Axonal Α7 Nicotinic Ach Receptors Modulate Presynaptic NMDA Receptor Expression and Structural Plasticity of Glutamatergic Presynaptic Boutons
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Axonal α7 nicotinic ACh receptors modulate presynaptic NMDA receptor expression and structural plasticity of glutamatergic presynaptic boutons Hong Lina,b,c, Stefano Vicinid, Fu-Chun Hsua,b,c, Shachee Doshia,b,c, Hajime Takanoa,b,c, Douglas A. Coultera,b,c,e, and David R. Lyncha,b,c,e,1 Departments of aNeurology and bPediatrics, eUniversity of Pennsylvania School of Medicine, Philadelphia, PA 19104; cChildren’s Hospital of Philadelphia, Philadelphia, PA 19104; and dDepartment of Physiology and Biophysics, Georgetown University School of Medicine, Washington, DC 20007 Edited* by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved August 3, 2010 (received for review May 27, 2010) In association with NMDA receptors (NMDARs), neuronal α7 nicotinic induced by NMDAR stimulation (excitotoxicity) (9, 10). nAChR ACh receptors (nAChRs) have been implicated in neuronal plasticity stimulation facilitates glutamatergic transmission at selected CNS as well as neurodevelopmental, neurological, and psychiatric disor- synapses and enhances a synapse selective form of LTP in the ders. However, the role of presynaptic NMDARs and their interaction amygdala (11). This facilitation is commonly mediated by pre- with α7 nAChRs in these physiological and pathophysiological events synaptic receptors and likely involves regulation of transmitter re- remains unknown. Here we report that axonal α7 nAChRs modulate lease, including the release of glutamate (12–14). nAChRs also presynaptic NMDAR expression and structural plasticity of glutama- regulate the downstream turnover of selected glutamate receptors tergic presynaptic boutons during early synaptic development. such as the AMPA receptor GluR1 subunit (15). Chronic inactivation of α7 nAChRs markedly increased cell surface Investigations of synaptic plasticity have concentrated on post- NMDAR expression as well as the number and size of glutamatergic synaptic mechanisms, especially on postsynaptic NMDA and axonal varicosities in cortical cultures. These boutons contained pre- AMPA receptors. Presynaptic NMDARs have recently been im- synaptic NMDARs and α7 nAChRs, and recordings from outside-out plicated in cortical synaptic function and plasticity (16). They exist pulled patches of enlarged presynaptic boutons identified functional at higher levels early in development, and are involved in regulation NEUROSCIENCE NMDAR-mediated currents. Multiphoton imaging of presynaptic of transmitter release and forms of LTD (16–20). However, the NMDAR-mediated calcium transients demonstrated significantly mechanisms controlling the expression of presynaptic NMDARs, larger responses in these enlarged boutons, suggesting enhanced how they affect synaptic development, and why they decrease with presynaptic NMDAR function that could lead to increased glutamate development are unknown. In the present study, we have identified fi release. Moreover, whole-cell patch clamp showed a signi cant in- a previously uncharacterized structural component of presynaptic crease in synaptic charge mediated by NMDAR miniature EPSCs but plasticity reflecting interactions of axonal α7nAChRsandpre- no alteration in the frequency of AMPAR miniature EPSCs, suggest- synaptic NMDARs in glutamatergic presynaptic bouton formation ing the selective enhancement of postsynaptically silent synapses during early synaptic development. upon inactivation of α7 nAChRs. Taken together, these findings in- dicate that axonal α7 nAChRs modulate presynaptic NMDAR expres- Results sion and presynaptic and postsynaptic maturation of glutamatergic Chronic Inactivation of α7 nAChR Increases Surface NMDAR Expression synapses, and implicate presynaptic α7 nAChR/NMDAR interactions and Numbers of Presynaptic Boutons Containing NMDARs in Cortical in synaptic development and plasticity. Cultures. To explore the possible interactions of nAChRs and NMDARs, we examined the effects of nicotine on cell surface ex- silent synapse | synaptic development | synaptic plasticity | alpha pression levels of NMDARs in cortical neurons. Nicotine markedly bungarotoxin | cytisine increased such levels (Fig. 1A). As nicotine is an agonist at both α4β2 and α7 nAChRs with a higher affinity for α4β2 receptors, we also s the major excitatory neurotransmitter systems in the CNS, investigated the subtype selective agents cytisine, α-bungarotoxin Athe nicotinic and glutamatergic systems have been implicated (α-BTX), and dihydro-β-erythrodine (DHβE). Cytisine is a full ag- in a variety of neurological, neurodevelopmental, and psychiatric onist at α7 and partial agonist at α4β2nAChRs.