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 indicate 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, but instead may act 200 200 ** through desensitization. The concentration–response curve of cy- % of control

% of control * 100 100 tisine on surface NMDAR expression demonstrated that the EC50 ∼ μ – 0 0 of cytisine is 200 M(Fig. S4), consistent with the dose response NR1 NR2ANR2BGluR1 GABA NR1 NR2A NR2B GABA curve of cytisine-evoked currents through α7 nAChR in cultured Fig. 1. Chronic inactivation of α7 nAChR, but not α4β2 nAChR, leads to neurons (21), further suggesting that cytisine-induced increases in marked increases in surface level of NR1, NR2, and GluR1 subunits in cortical surface NMDAR expression and presynaptic bouton number result neurons. Representative blots and quantiﬁcation showing surface level of from desensitization of α7nAChR.Takentogether,ourﬁndings

NR1, NR2A, NR2B, GluR1, and GABAAα1 in cortical cultures treated with suggest that chronic inactivation of α7 nAChR increases presynaptic nicotine (A), cytisine (B), α-BTX (C)orDHβE(D). Each experiment was repeated NMDAR expression and presynaptic boutons in cortical cultures. at least three times. Increased and Enlarged NR1-Positive Axonal Varicosities Are Glutamatergic Presynaptic Boutons. Previous work demonstrated an analogous the number and size of NR1-positive axonal varicosities or boutons pattern in developing cerebellar cultures in which NMDAR acti- in treated cortical cultures (Fig. 2). Axonal varicosities or boutons are vation increases the size of GABAergic presynaptic boutons swellings along an axon and are frequently the sites that release through presynaptic NMDARs (18). This suggests that a similar

NR1 DIC α-SYNAPT/MAP-2

G NR1-positive axonal varicosities 600 * p<0.05 **p<0.01 vs control control 500 **

A C E 400 ** 300

200 % of control 100 *

α -BTX CON CYT BTX NIC DHβE B D F

Fig. 2. Chronic inactivation of α7 nAChR increases number and size of NR1-positive axonal varicosities in cortical cultures. Representative images showing increased and enlarged NR1-positive axonal varicosities (B), DIC images of enlarged boutons (D), and α-synaptophysin–positive presynaptic terminals (F)in α-BTX–treated cultures compared with those boutons in control cultures (A, C, and E). Quantiﬁcation (G) showed that α-BTX, cytisine, but not DHβE or nicotine, increases the number of NR1-positive axonal varicosities in cortical cultures (n =3–5). (Scale bars, 20 μm.)

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1007397107 Lin et al. Downloaded by guest on October 2, 2021 NR1 vGLUT1 overlay positive (Fig. 3) but not GAD-65 positive. These vGLUT1-positive presynaptic boutons also contained the presynaptic terminal marker α-synaptophysin and were noted to be largely en passant boutons (discussed later here) along axonal shafts (Fig. 4). Fur- control thermore, quantitative measurement of size of boutons containing A B C both α-synaptophysin and vGLUT1 conﬁrmed the enlargement of NR1 vGLUT1 overlay glutamatergic boutons in cytisine- or α-BTX–treated cultures compared with those in control cultures (Fig. 4J′). α7 nAChRs also localized to α-synaptophysin– and vGLUT1-positive glutamatergic presynaptic boutons (Fig. 5). The ﬁndings provide structural evi- cytisine dence linking α7 nAChRs and presynaptic NMDARs in the growth D E F and formation of glutamatergic boutons. NR1 vGLUT1 overlay Presynaptic NMDAR Function Is Enhanced in Enlarged Glutamatergic Boutons and Likely Associated with Selective Enhancement of Post-

