrogen), 2 mM l-glutamine, and 100 units/ml penicillin–streptomycin. For sSIRPα-treated recordings (Fig. 2E–H) and AAV-infected cultures with activity manipulation (Fig. 9J–O), 50,000 cells were plated on poly-d-lysine–coated glass coverslips (12 mm) and grown in Neurobasal plus culture medium (Invitrogen) supplemented with B27 plus (Invitrogen), 2 mM l-glutamine, and 100 units/ml penicillin–streptomycin.

We used immunohistochemistry to determine the percentage of GABAergic neurons in our culture conditions and found only ∼5% of neurons are positive for GABAergic markers [parvalbumin (PV), somatostatin, calretinin].

Transfections were carried out using the calcium phosphate method (CalPhos Mammalian Transfection Kit, Clontech). At DIV3–4, cells were transfected with 1 μg of plasmids for 1 h. The transfected cells were further cultured until DIV18–24 in the same culture media.

sSIRPα was produced as previously described (Umemori and Sanes, 2008). Two nanometer of sSIRPα was delivered to cultured neurons according to the timing described in each experiment.

One micrometer 4-OH-tamoxifen (dissolved in ethanol) was added to cultures at DIV3–4, and mIPSCs were recorded at DIV18–24.

CamKII virus, pENN.AAV.CamKII.HI.GFP-Cre.WPRE.SV40, was a gift from James M. Wilson (Addgene viral prep #105551; http://n2t.net/addgene:105551; RRID:Addgene_105551). The high titer of virus used was 5 × 10¹² gc/ml. The low titer of virus used was 6.25 × 10¹¹ gc/ml. To calculate the percentage of infected cells with each viral titer, we divided the number of GFP-positive cells by the total number of cells as seen in bright field. The eSIRPα (extracellular domain of SIRPα) virus, AAV2/DJ-Syn-SirpaExt-p2A-mcherry at 5.1 × 10¹³ gc/ml, was made at the Boston Children's Hospital Viral Core. The GFP virus, AAV2/DJ-Syn-GFP at 2.64 × 10¹⁴ gc/ml, was made at the Boston Children's Hospital Viral Core.

Immunohistochemistry

Cultures were fixed with methanol for 3 min at −20°C and stained as described previously (Terauchi et al., 2010; Toth et al., 2013) for colocalization experiments (Fig. 1A–C) and sSIRPα-treated cultures (Fig. 2A–D). For the tamoxifen-treated experiment, cultures were fixed for 10 min in 4% paraformaldehyde (PFA)/4% sucrose. Briefly, coverslips were blocked in 2% BSA, 2% normal goat serum, and 0.1% Triton X-100 for 1 h, incubated with primary antibodies overnight at 4°C, secondary antibodies were applied for 1 h at room temperature, and coverslips were mounted with p-phenylenediamine.

Mouse brains were fixed for 24 h with 4% PFA in PBS, then sagittal sections of 20 μm thickness were cut in a cryostat (Leica), and then sections were stained the same way as the coverslips listed above.

Dilutions and sources of antibodies are anti-VGLUT1 (1:5,000; Millipore; AB5905), anti-VGAT (vesicular GABA transporter; 1:1,500; Synaptic Systems; 131003), anti-GFP (1:1,000; Aves Labs; FGP-1020), anti-Iba1 (1:200; FUJIFILM Wako Pure Chemical; 019-19741), polyclonal anti-SIRPα (against the SIRPα C-terminal domain; 1:200; Upstate Biotechnology; 06-729; Fig. 1), anti-SIRPα (against the SIRPα C-terminal domain; 1:200; BD Biosciences; 552371; Fig. 7), anti-somatostatin (1:1,000; Millipore; MAB354), anti-calretinin (1:1,000; Millipore; MAB1568), and anti-PV (1:1,000; Swant; 235). Secondary antibodies were all used at a dilution of 1:500. We used goat anti-rabbit 568, goat anti-rabbit 488, goat anti-chicken 488, donkey anti-rabbit 647, goat anti-rat FITC, and goat anti-guinea pig 568 (Invitrogen). Cultures/sections were routinely counterstained with DAPI (1 mg/ml) at a dilution of 1:1,000.

Imaging and quantification

Twelve bit images at a resolution of 1,376 × 1,032 pixels were acquired on an Olympus BX61 epifluorescence microscope using a 20× (221 × 166 μm) objective lens and an F-View II CCD Camera (Soft Imaging System). Alternatively, 12 bit images at a 1,024 × 1,024 pixel resolution were acquired on an Olympus confocal microscope (FV1000) using 40× objective lens with zoom 1.5× (211.8 × 211.8 μm) or on a Zeiss confocal microscope (LSM 700) with a 63× objective and no zoom (101.52 × 101.52 μm). All images for each experiment were acquired with identical exposure time and detector gain.

For sections stained for SIRPα, the average signal intensities of staining in the pyramidal cell layer and stratum radiatum layers were calculated with the MetaMorph software (Molecular Devices). The average signal intensity in the fimbria of the hippocampus was calculated and subtracted as the background. For images of cultured neurons, the staining intensity of the dendritic shaft was calculated and subtracted as the background. The puncta size, number, and intensity were quantified using either the MetaMorph or ImageJ software.

Electron microscopy

Mice were perfused transcardially with Karnovsky's fixative (Terauchi et al., 2010; Toth et al., 2013). Then, hippocampi were removed, 1 mm cubes from the stratum radiatum layer of the CA3 region were dissected, and then the cubes were processed for electron microscopy. Thin sections (70 nm) were cut and observed with a Philips CM100 electron microscope at 60 kV. Digital images were captured with a Hamamatsu ORCA-HR digital camera system operated with the Advanced Microscopy Techniques software.

Electrophysiology

Whole-cell patch-clamp and cell–attached recordings in cultures

Neurons were bathed in HEPES-buffered saline (HBS) at room temperature, containing the following (in mM): 119 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 30 glucose, and 10 HEPES, pH 7.4. For mIPSCs, this HBS was supplemented with 1 μM TTX, 10 μM CNQX, and 20 μM APV to isolate mIPSCs. For mIPSC recordings, whole-cell internal solution included the following (in mM): 135 CsCl₂, 1 EGTA, 5 MgCl₂, 4 ATP, 1 GTP, and 10 HEPES. For cell-attached, action potential recordings, recording pipettes contained 1 M NaCl and 25 mM HEPES. Recording pipettes had a resistance of 4–6 MΩ. Recordings were made with an Multiclamp 700B amplifier (Molecular Devices) and collected with Clampex 10.7 (Molecular Devices). Both mIPSCs and action potentials were analyzed using Minianalysis 6.0 (Synaptosoft).

Field slice preparation and recording

Mice were decapitated, and the hippocampi were isolated. Then, transverse slices (400 μm) were cut using a tissue chopper (Stoelting) and incubated at 25°C in a humidified chamber for at least 2 h before recording. Slices were then transferred to a recording chamber, maintained at 27–28°C and continuously perfused at a rate of 1.5 ml/min with oxygenated artificial cerebral spinal fluid (aCSF). aCSF contained the following (in mM): 119 NaCl, 2.5 KCl, 1 NaH₂PO₄, 26.3 NaHCO₃, 11 glucose, 1.3 MgSO₄, and 2.5 CaCl₂. Recording electrodes were pulled from borosilicate capillary glass (1.7 mm, o.d.; VWR International), filled with 3 M NaCl, and placed in the stratum radiatum layer of CA1. fEPSPs were stimulated using cluster electrodes (FHC) also placed in the stratum radiatum of CA1. Current was delivered with an ISO-flex stimulus isolation unit (AMPI). Recordings were made with a MultiClamp 700B amplifier, collected and analyzed using Clampfit 10.2 (Molecular Devices). An input/output (I/O) curve was obtained for each slice by increasing the stimulus intensity from 0.02 to 0.25 mA. For paired-pulse experiments, the intensity was set at 0.2 mA, which was the maximum response size. To obtain the paired-pulse ratio (PPR), we delivered two pulses with an interpulse interval from 25 to 200 ms.

Miniature postsynaptic potential and evoked inhibitory synaptic potential slice preparation and recording

Mice were decapitated, and then the brains were quickly removed. The 300 μm coronal slices were cut using a VT1200S vibratome (Leica). Slices were cut in an ice-cold solution (in mM: 206 sucrose, 2.8 KCl, 2 MgSO₄, 1 MgCl₂, 1.25 NaH₂PO₄, 1 CaCl₂, 10 glucose, 26 NaHCO₃, and 0.4 ascorbic acid). Slices were then put into aCSF for 1 h at room temperature. aCSF contains the following (in mM): 127 NaCl, 1.6 KCl, 1.24 KH₂PO₄, 1.3 MgSO₄, 2.4 CaCl₂, 10 glucose, and 26 NaHCO₃. All solutions were continuously bubbled with 95% O₂/5% CO₂. Neurons were visualized using a customized Scientifica/Olympus microscope. Data were obtained with a Multiclamp 700B amplifier, digitized with Digidata 1440A (Molecular Devices), and collected with Clampex 10.7. Whole-cell patch–clamp recordings were conducted with 4–6 MΩ pipette containing (for mEPSCs and eIPSCs) the following (in mM): 135 K-gluconate, 4 KCl, 10 HEPES, 4 Mg-ATP, 0.3 Na₂-GTP, and 10 Na₂-phosphocreatine. pH was adjusted to 7.2–7.3 with KOH. For mIPSCs, pipettes contained the following (in mM): 135 CsCl₂, 1 EGTA, 5 MgCl₂, 4 ATP, 1 GTP, and 10 HEPES, pH 7.2. Cells were held at −65 mV. aCSF was supplemented during recordings with 500 nM TTX and 50 µM picrotoxin (for mEPSC recordings) or 10 μM CNQX and 25 μM APV (for mIPSCs) and warmed to 32°C. mEPSCs and mIPSCs were analyzed using MiniAnalysis (Synaptosoft).

