Nicotine recruits glutamate receptors to postsynaptic sites

Abstract

Cholinergic neurons project throughout the nervous system and activate nicotinic receptors to modulate synaptic function in ways that shape higher order brain function. The acute effects of nicotinic signaling on long-term synaptic plasticity have been well-characterized. Less well understood is how chronic exposure to low levels of nicotine, such as those encountered by habitual smokers, can alter neural connections to promote addiction and other lasting behavioral effects. We show here that chronic exposure of hippocampal neurons in culture to low levels of nicotine recruits AMPA and NMDA receptors to the cell surface and sequesters them at postsynaptic sites. The receptors include GluA2-containing AMPA receptors, which are responsible for most of the excitatory postsynaptic current mediated by AMPA receptors on the neurons, and include NMDA receptors containing GluN1 and GluN2B subunits. Moreover, we find that the nicotine treatment also increases expression of the presynaptic component synapsin 1 and arranges it in puncta juxtaposed to the additional AMPA and NMDA receptor puncta, suggestive of increases in synaptic contacts. Consistent with increased synaptic input, we find that the nicotine treatment leads to an increase in the excitatory postsynaptic currents mediated by AMPA and NMDA receptors. Further, the increases skew the ratio of excitatory-to-inhibitory input that the cell receives, and this holds both for pyramidal neurons and inhibitory neurons in the hippocampal CA1 region. The GluN2B-containing NMDA receptor redistribution at synapses is associated with a significant increase in GluN2B phosphorylation at Tyr1472, a site known to prevent GluN2B endocytosis. These results suggest that chronic exposure to low levels of nicotine not only alters functional connections but also is likely to change excitability levels across networks. Further, it may increase the propensity for synaptic plasticity, given the increase in synaptic NMDA receptors.

Keywords: AMPA receptors; Glutamatergic; Hippocampus; NMDA receptors; Nicotine; Nicotinic; Synapses.

Figure 1

The GluA2 subunit is required for almost all of the evoked EPSC mediated by AMPA receptors. (A) Sample records of evoked EPSCs in a neuron from a control culture (left) or a culture receiving nicotine for 7 days (right) before and after initiating the exposure to 50 μM NASPM. (B) Quantification showing the proportion of evoked EPSC amplitude mediated by GluA2Rs (resistant to NASPM) as a fraction of total EPSC amplitude in control (Con) and nicotine-treated (Nic) cells (p ≥ 0.18; n = 9, 10). (C) Typical time course of AMPAR-EPSC in a neuron treated with NASPM (50 μM for 10 min). NASPM (50 μM) was bath-applied during the time indicated by the open bars.

Figure 2

Extended nicotine exposure increases surface expression of glutamate receptor subunits on hippocampal neurons in culture. (A) Neurons were treated with control medium (Con) or with medium containing nicotine (Nic) for 7 days and then biotinylated, solubilized, and immunoblotted for GluA2, GluN1, GluN2B, or GABA_A α1 receptors in aliquots of whole extract (left, total) or in the biotinylated fraction (right, surface). (B) Quantification of biotinylated surface components recovered on immunoblots from control (Con) and nicotine-treated (Nic) samples, normalized for the amount present in controls. GluA2, *p ≤ 0.02, n = 8; GluN1, *p ≤ 0.03, n = 5; GluN2B, *p ≤ 0.02, n = 5; GABA_A α1, p ≥ 0.9, n = 5. (C) Synapsin 1 in whole extracts from control (Con) and nicotine-treated (Nic) cultures probed on immunoblots (GAPDH for reference), quantified, and normalized for control values (right; *p ≤ 0.02; n=5).

