Glutamatergic synapse formation is promoted by α7-containing nicotinic acetylcholine receptors

Abstract

Glutamate is the primary excitatory transmitter in adult brain, acting through synapses on dendritic spines and shafts. Early in development, however, when glutamatergic synapses are only beginning to form, nicotinic cholinergic excitation is already widespread; it is mediated by acetylcholine activating nicotinic acetylcholine receptors (nAChRs) that generate waves of activity across brain regions. A major class of nAChRs contributing at this time is a species containing α7 subunits (α7-nAChRs). These receptors are highly permeable to calcium, influence a variety of calcium-dependent events, and are diversely distributed throughout the developing CNS. Here we show that α7-nAChRs unexpectedly promote formation of glutamatergic synapses during development. The dependence on α7-nAChRs becomes clear when comparing wild-type (WT) mice with mice constitutively lacking the α7-nAChR gene. Ultrastructural analysis, immunostaining, and patch-clamp recording all reveal synaptic deficits when α7-nAChR input is absent. Similarly, nicotinic activation of α7-nAChRs in WT organotypic culture, as well as cell culture, increases the number of glutamatergic synapses. RNA interference demonstrates that the α7-nAChRs must be expressed in the neuron being innervated for normal innervation to occur. Moreover, the deficits persist throughout the developmental period of major de novo synapse formation and are still fully apparent in the adult. GABAergic synapses, in contrast, are undiminished in number under such conditions. As a result, mice lacking α7-nAChRs have an altered balance in the excitatory/inhibitory input they receive. This ratio represents a fundamental feature of neural networks and shows for the first time that endogenous nicotinic cholinergic signaling plays a key role in network construction.

Figure 1.

Decrement in the number of glutamatergic synapses in α7KO mice. A, EMs showing synapses (arrows) in regions containing apical dendrites of CA1 pyramidal neurons in P8 hippocampal slices from WT and α7KO mice. Scale bar, 0.5 μm. B, Number of ultrastructurally identified synapses/μm² (≥150 images from 3 animals/genotype). C, Pictures showing a diagram of the hippocampus (upper left) with a box indicating the CA1 region used for imaging and low-magnification image of the CA1 region (lower left) with a box indicating the general area used for higher power images (right) of CA1 apical dendrite regions in P12 WT and α7KO mice. Slices were immunostained for glutamatergic synapses using the markers VGluT (blue), PSD-95 (red), and the images were merged (Merge) and thresholded (Thresh) to remove pixels of below-threshold intensity (arrows indicate examples of colocalization). Scale bars: 20 μm, left; 5 μm, right. D, Number of VGluT, PSD-95, and colocalized puncta/400 μm² for the indicated genotype in the CA1. E, Number of VGluT, PSD-95, and colocalized puncta/400 μm² at P12 on neurons in layer 5/6 of the cortex (3–4 cells/animal; 5–8 animals/genotype from ≥2 litters).

Figure 2.

Electrophysiological analysis showing reduced mEPSC frequency in α7KO mice compared with WTs. A, Patch-clamp recordings of spontaneous mEPSCs in CA1 pyramidal neurons from P12 WT and α7KO acute slices in TTX and gabazine. Inset, WT mEPSC with expanded time scale. B, Quantification of mEPSC frequency (left) and amplitude (right) at P12. C, Number of VGluT, PSD-95, and colocalized puncta/400 μm² in the CA1 region of P25 WTs and α7KOs analyzed as in Figure 1. D, Quantification of mEPSC frequency (left) and amplitude (right) at P25 for the indicated genotypes. Loss of the α7-nAChR gene product, but not the β2-nAChR gene product, correlates with reduced mEPSC frequency with no change in mEPSC amplitude, a result consistent with α7KOs having fewer functional glutamatergic synapses (≥50 events/cell, 11–19 cells from 4–7 animals/genotype).

Figure 3.

Nicotine in cell culture acts via α7-nAChRs to increase glutamatergic synaptic contacts. A, Control (Con) and nicotine-treated (Nic) cell cultures expressing GFP and surface stained for GluR1 receptors (red) and then intracellular PSD-95 (blue). Here and below, overlap of three colors appears as white. Scale bar, 10 μm. B, Quantification showing a significant increase in GluR1, PSD-95, and colocalized puncta in nicotine unless α7-nAChRs were blocked with either MLA or αBgt (n = 3–12 cultures, 5 cells/culture). C, Cultures immunostained for VGluT (red) and PSD-95 (green). The dashed white lines indicate the edges of a neurite; spines extending from the neurite are not demarcated. Scale bar, 5 μm. D, Quantification showing increased puncta and colocalization of VGluT and PSD-95 after nicotinic activation of α7-nAChRs. E, Cultures expressing GFP and immunostained first for surface α1-containing GABA_A receptors (red) and then for intracellular GAD-65 (blue). Scale bar, 10 μm. F, Quantification showing no changes in the numbers of GAD-65 and GABA_A receptor puncta (GABAR) or in their colocalization (n = 3–4 cultures; 5 cells/culture). Nicotinic stimulation appears to increase glutamatergic, but not GABAergic, synapses.

Figure 4.