For evoked IPSC recordings, sections were prepared as above except using aCSF containing the following (in mM): 118 NaCl, 2.5 KCl, 1.3 MgCl₂, 1.2 NaH₂PO₄, 2.5 CaCl₂, 10 glucose, and 26 NaHCO₃. To evoke IPSCs, two stimulating electrodes filled with 3 M NaCl were placed across the PY layer in CA1, and pyramidal cells were patched and held at 0 mV. The internal patch solution was the K-gluconate solution described above. aCSF was supplemented with APV and CNQX, but not TTX. Two stimuli from 0.1 to 0.8 mA were delivered with a 50 ms interstimulus interval, and the ratio of the peak amplitude of each stimulus was measured using Clampfit 10.7.

Axial resistance did not differ between genotypes in any of the recording experiments [axial resistances (Ω/cm), Fig. 1E,F, 25.54 ± 2.04 (control), 21.45 ± 1.53 (O.E.); Fig. 1I–L, 16.88 ± 2.72 (control), 15.73 ± 1.44 (KO); Fig. 2E–H, 17.71 ± 1.18 (control), 14.78 ± 1.07 (O.E.); Fig. 2N–Q, 13.04 ± 1.58 (control), 11.85 ± 0.66 (KO); Fig. 4A–D: 13.19 ± 0.91 (control), 14.71 ± 1.73 (KO); Fig. 4E–H, 13.19 ± 0.92 (control), 14.71 ± 1.70 (KO); Fig. 6B–F, 24.46 ± 2.37 (control), 19.31 ± 1.42 (KO); Fig. 6G–J, 18.35 ± 2.26 (control), 19.32 ± 3.05 (KO); Fig. 7B–D, 14.89 ± 0.95 (control), 17.35 ± 1.08 (KO); Fig. 7E–G, 19.03 ± 1.87 (control), 16.65 ± 2.00 (KO); Fig. 7K–M, 18.00 ± 1.45 (control), 15.45 ± 1.31 (KO); Fig. 8, 17.21 ± 2.08 (control), 19.84 ± 2.02 (KO); Fig. 9B–G, 20.04 ± 1.07 (control), 22.81 ± 1.18 (high titer), 18.07 ± 0.98 (low titer); Fig. 9J–O, 12.28 ± 0.64 (KO), 12.73 ± 0.72 (KO + inhibitors), 13.55 ± 1.09 (rescue), 10.88 ± 1.44 (rescue + inhibitors)].

Statistical analysis

All data are reported and graphed as mean ± standard error of the mean. Statistical analysis was conducted using the GraphPad Prism software or Sigma Plot Software and the tests used are reported in the figure legends. Statistical analysis was done with an unpaired t test, one-way ANOVA followed by a Tukey’s test, or a two-way ANOVA followed by a Sidak test as indicated in the figure legend. In our global KO experiment (Fig. 2), a Fisher's least significant difference test was used as a post hoc test. In the case of our cell-attached recordings, the data failed to meet normality standards according to Prism, so a Kruskal–Wallis test followed by a Dunn's test was performed. Significance was set at *p < 0.05 and is indicated in the figure legends. Our sample sizes (n's) are reported in the figure legends. Sample sizes were not predetermined but were similar to those reported in other publications in the field (Chubykin et al., 2007; Lin et al., 2008; Toth et al., 2013; Nagappan-Chettiar et al., 2018; Okur et al., 2024). All steps of the experiments were randomized to minimize the effects of confounding variables. This includes how mice were chosen for injections, order of cell culture treatments, etc. All electrophysiological experiments and analyses were done blind. Imaging was done in similar fashions among conditions: fields from brain sections were chosen randomly from the region of interest, and images of cell cultures were taken randomly from all areas of the culture.

Results

We have previously identified SIRPα as a synaptogenic molecule through biochemical purification (Umemori and Sanes, 2008). We initially focused on the synaptogenic role of SIRPα at excitatory synapses because SIRPα is more strongly localized at excitatory synapses than at inhibitory synapses (Fig. 1A–C; Toth et al., 2013; ∼75% at excitatory synapses vs ∼13% at inhibitory synapses, data from hippocampal cultures). We have shown that overexpression (OE) or deletion of SIRPα in a single hippocampal neuron increased or decreased the development of excitatory synapses, respectively (Toth et al., 2013; Nagappan-Chettiar et al., 2018), indicating that SIRPα is a driver of excitatory synaptogenesis. In this study, we first examined whether manipulation of SIRPα in a single hippocampal neuron influences inhibitory synaptogenesis. We found that SIRPα does not affect inhibitory synaptogenesis in a cell-autonomous manner based upon the following two experiments. First, we sparsely cotransfected plasmids expressing SIRPα and GFP (to label transfected cells; Fig. 1D) into cultured hippocampal neurons at DIV3 and recorded mIPSCs from GFP-positive putative pyramidal neurons at DIV18–24 (Fig. 1E), which is after robust GABAergic synaptogenesis has occurred in our culture system. When overexpressing SIRPα in this manner, we found that neither the frequency nor the amplitude of mIPSCs was changed compared with control cultures in which neurons were transfected with GFP alone (Fig. 1F–H). Thus, OE of SIRPα in a single neuron is not sufficient to drive inhibitory synaptic development. Second, we tested the opposite scenario, where SIRPα was deleted from a single neuron. To do so, we cultured hippocampal neurons from SIRPα^fl/fl (flox/flox) mice (Toth et al., 2013) and sparsely transfected the cultures with either GFP (control) or GFP + Cre (to inactivate SIRPα from individual neurons; Fig. 1I). After transfection at DIV3, we recorded mIPSCs at DIV18–24 from GFP-positive neurons. We observed no change in either mIPSC frequency or amplitude, again suggesting that SIRPα does not affect inhibitory synapses in a direct, cell-autonomous manner (Fig. 1J–L). Note that there is a difference in frequency between the control cells in the OE and KO experiments that is due to a change in the media used in the experiments (see Materials and Methods). The lack of effect when modulating SIRPα in a single cell is distinctly different from what we found with excitatory synapse development, where SIRPα does function cell-autonomously, and indicates that SIRPα itself is a driver of excitatory, but not inhibitory synaptic development.

Figure 1.