Figure 3

Nicotine, acting through α7-nAChRs, increases surface clusters of GluA2, internal clusters of synapsin1 clusters, and juxtaposition of the two on hippocampal neurons in culture. Neurons were incubated with nicotine for 7 days and then fixed and stained first without permeabilization for GluA2 and then co-stained after permeabilization for synapsin 1. (A) Images showing GluA2 (green), synapsin 1 (red), and co-localization (Co, yellow). Scale bars: 20 μm (left), 5 μm (right) for each set. (B) Quantification of GluA2 puncta number (left) and mean size (right). Including the α7-nAChR antagonist MLA in the 7-day nicotine incubation (Nic+MLA) prevented the nicotine from increasing either GluA2 or synapsin 1 puncta, or altering their co-localization. GluA2, *p ≤ 0.02; synapsin 1, *p ≤ 0.02; Co, **p ≤ 0.01; n = 20 for Con, 22 for Nic, 18 for Nic+MLA.

Figure 4

Nicotine pre-treatment increases GluN1 and GluN2B clusters on the surface and their co-localization with synapsin 1 puncta. (A) Images showing GluN1 (green), synapsin 1 (red), and co-localization (Co, yellow). Scale bars: 20 μm (left), 5 μm (right) for each set. (B) Quantification of GluN1 puncta number (left) and mean size (right). GluN1, *p ≤ 0.04; synapsin 1, **p ≤ 0.01; Co, *p ≤ 0.03, n = 21 for Con, 24 for Nic. (C) Quantification of GluN2B puncta number (left), and mean puncta size (right). GluN2B, **p ≤ 0.01; synapsin 1, **p ≤ 0.01; Co, *p ≤ 0.04; n = 17 for Con, 20 for Nic.

Figure 5

Nicotine pre-treatment increases the mean size of evoked EPSCs mediated by AMPA and NMDA receptors. (A) Representative AMPA and NMDA receptor-mediated EPSCs evoked in neurons after a 7-day exposure to 1 μM nicotine (Nic) or control media (Con). Arrow indicates stimulation with a bipolar extracellular electrode to elicit maximal EPSCs. (B) Quantification of AMPA receptor EPSCs (left), NMDA receptor EPSCs (middle), and their ratios (right). AMPA receptor EPSCs, ***p ≤ 0.0001; n = 64 for Con, 58 for Nic. NMDA receptor EPSCs, *p ≤ 0.04; n = 9 for Con, 16 for Nic. Ratios, p > 0.1; n = 9 for Con, 16 for Nic.

Figure 6

Chronic nicotine administration increases the incidence of tyrosine phosphorylation on GluN2B at position 1472. Left: immunoblots of GluN2B immunoprecipitated from control (Con) or nicotine-treated (Nic) cell extracts and then probed either with p-Tyr-1472-GluN2B antibodies to detect phosphorylation of GluN2B on tyrosine at position 1472 (upper) or with antibodies to total GluN2B protein (lower). Right: Amount of p-Tyr-1472-GluN2B immunostaining in Nic samples expressed as a % of that in controls (*p ≤ 0.02; n = 5).

Figure 7

Both pyramidal neurons and interneurons in the CA1 display increased E/I ratios for synaptic input, following extended exposure to nicotine. (A) Examples of a biocytin-filled pyramidal neuron (left) and an interneuron (right) in the CA1 region of a hippocampal slice maintained in organotypic culture for 10 days. Scale bar: 50 and 25 μm, for left and right panels, respectively. (B) Examples of patch-clamp recordings from a pyramidal neuron in a control (left) or nicotine-treated (right) slice clamped at −50 mV to detect glutamate-mediated EPSCs or at 0 mV to detect GABA-mediated IPSCs. Arrowheads indicate time of stimulation with a bipolar extracellular electrode to elicit maximal EPSCs. (C) Examples of patch-clamp recordings from interneurons in the CA1 treated as in panel B. (D) Ratios of evoked PSC peak amplitudes mediated by glutamatergic and GABAergic synapses for individual pyramidal neurons (left; **p < 0.01; n = 8 for Con, 8 for Nic) and interneurons in the CA1 (right; *p < 0.04; n = 7 for Con, 9 for Nic).