β-Neurexins Control Neural Circuits by Regulating Synaptic Endocannabinoid Signaling

Abstract

α- and β-neurexins are presynaptic cell-adhesion molecules implicated in autism and schizophrenia. We find that, although β-neurexins are expressed at much lower levels than α-neurexins, conditional knockout of β-neurexins with continued expression of α-neurexins dramatically decreased neurotransmitter release at excitatory synapses in cultured cortical neurons. The β-neurexin knockout phenotype was attenuated by CB1-receptor inhibition, which blocks presynaptic endocannabinoid signaling, or by 2-arachidonoylglycerol synthesis inhibition, which impairs postsynaptic endocannabinoid release. In synapses formed by CA1-region pyramidal neurons onto burst-firing subiculum neurons, presynaptic in vivo knockout of β-neurexins aggravated endocannabinoid-mediated inhibition of synaptic transmission and blocked LTP; presynaptic CB1-receptor antagonists or postsynaptic 2-arachidonoylglycerol synthesis inhibition again reversed this block. Moreover, conditional knockout of β-neurexins in CA1-region neurons impaired contextual fear memories. Thus, our data suggest that presynaptic β-neurexins control synaptic strength in excitatory synapses by regulating postsynaptic 2-arachidonoylglycerol synthesis, revealing an unexpected role for β-neurexins in the endocannabinoid-dependent regulation of neural circuits.

Figure 1. Conditional KO of β-neurexins impairs excitatory synaptic transmission

(A) β-Neurexins are expressed ~10–100 fold lower levels than α-neurexins. Data show relative α- and β-neurexin mRNA levels measured by quantitative RT-PCR (n=3 mice at P30). (B) Conditional KO (cKO) strategy for neurexin-1β (left) and neurexin-2β and -3β (right dashed box). All cKOs involve floxing the 5′ β-neurexin-specific exon and adding an N-terminal epitope tag (EGFP for neurexin-1β and -3β; HA-tag for neurexin-2β). (C) Immunoblots of tagged β-neurexins in cKO mice with antibodies to GFP (top) and to a conserved C-terminal neurexin epitope (bottom). Proteins in cortex homogenates (from 3-week old control and triple β-neurexin cKO mice; input) were immunoprecipitated with GFP antibodies (GFP-IP) to visualize the low-abundance β-neurexins in cKO mouse samples. (D) β-Neurexin KO does not alter morphological parameters in cultured cortical neurons. Left, representative images of neurons filled with Alexa Fluor 594 via the patch pipette; right, summary graphs of spine density. (E) β-Neurexin KO does not impair excitatory synapse density and size. Left, representative images; right, summary graphs of vGlut1-positive synapse density. (F–H) β-Neurexin KO in cultured cortical neurons impairs excitatory but not inhibitory synaptic transmission evoked by isolated action potentials (F, AMPAR-mediated EPSCs; G, NMDAR-mediated EPSCs; H, GABAR-mediated IPSCs). (I) β-Neurexin KO decreases the presynaptic release probability, measured via the MK-801 induced progressive block of NMDAR-mediated synaptic responses. Left, representative EPSC traces for the 1^st, 10^th, 25^th, and 125^th stimulus; center, mean ESPC amplitudes; right, summary graphs of decay constants. (J) β-Neurexin KO does not alter the readily-releasable vesicle pool as analyzed by stimulation with 0.5 M sucrose. Left, representative traces; right, total charge transfer. Data in D–J are means ± SEM (numbers of neurons/independent cultures examined are shown in graphs). Statistical analyses were performed by Student’s t-test (*p<0.05, **p<0.01, ***p<0.001). For additional data, see Figs. S1–S4.

Figure 2. Conditional KO of β-neurexins impairs presynaptic Ca²⁺-influx

(A) Design of Ca²⁺-imaging experiments. Left, schematic of the GCaMP5G-Syb2 fusion protein that acts as a presynaptic Ca²⁺-probe; right, flow diagram of experiments. (B) Representative Ca²⁺-imaging experiments. Left, sample images of presynaptic boutons containing GCaMP5G-Syb2 (green) that contact mCherry-containing dendritic spines (red; images were obtained before and after maximal stimulation to saturate Ca²⁺-transients [(ΔF_sat]; white circles = regions of interest [ROIs] for quantitative analysis by line scans [arrows]). Right, summary graphs of line scans through ROIs at before (top) and after maximal stimulation (ΔF_sat; bottom; green = presynaptic Ca²⁺-concentration; red = postsynaptic mCherry signal). (C) Presynaptic Ca²⁺-transients saturate after 20 stimuli and are blocked by TTX. Left, representative Ca²⁺-transients; right, summary graphs (-TTX, n=12 neurons; +TTX, n=2 neurons). (D) β-Neurexin KO impairs action potential-induced presynaptic Ca²⁺-influx in cultured cortical neurons (ΔCre = control; Cre = KO). Left, representative fluorescence traces of 10 stimuli applied at 50 Hz; right, scatter plots of individual bouton responses to 1, 5, and 10 stimuli. (E) Summary plot of mean Ca²⁺-transients after 1, 5, and 10 stimuli. Left, n=3 independent experiments with 2–4 boutons per neuron; right, summary graph of the mean linear slopes fitted through the 1, 5, 10 stimuli plots. (F) β-Neurexin KO does not alter levels or localization of presynaptic Ca²⁺-channels (representative images of cortical pyramidal neuron spines with sparse GFP expression that are stained for presynaptic P/Q-type (CaV2.1) or N-type (CaV2.2) Ca²⁺-channels). For quantitative analyses, see Figs. S4F–S4H. Data are means ± SEM. Statistical analysis was performed by Student’s t-test (*p<0.05, **p<0.01).