SIRPα primarily localizes to excitatory synapses and does not have a cell-autonomous effect on inhibitory synaptogenesis, and yet E/I balance is maintained in global SIRPα KO mice. A–C, Costaining of SIRPα (red) and presynaptic markers (green) in primary hippocampal cultures. SIRPα (red) colocalizes more strongly with excitatory VGLUT1 puncta (green; A) than inhibitory VGAT puncta (green; B). Scale bar, 5 µm. Quantification of the percentage of VGLUT1 or VGAT puncta that colocalize with SIRPα puncta (C). Far more SIRPα puncta colocalize with VGLUT1 than VGAT. D, Example image of a single transfected neuron (green) amidst a sea of nontransfected cells (as visualized with blue DAPI staining). Scale bar, 20 µm. E–H, Effects of single-cell overexpression (OE) of SIRPα + GFP (indigo in E) on mIPSCs in cultured wild-type hippocampal neurons. Cultures transfected only with GFP were used as controls. mIPSCs were recorded from GFP-positive (SIRPα-overexpressing) neurons. Sample traces are shown in F. No change in the mIPSC frequency (G) or amplitude (H) was detected by an unpaired t test, suggesting that OE of SIRPα in a single neuron does not drive inhibitory synaptogenesis. Frequency, p = 0.1498; t = 1.462; df = 51; n = 28 (control); 25 (OE) cells from six independent experiments. Amplitude, p = 0.233; t = 1.207; df = 51; n = 28 (control); 25 (OE) cells from six independent experiments. Scale bars, 10 pA, 100 ms. I–L, Effects of single-cell KO of SIRPα on mIPSCs. Hippocampal neurons were cultured from SIRPα^fl/fl mice, and transfection of Cre + GFP was used to sparsely delete SIRPα (green cell in I). Cultures transfected only with GFP were used as controls. mIPSCs were recorded from GFP-positive neurons. Sample traces are shown in J. No change in the mIPSC frequency (K) or amplitude (L) was detected by an unpaired t test. Frequency, p = 0.341; t = 0.958; df = 90; n = 49 (control); 43 (KO) cells from four independent experiments. Amplitude, p = 0.147; t = 1.461; df = 90; n = 49 (control); 43 (KO) cells from four independent experiments. Scale bars, 10 pA; 100 ms. M–U, Actin-Cre-ER::SIRPα^fl/fl mice were injected with tamoxifen at P14 to create a global KO of SIRPα (SIRPα KO^global), and then Shaffer collateral fEPSPs were measured in CA1 of the hippocampus at ~P29 (M). I/O curves (N–Q) and PPRs (R–U) from CA1 of SIRPα KO^global mice were compared with control mice either in the absence (O, S) or presence (Q, U) of picrotoxin to block inhibitory synapses. Control mice were either Actin-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice, injected with tamoxifen. Example traces are shown in N, P, R, and T. Scale bars, 0.25 mV, 10 ms (N, P); 0.5 mV, 20 ms (R, T). When inhibitory synapses are not blocked (no picrotoxin, O), the I/O curves from SIRPα KO^global mice are nearly identical to the control I/O curves. By a two-way ANOVA, there is not a significant genotype effect (p = 0.7727; F_(1,34) = 0.0848). n = 23 (control), 13 (KO) slices from 3 to 5 mice. When inhibitory synaptic transmission was blocked (picrotoxin; Q), control mice had a significantly higher I/O curve, suggesting stronger excitatory synaptic transmission in control compared with SIRPα KO^global mice. By a two-way ANOVA, there is a significant effect of genotype (*p = 0.0006; F_(1,220) = 12.05). n = 9 (control), 13 (KO) slices from 3 to 5 mice. Similarly, there is no change in PPR of SIRPα KO^global mice compared with control mice in the absence of picrotoxin (S), but SIRPα KO^global mice have significantly higher PPR (showing more facilitation) than control mice in the presence of picrotoxin (U), suggesting impaired presynaptic function at excitatory synapses. No picrotoxin PPR: by a two-way ANOVA, there is not a significant genotype effect (p = 0.4333; F_(1,293) = 0.6156). n = 23 (control), 16 (KO) slices from 3 to 5 mice. Picrotoxin PPR: by a two-way ANOVA, there is a significant effect of genotype (*p = 0.0014; F_(1,183) = 10.46). n = 9 (control), 13 (KO) slices from 3 to 5 mice. Figures P, Q, T, and U were recreated from data previously published (Toth et al., 2013).

Despite these findings, we had a hint that SIRPα inactivation could influence inhibitory synaptogenesis through the analysis of fEPSPs from global SIRPα KO (SIRPα KO^global) mice [Actin-Cre-ER::SIRPα^fl/fl mice, tamoxifen injection at P15; SIRPα is inactivated from all cells during excitatory synapse maturation; inactivation of SIRPα was previously confirmed by immunostaining (Toth et al., 2013)]. When we measured I/O curves of fEPSPs in CA1 of SIRPα KO^global mice at P28–35 (Fig. 1M) and compared them to control mice (Actin-Cre-ER::SIRPα^wt/wt or SIRPα^fl/fl littermate mice, tamoxifen injection at P15), we found no change in the I/O curves (Fig. 1N,O). However, when we added picrotoxin to block inhibition, we revealed a significantly decreased I/O curve in the SIRPα KO^global mice compared with control mice (Fig. 1P,Q; Toth et al., 2013). Similarly, when we measured paired-pulse facilitation (PPF) in the absence of picrotoxin, we did not see a difference between SIRPα KO^global and control mice (Fig. 1R,S). Not until we did the recordings in the absence of inhibition (with picrotoxin) did we clearly reveal an increase in PPF in SIRPα KO^global mice compared with control mice (Fig. 1T,U; Toth et al., 2013). These data suggest that (1) the presence of inhibition masks excitatory impairments in SIRPα KO^global mice, suggesting that global inactivation of SIRPα could also affect inhibitory synaptogenesis, and (2) E/I balance is maintained in the global absence of SIRPα.

Since SIRPα did not affect inhibitory synaptogenesis in a cell-autonomous manner (Fig. 1E–L), we postulated that global manipulations of SIRPα are necessary for SIRPα's potential effects on inhibitory synapses. To confirm this hypothesis, we first examined the effect of manipulating global levels of SIRPα in cultured hippocampal neurons. We tested whether the bath application of sSIRPα (soluble SIRPα = the extracellular portion of SIRPα) to cultured hippocampal neurons could promote GABAergic synaptic development (Fig. 2A–H). To do so, we first added sSIRPα to hippocampal cultures at DIV1, fixed the cultures at DIV11, and then immunostained for a presynaptic marker of inhibitory synapses, VGAT. There were significantly more VGAT puncta in the cultures treated with sSIRPα than control cultures (Fig. 2B,C). There was also a trend toward the increased VGAT puncta size in the sSIRPα-treated cultures (Fig. 2D; p = 0.131). To corroborate these data, we performed similar experiments, treating wild-type cultured hippocampal neurons with sSIRPα at DIV3 and recording mIPSCs at DIV14–18 (Fig. 2E,F). We found a significant increase in the frequency of sSIRPα-treated cultures (Fig. 2G), but no change in mIPSC amplitude (Fig. 2H). All together, these data support the idea that treatment with sSIRPα results in more inhibitory synapses. This contrasts with what we observed as the effect of sparse SIRPα OE (Fig. 1E–H). We then conducted the converse experiment by globally deleting SIRPα in culture (Fig. 2I–Q). To do so, we cultured hippocampal neurons from Actin-Cre-ER::SIRPα^fl/fl mice and added either vehicle control or tamoxifen (to delete SIRPα in all of the cultured cells) at DIV4. We also included cultures from SIRPα^fl/fl mice (without Cre) and added vehicle control or tamoxifen to control for the effects of tamoxifen on synaptic development. We infected cultures with a GFP-expressing virus to visualize the hippocampal neurons. Cultures were fixed and stained for VGAT and GFP at DIV14. In the case of the KO cultures (Tamoxifen+, Cre+), we found a significant decrease in the intensity, density, and size of VGAT puncta compared with at least one of the control conditions (Fig. 2J–M). To confirm that there are physiological consequences to the changes in VGAT puncta, tamoxifen-treated cultures were once again created by culturing hippocampal neurons from Actin-Cre-ER::SIRPα^fl/fl mice and adding either vehicle control or tamoxifen (to delete SIRPα in all of the cultured cells) at DIV4 (Fig. 2N). We then measured inhibitory activity at DIV18–24. There was no effect of tamoxifen in the absence of Cre. However, the creation of network-wide SIRPα KO by the addition of tamoxifen to Cre-positive cultures resulted in a significant decrease in both the frequency and amplitude of mIPSCs (Fig. 2O–Q). This again contrasts with the results from sparse SIRPα KO (Fig. 1I–L). Thus, in vitro, global, but not local, SIRPα manipulation alters the development of inhibitory synapses.

Figure 2.