α-BTX and DHβE disorders as well as learning and memory (1, 2). Glutamate exerts are specific antagonists at α7andα4β2 nAChRs, respectively. its effects through a series of postsynaptic receptors named for their Cytisine and α-BTX treatment markedly increased surface levels of prototypic agonists. The most common ionotropic receptors are the NMDAR subunits NR1, NR2A, NR2B, and the AMPAR GluR1 NMDA and AMPA receptors. The importance of NMDA receptor subunit in cortical cultures (Fig. 1 B and C), whereas DHβEhad (NMDAR)-mediated neural transmission is illustrated by its role in a smaller effect on surface levels of NMDARs (Fig. 1D). The surface models of synaptic plasticity such as long-term potentiation (LTP) level of the GABAAα1 subunit of GABA receptors was unchanged and long-term depression (LTD). One of the crucial biochemical – fi following exposure to any of these agents (Fig. 1 A D). mechanisms mediating these processes is traf cking of glutamate We then examined the distribution of glutamate receptors in receptors to and from the synapse/cell surface. Once these recep- cortical cultures. α-BTX or cytisine treatment markedly increased tors are placed into the membrane, they contribute to the genera- tion of additional biochemical or structural events leading to more permanent alterations in synaptic strength. Author contributions: H.L., S.V., F.-C.H., D.A.C., and D.R.L. designed research; H.L., S.V., Similarly, nicotinic ACh receptors (nAChRs) have crucial roles S.D., and H.T. performed research; S.V., F.-C.H., H.T., and D.A.C. contributed new reagents/ in a variety of CNS processes, including neuronal plasticity, nicotine analytic tools; H.L., S.V., F.-C.H., S.D., D.A.C., and D.R.L. analyzed data; and H.L. and D.R.L. addiction, Alzheimer’s disease, Down syndrome, and schizophrenia wrote the paper. (3–8). The α7andα4β2 subtypes are the predominant nAChRs in The authors declare no conflict of interest. the central nervous system. Interestingly, there are many inter- *This Direct Submission article had a prearranged editor. actions among both nAChR subtypes and glutamate receptors, 1To whom correspondence should be addressed. E-mail: [email protected]. particularly NMDARs, in physiological and pathological events. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. For example, nAChRs mediate neuroprotection against cell death 1073/pnas.1007397107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1007397107 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 A B transmitters and form synapses. We compared NR1-positive axonal CON NIC CON CYT varicosities (Fig. 2 A and B) and differential interference contrast NR2B NR2B (DIC) images of enlarged presynaptic boutons (Fig. 2 C and D)be- NR2A NR2A tween control and α-BTX–treated cultures. The presynaptic terminal NR1 NR1 marker α-synaptophysin was also present in the enlarged boutons GluR1 GluR1 (Fig. 2 E and F). Although NR1-positive presynaptic boutons were increased and enlarged in α-BTX– or cytisine-treated cultures, no GABAAα1 GABAAα1 fi β 700 * signi cant increases were observed in DH E- or nicotine-treated 600 CON cultures (Fig. 2G). The increases in surface NMDAR expression and CON 600 CYT NIC 500 * in presynaptic boutons were blocked by the transcriptional inhibitor * 500 400 ** actinomycin D and the translational inhibitor cycloheximide (Fig. S1). * 400 *** These data suggest that α-BTX– or cytisine-induced increases in pre- 300 fl 300 *** synaptic boutons containing NMDARs largely re ect presynaptic 200 % of control % of control 200 increases in surface NMDAR expression, whereas nicotine-induced 100 increases in surface NMDAR expression are not clearly associated 100 0 with presynaptic terminals. NR2B and GluR1 colocalized with NR1- NR1 NR2A NR2BGluR1 GABA 0 NR1 NR2ANR2BGluR1 GABA positive presynaptic boutons in treated cultures (Fig. S2), consistent with the α-BTX– or cytisine-induced increase in the surface levels of glutamate receptors largely reflecting enlarged axonal varicosities C D E β containing presynaptic glutamate receptors. CON α-BTX CON DH As α7 nicotinic receptors desensitize almost fully on chronic ex- NR2B NR2B posure to agonists such as cytisine, this, coupled with similar effects NR2A NR2A of α-BTX, suggests that the increase in NMDAR levels results from NR1 α7 inactivation (either by desensitization or blockade). The time NR1 GluR1 course of events was consistent with inactivation of α7beingthe GABAAα1 GABAAα1 primary site action of cytisine, as brief (3 min) application of cytisine 600 600 slightly decreased NMDAR levels, whereas more prolonged ap- CON CON ** BTX 500 DHβE plication (3–24 h) increased levels (Fig. S3A). Coapplication of ei- ** 500 * ther α-BTX or DHβE had no effect on cytisine-induced changes in 400 400 ** NMDARs (Fig. S3 B and C), suggesting that cytisine does not act as 300 300 an agonist at either subtype in this paradigm,