Figure 3. Neurexin-1β, but not neurexin-1α, and the CB1R antagonist AM251 rescue impaired spontaneous mini release in β-neurexin KO neurons

(A–D) β-Neurexin KO impairs mini release at excitatory but not inhibitory synapses (A & B, mESPCs; C & D, mIPSCs) in cortical neurons from triple β-neurexin cKO mice expressing inactive (ΔCre) or active Cre-recombinase (Cre). A & C, representative traces (left), cumulative distributions of inter-event intervals (right), and mean event frequencies (inset); B & D, average individual events (left), cumulative distributions of event amplitudes (right), and mean event amplitudes (inset). (E) Neurexin-1β without an insert in SS#4 (−SS4), but not with an insert (+SS4), rescues the decreased mEPSC frequency in β-neurexin KO neurons. (F) Neurexin-1α without an insert in SS#4 (−SS4) fails to rescue the decreased mEPSC frequency in β-neurexin KO neurons. (G) Blocking CB1Rs with AM251 reverses the decrease in mEPSC frequency in β-neurexin KO neurons. Left, representative traces; center, cumulative distributions of event frequencies with insets showing mean frequencies; right, summary graphs of AM251-induced changes. (H) Activating CB1Rs with WIN (WIN 55,212–2 mesylate) depresses mini release significantly more in control (ΔCre) than in β-neurexin KO neurons (Cre). Figure design is analogous to that of G. Data are means ± SEM; numbers of neurons/independent cultures examined are shown in the graphs. Statistical analyses were performed using Student’s t-test (*p<0.05, **p<0.01, ***p<0.001). For additional data, see Figs. S4 and S5.

Figure 4. Blocking postsynaptic 2-AG synthesis rescues presynaptic β-neurexin KO phenotype in cultured neurons

(A) Diagram of endocannabinoid signaling pathways. Two distinct endocannabinoids (2-arachidonoylglycerol [2-AG] and anandamide [AEA, N-arachidonoylethanolamine]) are synthesized by different postsynaptic enzymes; both act on presynaptic CB1Rs (abbreviations: PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; DAGL, diacylglycerol lipase; NAT, N-acyltransferase; PE, phosphatidylethanolamine; NAPE, N-arachidonoyl phosphatidylethanolamine; PLD, phospholipase D). Modes of action of the 2-AG synthesis inhibitor U73122 and the CB1R agonist WIN and antagonist AM251 are indicated. (B–D) CB1R levels are similar in cortical neurons from triple β-neurexin cKO mice expressing inactive (ΔCre) or active Cre-recombinase (Cre). B, representative immunoblots with antibodies to CB1R, CaV2.1 and CaV2.2 Ca²⁺-channels, GluR1, NR1 (an NMDAR subunit), and α-neurexins; C & D, immunocytochemistry quantifications of CB1R levels (C) and CB1R localization (D); note that neurons were sparsely transfected with EGFP for visualization of neuronal morphology. (E & F) 2-AG causes a larger depression of mini release in control than in β-neurexin KO neurons (E), whereas anandamide is ineffective likely because it is only a partial agonist (F). Left, representative traces; center, cumulative distributions of mEPSC inter-event intervals (insets = mean frequencies); right, summary graphs of anandamide- or 2-AG-induced changes. (G) Selective postsynaptic block of 2-AG synthesis by U73122 in the patch pipette rescues decreased mini release in β-neurexin KO neurons. Left, experimental set-up and representative mEPSC traces; right, bar diagrams of mEPSC frequencies and amplitudes. (H & I) Selective postsynaptic block of 2-AG synthesis by U73122 prevents CB1R activation in β-neurexin KO neurons as measured by blocking CB1Rs with bath applied AM251 (H) or activating CB1Rs with bath-applied 2-AG (I). Left, representative traces; center, cumulative distributions of mEPSC inter-event intervals (insets = mean frequencies); right, summary graphs of AM251- or 2-AG-induced changes in mEPSC frequencies. Data are means ± SEM; numbers of neurons/independent cultures examined are shown in graphs. Statistical analyses were with Student’s t-test (*p<0.05, **p<0.01, ***p<0.001).