Global increases or decreases in SIRPα impact inhibitory synaptic development in hippocampal cultures. A–D, Soluble SIRPα (sSIRPα) was added to wild-type cultured hippocampal neurons from DIV1–11 (A), and then cultures were stained for VGAT. Example images of VGAT staining (B). Scale bar, 5 µm. VGAT staining was quantified for density (C) and size (D) of VGAT puncta on hippocampal neurons. The density of VGAT puncta was significantly increased, and the size of VGAT puncta had a trend toward an increase with the addition of sSIRPα. Density, *p = 0.015; t = 2.932; df = 10; size, p = 0.131; t = 1.645; df = 10; by an unpaired t test, n = 6 (control), 6 (sSIRPα) images from three independent experiments. E–H, sSIRPα was added to wild-type cultured hippocampal neurons at DIV3 (E), and then mIPSCs were recorded in cultured neurons between DIV14–18. Example traces from control and sSIRPα-treated cultures (F). Scale bars, 5 pA, 25 ms. Treatment with sSIRPα significantly increased the frequency (G), but not the amplitude of mIPSCs (H). Frequency, *p = 0.022; t = 2.318; df = 158; amplitude, p = 0.459; t = 0.743; df = 158; by unpaired t test, n = 83 (control), 77 (sSIRPα) neurons from three independent cultures. I–M, Effect of deleting SIRPα from all cells in a hippocampal culture dish. Global KO of SIRPα was accomplished by adding tamoxifen to cultures from CAG-Cre-ER::SIRPα^fl/fl mice at DIV4 (I); at the same time, cultures were infected with GFP to allow for visualization of neurons. Simultaneously, a set of cultures from Cre-negative SIRPα^fl/fl mice were also either treated with tamoxifen (Tam) or vehicle control. Cultures were then fixed at DIV14 and stained for GFP and VGAT (to label GABAergic presynaptic terminals). Sample images shown in J. VGAT immunostaining was then quantified for intensity (K), density (L), and size (M). The first two left-most bars in each graph are control cultures from Cre-negative SIRPα^fl/fl cultures [from left (Bar 1) to right (Bar 2): (Tam−, Cre−), (Tam+, Cre−)]. The right two bars on each graph are from CAG-Cre-ER::SIRPα^fl/fl cultures [from left (Bar 3) to right (Bar 4): control (Tam−, Cre+), KO (Tam+, Cre+)]. KO cultures (Tam+, Cre+) had significantly dimmer puncta, fewer puncta, and smaller size. Intensity: one-way ANOVA followed by a Dunnett's multiple-comparison test, *p = 0.013 (Tam−/Cre+ vs KO); *p = 0.004 (Tam−/Cre− vs KO); *p = 0.018 (Tam+/Cre− vs KO); F_(3,93) = 3.853. n = 26 (Tam−, Cre−), 9 (Tam+, Cre−), 33 (Tam+, Cre−), and 29 (Tam+, Cre+) cells from three independent experiments. Density: one-way ANOVA followed by a Dunnett's multiple-comparison test, *p = 0.011 (Tam+/Cre− vs KO); F_(3,94) = 3.243. n = 26 (Tam−, Cre−), 9 (Tam+, Cre−), 33 (Tam+, Cre−), and 30 (Tam+, Cre+) cells from three independent experiments. Size: one-way ANOVA followed by a Dunnett's multiple-comparison test, *p = 0.0004 (Tam−/Cre− vs KO); F_(3,93) = 5.931. n = 26 (Tam−, Cre−), 9 (Tam+, Cre−), 33 (Tam+, Cre−), and 29 (Tam+, Cre+) cells from three independent experiments. N–Q, Effect of deleting SIRPα from all cells in a hippocampal culture dish on mIPSCs. Global KO of SIRPα was accomplished by adding tamoxifen to cultures from Actin-Cre-ER::SIRPα^fl/fl mice at DIV4. mIPSCs were recorded at DIV18–21 (N). Example traces from either a control culture (Actin-Cre-ER::SIRPα^fl/fl without Tamoxifen) or a SIRPα Global KO culture (O). Scale bars, 10 pA, 25 ms. The first two left-most bars in each graph are control cultures from Cre-negative SIRPα^fl/fl cultures [from left (Bar 1) to right (Bar 2): (Tam−, Cre−), (Tam+, Cre−)]. The right two bars on each graph are from Actin-Cre-ER::SIRPα^fl/fl cultures [from left (Bar 3) to right (Bar 4): control (Tam−, Cre+), KO (Tam+, Cre+)]. When tamoxifen (Tam) was delivered to a dish of Cre-positive cultured hippocampal neurons to delete SIRPα in all cells (KO), both the frequency (P) and amplitude (Q) of mIPSCs were decreased. The addition of tamoxifen to Cre-negative cultures had no effect on the mIPSCs, indicating that tamoxifen itself does not impact inhibitory synaptic transmission. The number with arrow represents the number of points above the graph. Frequency: one-way ANOVA followed by a Tukey’s test, *p = 0.0006 (Tam+/Cre− vs KO); *p = 0.007 (Tam−, Cre+ vs KO); F_(3,153) = 3.143. Amplitude: one-way ANOVA followed by an uncorrected Fisher's least significant difference test, *p = 0.043; F_(3,153) = 2.781. n = 24 (Tam−, Cre−), 23 (Tam+, Cre−), 54 (Tam+, Cre−), and 56 (Tam+, Cre+) cells from three independent experiments.

To confirm that the global deletion of SIRPα also impairs inhibitory synaptogenesis in the hippocampus in vivo, we again utilized the Actin-Cre-ER::SIRPα^fl/fl mice. We injected tamoxifen to delete SIRPα at either P0, P15, or P30 and then fixed the brains and immunostained for VGAT 14 d later (i.e., at P14, P29, or P44, respectively). These time periods were chosen because we have previously identified the critical period for SIRPα's effects on excitatory synapses: inactivation of SIRPα at P15–P29, but not at P0–P14 or P30–P44, decreased excitatory synapse formation in the hippocampus (Toth et al., 2013). While inactivating SIRPα at either P0–P14 or P30–P44 had no effect on inhibitory presynaptic development in CA3 of the hippocampus, as assessed by VGAT staining, inactivation of SIRPα at P15–P29 resulted in a significant decrease in VGAT staining (Fig. 3A–L). To further explore the role of SIRPα in inhibitory synaptogenesis, we injected Actin-Cre-ER::SIRPα^fl/fl mice at P15 with tamoxifen to delete SIRPα and then performed electron microscopic analysis of inhibitory (symmetric) synapses at P29. The lack of SIRPα resulted in a decrease in both the clustering of synaptic vesicles to active zones, and the number of these vesicles that were docked at symmetric synapses (Fig. 3M–O), further demonstrating that the global inactivation of SIRPα during the period of excitatory synaptic maturation impairs inhibitory synaptogenesis as well as excitatory synaptogenesis.

Figure 3.

Global deletion of SIRPα during the developmental period corresponding to excitatory synaptic maturation impairs GABAergic synaptic development in vivo. A–D, SIRPα was deleted globally during early synaptic development by delivery of tamoxifen at P0 to Actin-Cre-ER::SIRPα^fl/fl mice (SIRPα KO^P0–14), and sections of the hippocampal CA3 region were stained for VGAT at P14. Control mice were either Actin-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice, injected with tamoxifen. Sample images are shown in A. Scale bar, 25 µm. The intensity of VGAT staining (normalized to control levels; B) and the density (C) and size (D) of VGAT puncta were quantified in both the pyramidal (PY) and stratum radiatum (SR) layers. There was no change in VGAT intensity, density, or size of puncta. Intensity: unpaired t test [n = 11 (control), 11 (KO) sections from 3 to 5 mice; PY, p = 0.254; t₍₂₀₎ = 1.176; SR, p = 0.315; t₍₂₀₎ = 1.030]. Density: unpaired t test [n = 9 (control), 12 (KO) sections from 3 to 5 mice; PY, p = 0.097; t₍₁₉₎ = 1.746; SR, p = 0.460; t₍₁₉₎ = 0.755). Size, unpaired t test (n = 9 (control), 12 (KO) sections from 3 to 5 mice; PY, p = 0.893; t₍₁₉₎ = 0.137; SR, p = 0.292; t₍₁₉₎ = 1.085]. PY, pyramidal cell layer; SR, stratum radiatum; SL, stratum lucidum. Scale bar, 30 µm. E–H, SIRPα is deleted during the maturation phase of excitatory synaptic development by delivery of tamoxifen at P15 to Actin-Cre-ER::SIRPα^fl/fl mice (“SIRPα KO^global mice”). Brains were then collected at P29, and the hippocampi were stained for VGAT. Control mice were either Actin-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice, injected with tamoxifen. Sample images of CA3 are shown in E. The intensity of VGAT staining (normalized to control mice; F) and the density (G) and size (H) of VGAT puncta were quantified in the PY and SR layers. There was a significant decrease in VGAT intensity, density, and puncta size in the SIRPα KO^global mice. Intensity: unpaired t test [n = 21 (control), 21 (KO) sections from 4 to 6 mice) PY, *p = 0.001; t₍₄₀₎ = 3.484; SR, *p < 0.0001; t₍₄₀₎ = 6.136]. Density: unpaired t test [n = 25 (control), 20 (KO) sections from 4 to 6 mice]; PY, p = 0.121; t₍₄₃₎ = 1.580; SR, *p = 0.036; t₍₄₃₎ = 2.164. Size: unpaired t test [n = 25 (control), 20 (KO) sections from 4 to 6 mice]; PY, *p = 0.0005; t₍₄₃₎ = 3.752; SR, *p = 0.025; t₍₄₃₎ = 2.324. I–L, SIRPα is deleted during the maintenance phase of excitatory synaptic development by delivering tamoxifen at P30 to Actin-Cre-ER::SIRPα^fl/fl mice and then measuring VGAT intensity at P44 (SIRPα KO^P30–44). Control mice were either Actin-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice, injected with tamoxifen. Sample images of CA3 are shown in I. The intensity of VGAT staining (normalized to control levels; J) and the density (K) and size (L) of VGAT puncta were quantified in the PY and SR layers. There is no effect on VGAT intensity, density, or size. Intensity: unpaired t test, [n = 10 (control); 16 (KO) sections from 4 to 6 mice; genotype factor]; PY, p = 0.253; t₍₂₄₎ = 1.170; SR, p = 0.168; t₍₂₄₎ = 1.422. Density: unpaired t test [n = 13 (control); 11 (KO) sections from 4 to 6 mice; genotype factor]; PY, p = 0.722; t₍₂₂₎ = 0.360; SR, p = 0.375; t₍₂₂₎ = 0.906. Size: unpaired t test [n= 12 (control); 11 (KO) sections from 4 to 6 mice; genotype factor]; PY, p = 0.904; t₍₂₂₎ = 0.122; SR, p = 0.649; t₍₂₂₎ = 0.462. M–O, EM analysis of symmetric (inhibitory) synapses in CA3 of the hippocampus at P29 after delivering tamoxifen at P15 to control or Actin-Cre-ER::SIRPα^fl/fl mice (SIRPα KO^global mice). Control mice were either Actin-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice, injected with tamoxifen. Sample images are shown in M. Quantification of EM analysis of symmetric (inhibitory) synapses found a significant decrease in both the number of synaptic vesicles (SVs) within 400 nm of the active zone (N) and the number of docked vesicles (O) in CA3 of the hippocampus. Unpaired t test for N: *p = 0.0027; t = 3.292; df = 28; n = 13 (control); 17 (KO) synapses from three mice. Unpaired t test for O: *p = 0.0063; t = 2.953; df = 28; n = 13 (control), 17 (KO) synapses from 3 mice. Scale bar, 100 nm.