Nicotine-induced increases in the number of functional glutamatergic synapses in cell culture. Rat hippocampal cultures were incubated 7 d (7d) in nicotine (Nic) with or without blockers (MLA, DHβE), rinsed to remove nicotine, and then incubated 1 h in MLA/DHβE. As a control, some cultures received nicotine only during the last hour of the 7 d incubation (1 h Nic). A, Patch-clamp recording of spontaneous mEPSCs in TTX and gabazine. B, Quantification of mEPSC frequency (15–20 cells from 5–8 cultures per condition). C, Images of cells in culture having received the indicated treatment, then incubated with MLA/DHβE for 1 h followed by labeling with a 1 min incubation in FM4–64 (red) plus 50 mm KCl to reveal active synapses on dendrites and spines of cells expressing GFP. Scale bar, 10 μm. D, Quantification of FM4–64-labeled puncta. Baseline control (7d Nic/noK): KCl omitted from FM4–64 incubation (n = 3–6 cultures; 5–7 cells/culture).

Figure 5.

Nicotine induction of glutamatergic synapses in organotypic slice culture. A, EMs showing examples of synapses (arrows) in CA1 region of P4 hippocampal slices incubated in culture 4 d with (Nic) and without (Con) 1 μm nicotine. Scale bar, 1 μm. B, Quantification of ultrastructurally identified synapses per unit area (from 150–250 images/condition from 3–6 animals). C, Hippocampal slices from P0–P1 mice, maintained in culture for 4–7 d, were treated with vehicle (Con), nicotine (Nic), nicotine plus MLA (Nic/MLA), or nicotine plus DHβE (Nic/DHβE) for the last 4 d and then immunostained for VGluT (blue) and PSD-95 (red), and the images merged (Merge) and thresholded (Thresh) as in Figure 1C. Quantification of the number of indicated puncta/400 μm² in the CA1 region was performed as in Figure 1D for the drug conditions indicated. Scale bar, 5 μm. D, Images and quantification obtained as in C except that slices were coimmunostained for VGluT and GluR1 (6 cultures/condition). Nicotinic stimulation of α7-nAChRs (blocked by MLA), but not β2*-nAChRs (blocked by DHβE), increases the number of glutamatergic synapses identified immunohistochemically, and the increases are reflected in the total number of synapses per unit area identified ultrastructurally.

Figure 6.

Relevance of local α7-nAChRs for glutamatergic synapse formation. A, Apical dendrite regions of P12 hippocampal CA1 expressing either lentiviral α7-Scr (upper) or α7-RNAi (lower) after intracranial injection in vivo (GFP) and immunostaining for VGluT (blue) and PSD-95 (red) shown together (Merge) and after thresholding (Thresh; arrows indicate examples of colocalization). Scale bar, 5 μm. B, Quantification of VGluT, PSD-95, and colocalized puncta/400 μm² in P12 hippocampal CA1. C, Quantification in P12 visual cortex layer 5/6. Results are normalized to values obtained in adjacent regions lacking viral infection and demonstrate that α7-nAChRs within the local area are required for neurons to acquire WT levels of glutamatergic synapses. D, Quantification of VGluT, PSD-95, and colocalized puncta in α7KOs expressing the indicated constructs, showing that the α7-RNAi construct has no off-target effects as defined by changes induced in α7KOs (3 fields/animal; 4–8 animals/condition).

Figure 7.

Cell-autonomous requirement for α7-nAChRs to promote glutamatergic synaptogenesis. A, Isolated dendrites of P12 CA1 pyramidal neurons on the periphery of the virally infected area, expressing either α7-Scr (left) or α7-RNAi (right) and immunostained for VGluT (blue), PSD-95 (red), merged (Merge), and thresholded (Thresh). Arrows indicate examples of colocalized puncta. Scale bar, 10 μm. B, Quantification of puncta on isolated dendrites (from 3–4 cells/animal, 4–8 animals/condition). Downregulation of the α7-nAChR gene product in isolated neurons reduces the number of glutamatergic synapses formed on the neuron, demonstrating a requirement for expression of the receptors by the postsynaptic cell for normal levels of glutamatergic innervation.

Figure 8.

Decreased ratio of glutamatergic/GABAergic input in α7KO neurons. A, CA1 apical dendrite regions in P12 WT and α7KO mice immunostained for VGAT (blue), GABA_A α1 receptor (red), merged (Merge), and thresholded (Thresh). Scale bar, 5 μm. B, Number of VGAT, GABA_A α1 receptor, and colocalized puncta/400 μm² in P12 (3 fields/animal; 3 animals/genotype). C, Evoked glutamatergic PSCs recorded at −70 mV and GABAergic PSCs recorded at 0 mV (aligned by stimulus artifact) in WT and α7KO CA1 at P12. D, Ratios of evoked PSC peak amplitudes mediated by glutamatergic and GABAergic synapses for individual neurons at P12 (left) and P25 (middle; Glut/GABA PSC; 5–11 cells from 3–6 animals/genotype). Right, Ratios in WT neurons before (Con) and after adding MLA (MLA), showing that the responses are not influenced by acute endogenous α7-nAChR signaling during the recordings (5 cells from 3 animals). E, No differences were seen in PPR for evoked EPSCs amplitudes in α7KO versus WT neurons. Two EPSCs evoked in a WT CA1 pyramidal neuron clamped at −70 mV while stimulating the Schaffer collateral input twice, 100 ms apart (vertical line indicates stimulus artifact). F, Ratio of the second EPSC amplitude divided by the first (PPR) for the indicated genotype at the indicated age (5–11 cells from 3–6 animals per genotype and age).