Figure 5. Presynaptic KO of β-neurexins in CA1 pyramidal neurons decreases synaptic strength at burst-firing neuron synapses in the subiculum

(A) Experimental design. Left, diagram of stereotactic injections into the CA1 region; center, representative images of slices from stereotactically injected mice at P35 to visualize AAV infections (slices with <90% EGFP expression in the CA1 region were rejected); right, electrophysiological recording configuration in acute subiculum slices (Aoto et al., 2013). (B) Identification of regular- and burst-firing pyramidal subiculum neurons in current-clamp mode. Left and right, representative traces; center, summary graph of the initial spiking frequency. (C & D) Input/output (I/O) relations of AMPAR-mediated EPSCs elicited by stimulation of CA1-derived axons and recorded in regular- (C) or burst-firing subiculum neurons (D). Left, summary plots with representative traces on top; right, summary graphs of fitted linear input/output slopes. (E & F) Paired-pulse ratio (PPR) measurements of AMPAR EPSCs in regular- (E) and burst-firing subiculum neurons (F). Left, summary plots of PPRs vs. inter-stimulus intervals with representative traces on top; right, summary graphs of PPRs at 100 ms inter-stimulus intervals. (G) Only burst-firing but not regular-firing subiculum neurons exhibit tonic endocannabinoid signaling in wild-type mice. EPSCs were elicited at 0.1 Hz before and after bath-application of the CB1R antagonist AM251. Left, representative EPSC traces; center, plots of relative EPSC amplitudes and AM251-induced EPSC amplitude changes in individual neurons; right, summary graphs of AM251-induced EPSC amplitude changes. (H) Presynaptic β-neurexin KO increases tonic endocannabinoid signaling in burst-firing subiculum neurons. Experiments were performed as in G, except that burst-firing neurons were analyzed in slices from β-neurexin cKO mice with presynaptic expression of ΔCre- or Cre-EGFP in the CA1 region (see A). Data are means ± SEM; numbers of neurons/mice examined are shown in the summary graphs. Statistical analysis was by paired Student’s t-test for single cell plots, and unpaired Student’s t-test for comparisons in other summary graphs (*p<0.05, **p<0.01, ***p<0.001).

Figure 6. Presynaptic KO of β-neurexins selectively impairs LTP at burst-firing subiculum neurons by enhancing basal endocannabinoid activity

(A) KO of β-neurexins does not change LTP of CA1 EPSCs onto regular-firing subiculum neurons. LTP was induced by 4 × 100 Hz/1 s stimulation with 10 s intervals in current-clamp mode at resting potential in acute slices from CA1-region specific β-neurexin KO mice obtained as described in Fig. 5A. Left, representative traces; center, average EPSC amplitudes (1 min bins); right, summary graphs of mean LTP magnitude 50–60 min after induction. (B) Same as (A), except that burst-firing subiculum neurons were analyzed. (C & D) Same as (B), except that the effect of the CB1R antagonist AM251 on LTP was examined in slices from mice injected with inactive Cre-recombinase (C) or with active Cre-recombinase (D). (E & F) Same as (C & D), except that the effect of the phospholipase C inhibitor U73122 introduced into the postsynaptic neuron via the patch pipette was examined. Data shown are means ± SEM; numbers of neurons/mice examined are shown in the graphs. Statistical analysis was performed by Student’s t-test (* p<0.05, **p<0.01).

Figure 7. Conditional KO of β-neurexins in the hippocampal CA1 region impairs contextual memory: Model for β-neurexin action

(A) Design of behavioral experiments (after Xu et al., 2012). (B) Representative coronal images illustrating expression of Cre-EGFP in the CA1 region of the hippocampus after stereotactic injection (top) and zoomed CA1 image (bottom). (C & D) Analysis of ΔCre- or Cre-injected mice in open field (C) and fear-conditioning tests (D). Open field behavior (analyzed in three 5 min segments) was quantified as spatial confinement (C, left), low mobility bouts (C, center), and total distance traveled (C, right). Fear-conditioning training exposed mice to three 30 s tones ending with 2 s electrical footshocks separated by 1 min intervals (D; left graphs, cumulative distributions; right summary graphs, mean fear-conditioning memory as measured by freezing). Data are means ± SEM; numbers of mice examined are shown in the graphs. Statistical analysis was performed by Student’s t-test (* p<0.05). (E) Model for β-neurexin action. In wild-type excitatory synapses (left), presynaptic β-neurexins regulate endocannabinoid signaling by controlling postsynaptic 2-AG synthesis, possibly via trans-synaptic interaction with postsynaptic neuroligin isoforms that exclusively bind to β- but not α-neurexins lacking an insert in SS#4. In excitatory β-neurexin KO synapses (right), 2-AG synthesis is disinhibited, CB1Rs are activated, and synaptic strength is decreased; moreover, in burst-firing subiculum neurons LTP is blocked which may be responsible for the impairment in contextual memory.