While we had previously reported that VGLUT1 puncta were significantly decreased in SIRPα KO^global mice (Toth et al., 2013), that analysis did not include physiological support. Thus, to confirm that there is impaired excitatory synaptic transmission in the SIRPα KO^global mice, we again injected Actin-Cre-ER::SIRPα^fl/fl mice with tamoxifen at P15 to delete SIRPα (SIRPα KO^global mice) and recorded mEPSCs from CA1 pyramidal neurons at P28–P35. We found that in the absence of SIRPα, there was a significant decrease in mEPSC frequency, but not amplitude (Fig. 4A–D). To further confirm that inhibition was similarly functionally altered in these mice, we also recorded mIPSCs from the SIRPα KO^global mice. Consistent with the histological data described above, the frequency and amplitude of mIPSCs were significantly decreased in the SIRPα KO^global mice (Fig. 4E–H). Altogether, these results confirm that global inactivation of SIRPα, both in vitro and in vivo, affects excitatory and inhibitory synapse development.

Figure 4.

Global KO of SIRPα impacts excitatory and inhibitory synaptic transmission. A–D, mEPSCs were recorded from CA1 pyramidal neurons in SIRPα KO^global mice at P28–P35 (the diagram shown in A). Control mice were either Actin-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice, injected with tamoxifen. Sample traces are shown in B. There was a significant decrease in the frequency (C), but not the amplitude (D) of mEPSCs by an unpaired t test. Frequency: *p = 0.0234; t = 2.318; df = 69. Amplitude: p = 0.2796; t = 1.09; df = 69. n = 46 (control), 25 (KO) cells from 3 to 6 mice. Scale bars, 10 pA, 100 ms. E, F, Tamoxifen was injected into Actin-Cre-ER::SIRPα^fl/fl mice at P15 (SIRPα KO^global mice), and mIPSCs were recorded from CA1 pyramidal neurons at P28–P35 (the diagram shown in E). Control mice were either Actin-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice, injected with tamoxifen. Sample traces are shown in F. There was a significant decrease in both the frequency (G) and amplitude (H) of mIPSCs by an unpaired t test. The number with the arrow represents the number of points above the graph. Frequency: *p = 0.0292; t = 2.271; df = 36. Amplitude, *p = 0.0035; t = 3.118; df = 36. n = 18 (control), 20 (KO) cells from 6 mice. Scale bars, 10 pA, 100 ms.

To this point, we have focused primarily on frequency and amplitude data from our mIPSC recordings. To deepen our understanding of the synaptic changes triggered by manipulation of SIRPα expression, we analyzed the kinetics of mIPSCs reported above. Kinetics were analyzed for synaptic charge transfer, the 10–90% rise time, and the first decay time constant (a two exponential curve was used to fit the traces). Previous reports showed that a faster rise and shorter decay are associated with synapses from older mice (Moss et al., 1992; Draguhn and Heinemann, 1996; Hollrigel and Soltesz, 1997; Cohen et al., 2000). In the case of sSIRPα-treated cultures (related to Fig. 2E–H), cultures treated with sSIRPα had a significantly longer decay time constant compared with control cultures, although charge transfer and rise time were unchanged (Fig. 5A–D). In the case of global KO of SIRPα in hippocampal cultures (related to Fig. 2N–Q), a significant decrease in charge transfer and rise time was seen between control and KO cultures, although no change in the decay time constant was evident (Fig. 5E–H). In SIRPα KO^global mice (related to Fig. 4E–H), similar to the global KO in culture above, there was a significant decrease in rise time and charge transfer, but no change in decay time constant when SIRPα KO^global mice were compared with control mice (Fig. 5I–L). These data suggest that in the global absence of SIRPα, mIPSC kinetics show characteristics of older animals. In contrast, with sSIRPα application, the mIPSC kinetics are reminiscent of young animals.

Figure 5.

Inhibitory responses have altered kinetics and PPRs when SIRPα expression is globally manipulated. A–D, The kinetics of mIPSCs from hippocampal cultures treated with either vehicle or with sSIRPα (A) were analyzed for synaptic charge transfer (B), 10–90% rise time (C), and the first decay time constant (D). Treatment with sSIRPα results in increased decay time constant. The number with arrow represents the number of points above the graph. Charge transfer: p = 0.605; t = 0.519; df = 159 by unpaired t test; n = 83 (control); 78 (sSIRPα) neurons from three independent cultures; rise time, p = 0.389; t = 0.864; df = 159; n = 83 (control); 78 (sSIRPα) neurons from three independent cultures; decay, *p = 0.020; t = 2.354; df = 151; n = 78 (control); 75 (sSIRPα) neurons from three independent cultures. E–H, Kinetics of mIPSCs from global KO cultures (E). Control cultures were from Actin-Cre-ER::SIRPα^fl/fl cultures without tamoxifen, and global KO cultures were from Actin-Cre-ER::SIRPα^fl/fl cultures with tamoxifen. mIPSC charge transfer was decreased in SIRPα KO cultures (F). The 10–90% rise time was also decreased in SIRPα KO cultures (G). The decay time constant was unaltered (H). The number with the arrow represents the number of points above the graph. Significance determined by an unpaired t test. Charge transfer, *p = 0.015; t = 2.46; df = 108; n = 54 (control); 56 (KO) from three independent experiments; rise time, *p = 0.029; t = 2.22; df = 108; n = 54 (control); 56 (KO) from three independent experiments; decay, p = 0.944; t = 0.071; df = 91; n = 52 (control); 41 (KO) from three independent experiments. I–L, Kinetics of mIPSCs from global KO mice (I). Control mice were either Actin-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice, injected with tamoxifen. mIPSC synaptic charge transfer was decreased in SIRPα KO^global mice (J). The 10–90% rise time was also decreased in SIRPα KO^global mice (K). The decay time constant was unaltered (L). The number with arrow represents the number of points above the graph. Significance determined by an unpaired t test. Charge transfer, *p = 0.019; t = 2.42; df = 64; n = 36 (control); 30 (KO) from 4 to 6 mice; rise time, *p = 0.046; t = 2.03; df = 64; n = 36 (control); 30 (KO) from 4 to 6 mice; decay, p = 0.124; t = 1.56; df = 64; n = 36 (control); 30 (KO) from 4 to 6 mice. M–O, IPSCs were evoked with a 50 ms interstimulus interval in global SIRPα KO mice. Control mice were either CAG-Cre-ER::SIRPα^wt/wt mice or SIRPα^fl/fl mice, injected with tamoxifen. The diagram shown in M and sample traces in N. Scale bars, 5 pA (vertical), 50 ms (horizontal). There was a significant decrease in the PPR (O) by unpaired t test. PPR: *p = 0.011; t = 3.29; df = 8; n = 6 (control); 4 (KO) from three mice per genotype.

To further investigate the maturity of GABAergic synapses, we examined the PPR in SIRPα KO^global mice. IPSCs were evoked using electrical stimulation delivered with a paired-pulse interval of 50 ms (Fig. 5M). We found that the PPR in the SIRPα KO^global mice was significantly decreased compared with control mice (Fig. 5N,O). Previous reports showed that PPR facilitates in younger animals, while it tends to depress in adult mice. Therefore, the PPR data suggest that in the global absence of SIRPα, it shows characteristics of older animals, which is consistent with the mIPSC kinetics data.

Having shown that global manipulations of SIRPα expression affect inhibitory synaptic development, we next asked how the changes in inhibition are occurring. We first tested if they are the result of excitatory changes onto inhibitory interneurons that form inhibitory synapses onto pyramidal neurons. We postulated that if the development of excitatory synapses on inhibitory interneurons is regulated by SIRPα, like those on pyramidal neurons (Fig. 4A–D; Toth et al., 2013), then, when SIRPα is deleted in interneurons, these interneurons would have a decrease in excitatory synapses onto them (Fig. 6A). This theoretical decrease in excitatory synapses could then lead to a decrease in inhibitory output from the inhibitory interneurons back onto the pyramidal neurons (Fig. 6A). Indeed, when we crossed SIRPα^fl/fl mice with Dlx5/6-Cre mice to delete SIRPα specifically within hippocampal interneurons (SIRPα KO^interneuron) and then patched interneurons around the pyramidal cell layer of CA1 at P28–P35 (Fig. 6B,C), we found a significant decrease in mEPSC frequency (Fig. 6D,E). mEPSC amplitude remained unchanged (Fig. 6D,F). This is exactly what we saw in excitatory pyramidal neurons lacking SIRPα (Fig. 4A–D). Thus, deletion of SIRPα results in decreased excitatory synapses onto the neuron lacking SIRPα, whether that neuron is an excitatory pyramidal neuron or an inhibitory interneuron. Given the decreased excitatory drive onto the interneurons lacking SIRPα, we next investigated the second part of our hypothesis that this decreased excitatory drive onto the interneurons would subsequently lead to a decreased inhibitory output from those neurons back onto pyramidal neurons (Fig. 6A, right). To measure inhibitory output, we used the same SIRPα KO^interneuron mice, but this time patched pyramidal neurons in CA1 and recorded mIPSCs (Fig. 6G). We found that neither mIPSC frequency nor amplitude was changed in SIRPα KO^interneuron mice (Fig. 6H–J), so even though the interneuron was receiving less excitatory input (Fig. 6B–F), the inhibitory output remained unchanged. Therefore, the second part of our hypothesis was incorrect, and instead, our results indicate that interneuron expression of SIRPα is not critical for the development of inhibitory synapses onto pyramidal neurons.

Figure 6.

Inactivation of SIRPα in interneurons impairs excitatory synaptic input onto them, but not subsequent inhibitory synaptic output onto pyramidal neurons. A, Proposed hypothesis about how SIRPα could be regulating inhibitory synaptogenesis. Left panel (wild-type): in the mouse, both excitatory pyramidal neurons (PN) and inhibitory interneurons (IN) express SIRPα, and the SIRPα-positive interneurons make connections (shown in red) onto the pyramidal neurons. The SIRPα-positive interneurons are also receiving excitatory input (shown in turquoise). Right panel (SIRPα Interneuron KO): we hypothesize that if SIRPα is deleted from interneurons, they will have decreased excitatory input onto them (which is what happens with excitatory synapses onto pyramidal neurons). This is depicted on the left in the SIRPα interneuron KO panel (fewer turquoise inputs than in wild-type panel). This decreased excitation onto interneurons would then result in a decreased inhibitory output from these interneurons onto pyramidal neurons, depicted on the right (red connections). B–F, Examining if SIRPα affects excitatory synapse development onto inhibitory interneurons. Diagram depicting as follows: SIRPα was deleted from interneurons by crossing SIRPα^fl/fl mice with Dlx5/6 Cre mice (SIRPα KO^interneuron), and then mEPSCs were recorded from inhibitory interneurons around the pyramidal cell layer of CA1 at P28–P35 (B). The area we targeted for recording is shown in C; interneurons were targeted from the PY layer or within ∼50 µm of the PY layer. Scale bar, 10 µm. Sample traces are shown in D. Scale bars, 10 pA, 100 ms. The frequency (E), but not the amplitude (F), of mEPSCs was decreased when SIRPα was deleted from interneurons by an unpaired t test. The number with the arrow represents the number of points above the graph. Frequency: *p = 0.034; t = 2.151; df = 94. Amplitude: p = 0.2231; t = 1.226; df = 94. n = 54 (control), 42 (KO) cells from 6 to 7 mice. G–J, Testing the second part of our interneuron hypothesis by measuring mIPSCs from pyramidal neurons. The diagram depicting as follows: mIPSC recordings in CA1 pyramidal neurons when SIRPα was deleted from interneurons (SIRPα KO^interneuron) at P28–P35 (G). Sample traces are shown in H. Scale bars, 10 pA, 100 ms. Neither the frequency (I) nor amplitude (J) was changed based on an unpaired t test, suggesting that expression of SIRPα in inhibitory interneurons is not necessary for inhibitory synaptic development. Frequency: p = 0.8862; t = 0.1435; df = 82. Amplitude: p = 0.8092; t = 0.2423; df = 82. n = 50 (control), 34 (KO) cells from 4 to 6 mice. PN, pyramidal neuron; IN, interneuron; MG, microglia; AST, astrocyte.

In SIRPα KO^global mice, SIRPα is inactivated in all cells, including both neurons and glia. Since SIRPα is also expressed by glia, we next asked whether glia are the source of the SIRPα regulating inhibitory synaptogenesis. Both microglia and astrocytes express high levels of SIRPα (Zhang et al., 2014) and can regulate synaptic development (Ullian et al., 2001; Christopherson et al., 2005; Schafer et al., 2012) and so are well situated to regulate inhibitory synaptogenesis in the hippocampus. We first focused on microglial SIRPα (Fig. 7A). Microglial SIRPα was deleted by crossing SIRPα^fl/fl mice with mice expressing Cx3cr1-Cre-ER and injecting with tamoxifen at P14 (SIRPα KO^microglia mice). We then recorded mIPSCs from pyramidal neurons at P28–P35. There was no difference between the frequency or amplitude of mIPSCs of control and SIRPα KO^microglia mice (Fig. 7B–D). Previous study suggests that microglial SIRPα regulates excitatory synaptogenesis, reporting a decrease in excitatory synaptic transmission when SIRPα is deleted at P0 from microglia (Ding et al., 2021). Therefore, we also recorded mEPSCs in our SIRPα KO^microglia mice, which were injected with tamoxifen at P14. No change between mEPSC frequency and amplitude was detected in SIRPα KO^microglia mice (Fig. 7E–G). We confirmed KO of SIRPα by immunostaining for SIRPα and Iba1 (a microglia marker). Clear SIRPα staining is seen in both control and SIRPα KO^microglia mice (Fig. 7H), but significantly less SIRPα staining is seen within Iba1-positive microglia (Fig. 7H,I), confirming the microglial deletion of SIRPα. Thus, our results are different from those previously reported; the reason for this discrepancy is likely the timing of when SIRPα was deleted. In their paper, Ding et al. deliver tamoxifen at P0, and we deliver tamoxifen at P14. Ding et al. found that expression of SIRPα in microglia peaks during the first postnatal week and then declines. Taken together with our data, this may suggest that microglial SIRPα is important for synaptic development during the first postnatal week, but not at later ages.

Figure 7.

Deletion of SIRPα from glia does not influence inhibitory synaptic development. A–D, Examining the importance of microglial SIRPα in inhibitory synaptic development. The diagram depicting as follows: SIRPα^fl/fl mice were crossed with Cx3cr1-Cre-ER mice, and tamoxifen was delivered at P14 to delete SIRPα from microglia (SIRPα KO^microglia); then mIPSCs were recorded from CA1 pyramidal neurons at P28–P35 (A). Sample traces are shown in B. Scale bars, 10 pA, 100 ms. Neither the frequency (C) nor the amplitude (D) of mIPSCs recorded from CA1 pyramidal neurons was affected by the deletion of microglial SIRPα based on an unpaired t test. Numbers with arrows represent the number of data points above the graph. Frequency: p = 0.779; t = 0.280; df = 199. Amplitude: p = 0.627; t = 0.486; df = 199. n = 110 (control), 91 (KO) cells from 6 to 7 mice. E–G, Examining the importance of microglial SIRPα in excitatory synaptic development. mEPSCs were recorded from CA1 pyramidal neurons in SIRPα KO^microglia mice at P28–P35. Sample traces are shown in E. Scale bars, 10 pA, 100 ms. Neither the frequency (F) nor the amplitude (G) of mIPSCs recorded from CA1 pyramidal neurons was affected by the deletion of microglial SIRPα based on an unpaired t test. Numbers with arrows represent the number of data points above the graph. Frequency: p = 0.824; t = 0.224; df = 43. Amplitude: p = 0.230; t = 1.217; df = 43. n = 20 (control), 25 (KO) cells from three mice. H, I, Confirmation of deletion of microglial SIRPα in SIRPα KO^microglia mice. Sections were stained with Iba1 (microglial marker) and SIRPα (H), and the amount of SIRPα in the Iba1 cell body was quantified (I). There was significantly less SIRPα in SIRPα KO^microglia mice by unpaired t test. *p = 0.0001; t = 4.391; df = 29; n = 16 (control), 15 (KO) cells. J–M, Examining the importance of astrocytic SIRPα. The diagram showing as follows: SIRPα^fl/fl mice were crossed with Aldh1l1-Cre mice (SIRPα KO^astrocyte); then mIPSCs were recorded from CA1 pyramidal neurons at P28–P35 (J). Sample traces are shown in K. Scale bars, 10 pA, 100 ms. Neither the frequency (L) nor the amplitude (M) of mIPSCs recorded from CA1 pyramidal neurons was affected by the deletion of astrocytic SIRPα based on an unpaired t test. The number with the arrow represents the number of points above the graph. Frequency: p = 0.8438; t = 0.1973; df = 157. Amplitude: p = 0.8092; t = 0.2423; df = 157. n = 113 (control), 46 (KO) from 4 to 8 mice.

Since astrocytes also express SIRPα, we next examined the role of astrocytic SIRPα by crossing SIRPα^fl/fl mice with Aldh1l1-Cre mice to delete SIRPα in astrocytes (SIRPα KO^astrocyte) and recording mIPSCs from CA1 pyramidal neurons (Fig. 7J). Similar to SIRPα KO^microglia mice, neither frequency nor amplitude in SIRPα KO^astrocyte mice was altered compared with control littermates (Fig. 7K–M). Taken together, these data suggest that glial SIRPα is not an important regulator of inhibitory synaptic development in the hippocampus.

Having eliminated interneurons and glia as the source of the SIRPα regulating inhibitory synaptogenesis, we analyzed the role of SIRPα in the pyramidal neurons themselves. We crossed SIRPα^fl/fl mice with Vglut1-Cre mice to delete SIRPα in all excitatory neurons within the hippocampus (SIRPα KO^excitatory). Upon recording mIPSCs from CA1 pyramidal neurons at P28–P35 (Fig. 8A), we found a significant decrease in both the mIPSC frequency and amplitude in the SIRPα KO^excitatory mice (Fig. 8B–D), replicating the results from the global KO of SIRPα (SIRPα KO^global; Fig. 4E–H). There is also a trend toward a decreased charge transfer, rise time, and decay time in the SIRPα KO^excitatory mice (Fig. 8E–G). This indicates that pyramidal neurons are the source of the SIRPα regulating inhibitory synaptogenesis.

Figure 8.

Inhibitory synapse development is impaired when SIRPα is deleted from pyramidal neurons. A–G, Probing the role of excitatory, pyramidal SIRPα in inhibitory synaptic development. The diagram depicting as follows: SIRPα was deleted from pyramidal neurons by crossing SIRPα^fl/fl mice with Vglut1-Cre mice (SIRPα KO^excitatory), and mIPSCs were recorded from CA1 pyramidal neurons at P28–P35 (A). Sample traces are shown in B. Scale bars, 10 pA, 50 ms. Both the frequency (C) and amplitude (D) of mIPSCs were decreased upon deletion of SIRPα from excitatory pyramidal neurons by an unpaired t test. While not significant, there was also a trend toward a decrease in synaptic charge transfer (E), 10–90% rise time (F), and primary decay time constant (G) in SIRPα^excitatory mice compared with control mice. The number with the arrow represents points above the graph. Frequency: *p = 0.0429; t = 2.045; df = 127. n = 79 (control), 50 (KO) cells from 5 to 7 mice. Amplitude: *p = 0.0253; t = 2.263; df = 127. n = 79 (control), 50 (KO) cells from 5 to 7 mice. Charge transfer: p = 0.137; t = 1.478; df = 127. n = 79 (control), 50 (KO) cells from 5 to 7 mice. Rise time, p = 0.0706; t = 1.823; df = 128. n = 80 (control), 50 (KO) cells from 5 to 7 mice. Decay: p = 0.0583; t = 1.975; df = 49. n = 31 (control), 20 (KO) cells from 5 to 7 mice.

Because pyramidal SIRPα clearly regulates inhibitory synaptogenesis, but not in a cell-autonomous manner (Figs. 1, 2), we tested the idea that inhibitory synaptogenesis is being regulated by the level of excitation within the network. We addressed this hypothesis by changing the percentage of pyramidal neurons in which SIRPα is inactivated. For this, we returned to cultured hippocampal neurons from SIRPα^fl/fl mice and used AAV-Cre driven by the CamKII promoter to delete SIRPα from pyramidal neurons. We used both a relatively high (5 × 10¹² gc/ml) and a relatively low (6.25 × 10¹¹ gc/ml) titer of virus (Fig. 9A). In the case of the high titer, most cells were infected (71 ± 25.1%). In the case of a lower titer virus, only 24 ± 16.4% of the cells were infected. When we used a high titer of virus, we replicated our findings from the SIRPα KO^excitatory mice, with decreases in both the frequency and amplitude of mIPSCs (Fig. 9B–D). There is also a significant decrease in charge transfer between control and high titer conditions, a trend toward decreased rise time, and no change in the decay time in the high titer conditions (Fig. 9E–G). For cultures infected at a low titer, mIPSC frequency, amplitude, and kinetics recorded from SIRPα KO neurons remained unaffected as compared with controls (Fig. 9B–G). These data demonstrate that SIRPα must be deleted from most pyramidal neurons to affect inhibitory synaptogenesis, suggesting that developing inhibitory synapses are sensitive to network levels of excitatory activity.

Figure 9.

Inhibitory synapse development is homeostatically regulated by circuit-level excitatory activity in pyramidal neurons. A–G, Using AAV expression of Cre to regulate the number of neurons lacking SIRPα. Cultured hippocampal neurons from SIRPα^fl/fl mice were infected with AAV expressing either GFP (control), a high titer of Cre (∼71% of pyramidal neurons missing SIRPα), or a low titer of Cre (∼24% of pyramidal neurons missing SIRPα). Infection occurred at DIV4. Cre was driven by the CamKII promoter (A). mIPSCs were measured in these cultures at DIV18–24 (B–G). Sample traces are shown in B. Scale bars, 10 pA, 50 ms. In the high titer case, there was a significant decrease in mIPSC frequency (C), decrease in amplitude (D), a significant decrease in charge transfer (E), trend toward a decrease in 10–90% rise time (F), and no change in the decay time constant (G) by a one-way ANOVA followed by a Tukey’s test. Low titer virus did not affect either any of these measures of mIPSCs. The number with the arrow represents the number of points above the graph. Frequency: *p = 0.0362; F_(2,231) = 3.367. Amplitude: *p < 0.0001; F_(2,231) = 11.35. Charge transfer: p = 0.013; F_(2,231) = 4.424. Rise time: p = 0.107; F_(2,231) = 2.253. Decay: p = 0.051; F_(2,231) = 3.027. n = 83 (control), 98 (high titer), and 53 (low titer) cells from eight independent experiments. H, I, Alteration of the cell firing rate in hippocampal cultures lacking SIRPα in most cells by cell-attached recordings from control cultures, high titer cultures, and low titer cultures (A). Sample traces are shown in H. Scale bars, 100 pA, 400 ms. There was a significant decrease in the frequency of action potentials in the high titer condition (I). *p = 0.023 by a Kruskal–Wallis ANOVA (H = 7.503; df = 2) followed by Dunn's test. n = 46 (control), 52 (high), and 68 (low) cells from four independent experiments. The number with the arrow represents the number of points above the graph. J–O, Manipulation of both SIRPα expression and activity levels in cultured hippocampal neurons. Cultured hippocampal neurons from SIRPα^fl/fl mice were infected with AAV expressing Cre (KO, same as the high titer KO of SIRPα above) or both Cre and the extracellular portion of SIRPα (eSIRPα; rescue). Some cultures were treated for 48 h with inhibitors (CNQX, APV, and picrotoxin) to block all synaptic activity. mIPSCs (only in the presence of CNQX and APV) were then recorded at DIV15–18 (J). Sample traces are shown in K. Scale bars, 10 pA, 50 ms. There is an increase in mIPSC frequency when eSIRPα was added to cultures in the absence of activity inhibitors (L, *p = 0.007 by an unpaired t test); this increase was abolished by activity blockade (M; it rather showed a decrease). There is also an increase in amplitude in cultures infected with eSIRPα (N) that is not blocked by inhibitor treatment (O). The number with the arrow represents the number of points above the graph. Frequency: unpaired t test for control treatment, *p = 0.007; t = 2.727; df = 116; unpaired t test for inhibitor treatment, *p = 0.017; t = 2.346; df = 104. Amplitude: unpaired t test for control treatment, *p = 0.027; t = 2.244; df = 116; unpaired t test for inhibitor treatment, *p = 0.008; t = 2.698; df = 104. n = 58 (KO), 59 (KO + inhibitors), 60 (rescue), 47 (rescue + inhibitors) cells from seven independent experiments.

To directly measure the amount of excitatory activity at the network level, we performed cell-attached recordings from cultured hippocampal neurons with the same viral infections as the previous experiment, using action potential frequency as a proxy for the overall level of excitation within the culture network. We see that in high titer-infected cultures, but not low titer-infected cultures, the frequency of action potentials is decreased compared with control cultures (Fig. 9H,I). These data further support the notion that network levels of excitatory activity, but not the number of excitatory synapses onto an individual neuron, drive the development of inhibitory synapses.

To further test the idea that inhibitory synaptogenesis is being regulated by the global levels of activity (i.e., the excitatory activity within the network), we manipulated both SIRPα expression and network activity (Fig. 9J). We first deleted SIRPα from hippocampal cultures at DIV3 by infecting cultures with Cre (equivalent to the high titer condition above). Then, to one set of coverslips, we tried to rescue SIRPα deletion by infecting the cultures with an AAV expressing the extracellular domain of SIRPα (eSIRPα) at DIV8. We then blocked synaptic activity in a subset of the cultures at DIV12 with an inhibitor cocktail (10 µM CNQX, 25 µM APV, and 50 µM picrotoxin) and recorded mIPSCs from pyramidal neurons at DIV15–18 (Fig. 9K–O). Expression of eSIRPα increased mIPSC frequency and amplitude compared with KO cultures (Fig. 9L,N). However, the increase in frequency was not seen in conditions where synaptic transmission was blocked (Fig. 9M,O). This inability of eSIRPα to rescue inhibitory synaptic transmission is in contrast to its role at excitatory synapses (Toth et al., 2013) and supports the idea that it is the level of network activity that plays a critical role in inhibitory development, and a role of SIRPα is to set the level of excitation.

Discussion

Here we explored how E/I balance evolves during synapse development, by examining the role of the excitatory synaptogenic molecule SIRPα in the development of inhibitory synapses within the murine hippocampus. We found the following: (1) single-cell manipulation of SIRPα did not affect inhibitory synapses, (2) global manipulation of SIRPα regulated the development of inhibitory synapses, (3) E/I balance was maintained in the global absence of SIRPα, (4) SIRPα from pyramidal neurons, but not interneurons or glia, was the source of SIRPα that was regulating inhibitory synaptogenesis, and (5) this regulation of inhibitory synaptic development was through the regulation of network levels of excitatory activity rather than through the direct action of SIRPα in individual cells. Altogether, we propose that inhibitory synaptic development is sensitive to the amount of excitatory drive within the hippocampal network and not the single-cell–level excitatory input. The sensitivity to circuit-level excitatory activity allows the network to develop appropriate E/I balance in the hippocampal network.

While E/I balance has long been postulated to be critical in a healthy functioning brain, the concept has been evolving from E/I balance as a single entity to a flexible framework that varies across developmental time, plasticity state, and brain region (He and Cline, 2019; Papatheodoropoulos, 2025). Furthermore, modeling studies suggest that alterations to distinct parts of an interconnected E/I network are likely to have different and unequal outcomes (O’Donnell et al., 2017). One potential way to achieve E/I balance is through homeostatic plasticity mechanisms. Like E/I balance, our understanding of these mechanisms is also evolving. A recent study suggests that in response to activity deprivation, multiple forms of homeostatic compensation are seen, including both network level and cell intrinsic changes (Lakhani et al., 2025). Our data suggest that SIRPα may be one way in which homeostatic changes and inhibitory synaptic plasticity are triggered, in that the presence of SIRPα drives the development of excitatory synapses, which leads to an increase in network activity, and subsequently this increased network activity results in an increase in inhibitory synapses.

In addition to the compensation of decreased excitation within networks lacking SIRPα, a different type of homeostatic compensation was also seen with global activity manipulation. During development, previous publications showed that PPR and rise/decay time constants of mIPSCs decrease from postnatal week 1 to postnatal week 3 in rodents, while the frequency of mIPSCs increases during that period (Hollrigel and Soltesz, 1997; Cohen et al., 2000; Okada et al., 2000). In CA1, the amplitude of mIPSCs is shown to decrease during development (Cohen et al., 2000). Therefore, our data suggest that in the global absence of SIRPα, mIPSC frequency showed a characteristic of young animals, while PPR and mIPSC kinetics/amplitude showed characteristics of older animals. In contrast, sSIRPα application resulted in mIPSC frequency reminiscent of older animals, while the mIPSC kinetics are reminiscent of young animals. These results suggest that in SIRPα KO neurons, there are fewer inhibitory synapses (as suggested from decreased mIPSC frequency and VGAT puncta density), while the synapses remaining show more mature phenotypes (as suggested from decreased PPR and mIPSC kinetics). In neurons with sSIRPα applied, the opposite is true. The pairing of decreased synaptic number with more mature synaptic transmission or increased synaptic number with more immature synapses may represent compensatory or homeostatic mechanisms within the developing inhibitory network. Additionally, from P14 to P28 is the time of excitatory synapse refinement in CA1 (Yasuda et al., 2011), which may include the maintenance and strengthening of strong synapses and the elimination of weak synapses. Similar mechanisms may be present for inhibitory synapses: when there are fewer inhibitory synapses at the time of synapse refinement, those remaining synapses may be strengthened.

What remains an intriguing question for future research is how the level of global activity is being read out at the cellular level. We showed here that decreased excitation onto inhibitory interneurons is not sufficient to alter inhibitory drive (Fig. 6), so the calculation of homeostatic compensation does not appear to be occurring within the interneurons themselves. One possibility for why no changes in output were seen in the interneurons lacking SIRPα is that other alterations were also happening in the interneurons. For example, interneurons with decreased excitatory drive could also adjust their output not at the synaptic level but by altering their intrinsic excitability or firing rate. Network-level changes in excitatory activity may be necessary to trigger the expression of activity-dependent transcription factors, such as Npas4, in pyramidal neurons that promote the development of inhibitory synapses (Lin et al., 2008). The tuberous sclerosis complex–mTOR signaling pathway and downstream transcriptional program also appears to be a critical regulator of inhibitory input onto excitatory neurons (Bateup et al., 2013). Understanding how the interplay between excitatory and inhibitory components of circuit occurs mechanistically remains a critical question for future research.

During development, timing may be another critical factor for regulating the E/I balance. While homeostatic forces are likely involved in inhibitory synaptogenesis (Hengen et al., 2013), as shown here, a change in excitation does not always result in a change in inhibition. For example, there are many molecules, like FGF22, NGL1, and Slitrk1/5, that can promote excitatory synaptogenesis, seemingly without affecting the development of inhibitory synapses (Graf et al., 2004; Chubykin et al., 2007; Terauchi et al., 2010; Kang et al., 2016; Li et al., 2017). It is of note that these studies analyzed synapse development at an earlier stage, such as P0–P14. Thus, it is possible that there could be changes to inhibition later during development. We have shown here that SIRPα only regulates inhibitory synaptogenesis during a developmental time window corresponding to the period of excitatory synaptic maturation in the developing hippocampus. Indeed, other studies also demonstrated that activity manipulation must be done during a critical period to influence inhibitory synaptogenesis (Chattopadhyaya et al., 2004; Huang et al., 2004).

Given the strong hypothesis that an imbalance in excitation and inhibition could underlie or contribute to neuropsychiatric disorders (Sohal and Rubenstein, 2019), many studies examined the factors that affect this balance. Often, these studies have focused on the regulation of interneurons themselves. Indeed, alterations in the number of interneurons result in an imbalanced network (Sohal and Rubenstein, 2019). The number of interneurons seems to be an important driver of network maturation, and this maturation contributes to setting the adult level of E/I balance (Xing et al., 2021). The maturation of PV interneurons and the perineuronal nets surrounding them seem to be of particular importance for E/I balance in the mature brain as perturbations to PV neurons result in network and behavioral impairments (Takesian and Hensch, 2013; Selimbeyoglu et al., 2017). In the adult somatosensory cortex, increased neuronal network activity triggers the release of BMP2 from pyramidal neurons to adjust the excitatory innervation onto and excitability of PV interneurons, which serves to maintain the E/I balance (Okur et al., 2024). Interestingly, this BMP2-dependent effect appears to be only observed in adult and not in juvenile mice, suggesting that different mechanisms exist between developing and mature brains to control E/I balance. Indeed, we found here that during development, altering the excitation onto interneurons was not sufficient to alter their output onto pyramidal neurons (Fig. 5). It is possible that during development, changes in network activity regulate the factors from pyramidal neurons that directly adjust the innervation of inhibitory inputs onto pyramidal neurons to control the E/I balance. During development, the hippocampal circuit dynamics, short-term plasticity, and neuronal firing patterns change, and as the circuit matures, the triggering of homeostatic mechanisms appears to be altered (Huupponen et al., 2007; Jia et al., 2022; Weir et al., 2023). Understanding the dynamic relationships between the timing, molecular signaling, and homeostatic modulation of inhibitory synapse development is particularly important given the prevalence of E/I imbalance in many neurodevelopmental disorders (Duman et al., 2019; Lopatina et al., 2019; Liu et al., 2021; Nomura, 2021).

Footnotes

We thank Anna B. Toth and Lily Y. Zhang for their help with histological analysis and for their technical contributions; Emily Durlacher, Lisa Kim, Chloe DiScipio, and Elizabeth Strang for the mouse colony management; Sivapratha Nagappan-Chettiar for the critical reading of the manuscript; and the Boston Children's Hospital Viral Core. This work was supported by the NIH/NIMH Grant MH111647 and the NIH/NINDS Grant NS092578 (to H.U.). The diagrams in the figures were created using Biorender.com.
The authors declare no competing financial interests.
Correspondence should be addressed to Hisashi Umemori at hisashi.umemori{at}childrens.harvard.edu.

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Network Activity Shapes Inhibitory Synaptic Development in the Mouse Hippocampus

Abstract

Significance Statement

Introduction

Materials and Methods

Mice

Primary hippocampal cultures and treatment

Immunohistochemistry

Imaging and quantification

Electron microscopy

Electrophysiology

Whole-cell patch-clamp and cell–attached recordings in cultures

Field slice preparation and recording

Miniature postsynaptic potential and evoked inhibitory synaptic potential slice preparation and recording

Statistical analysis

Results

Discussion

Footnotes

References

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Main menu

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Search

Network Activity Shapes Inhibitory Synaptic Development in the Mouse Hippocampus

Abstract

Significance Statement

Introduction

Materials and Methods

Mice

Primary hippocampal cultures and treatment

Immunohistochemistry

Imaging and quantification

Electron microscopy

Electrophysiology

Whole-cell patch-clamp and cell–attached recordings in cultures

Field slice preparation and recording

Miniature postsynaptic potential and evoked inhibitory synaptic potential slice preparation and recording

Statistical analysis

Results

Discussion

Footnotes

References

In this issue

Citation Manager Formats

Jump to section

Keywords

Responses to this article

Jump to comment:

Related Articles

Cited By...

More in this TOC Section

Research Articles

Development/Plasticity/Repair