α -BTX synaptically Silent Synapses. We then assessed whether presynaptic NMDARs in the enlarged boutons are functional. Direct voltage G H I clamp recordings from outside-out pulled membrane patches of enlarged boutons revealed single channel currents in response to Fig. 3. Increased and enlarged NR1-positive axonal varicosities are gluta- μ − matergic presynaptic boutons. (A–C) NR1 and vGLUT1 immunoreactivities in NMDA (5 M) application at a holding potential of 60 mV, control cultures. (D–I) Colocalization of glutamatergic presynaptic terminal demonstrating the presence of functional NMDARs in the enlarged marker, vesicular glutamate transporter 1 (vGLUT1), with NR1-positive axonal boutons (Fig. 6A). In addition, calcium imaging using Fura-2 as the varicosities in cytisine- or α-BTX–treated cultures. (Scale bars as indicated.) calcium indicator was conducted using two-photon microscopy and 780-nm excitation; at this wavelength, ﬂuorescence emission decreases upon calcium entry. In the presence of tetrodotoxin and control mechanism might exist in cortical neurons. To assess the voltage-dependent calcium channel blockers, bath application of neurotransmitter identity in NR1-positive presynaptic boutons, we NMDA produced a greater decrease in ﬂuorescence intensity in examined the immunoreactivities of GAD-65 (a GABAergic neu- boutons of α-BTX– or cytisine-treated cultures than those in control NEUROSCIENCE ronal marker), the vesicular glutamate transporter 1 (vGLUT1, cultures (Fig. 6B). These results indicate that presynaptic NMDAR- a glutamatergic presynaptic terminal marker), and α-synaptophysin mediated calcium entry is increased in boutons of treated cultures, (a presynaptic terminal marker) in cortical cultures. The increased suggesting that presynaptic NMDAR function is enhanced in the and enlarged NR1-positive presynaptic boutons were vGLUT1 enlarged glutamatergic boutons.

SYNAPT vGLUT1 overlay A' B' control A B C C' SYNAPT vGLUT1 overlay D' E' cytisine D E F F' SYNAPT vGLUT1 overlay G' H' α -BTX G H I I' bouton size GAP-43 vGLUT1 overlay J' 350 *** *** 300 250 200 150

α -BTX 100

% of control 50 0 JLK CON CYT BTX

Fig. 4. Glutamatergic presynaptic boutons containing α-synaptophysin are significantly enlarged and are largely en passant boutons. (A–C and A′–C′) α-Synapto- physin and vGLUT1 immunoreactivities in control cultures. (D–I and D′–I′) Colocalization of α-synaptophysin in increased and enlarged vGLUT1-positive glutamatergic presynaptic boutons in cytisine- or α-BTX–treated cultures. (J–L) Glutamatergic presynaptic boutons (vGLUT1 as glutamatergic presynaptic terminal marker) along axons (GAP-43 as axonal marker) in α-BTX–treated cultures. (Scale bars as indicated.) (J′) Quantitative measurement of bouton size, showing significant enlargement of glutamatergic boutons containing both α-synaptophysin and vGLUT1 in cytisine-treated (1.21 ± 0.05 μm2, n =61,P < 0.001 vs. control) or α-BTX–treated (1.23 ± 0.06 μm2, n =60,P < 0.001 vs. control) cultures compared with those in control cultures (0.39 ± 0.04 μm2, n = 31). n, Number of boutons in three to four fields from different cultures in each group. (Scale bars, 5 μm.)

Lin et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 vGLUT1 α7 overlay A B TTX+Mg free CON CON BTX BTX control A CB 10pA 3s 10pA 5s AMPAR mEPSCs NMDAR mEPSCs vGLUT1 α7 overlay *** 1.25 800 )zH(ycneuqerF 1.0 )s.Ap(egrahc c it 600 p

0.75 a nys

cytisine 400 0.5 D FE 0.25 200 vGLUT1 α7 overlay 0.0 0 CON B/C CON B/C

Fig. 7. Selective enhancement of postsynaptically silent synapses upon in- α α -BTX activation of 7nAChR.(A) Whole-cell voltage-clamp recordings illustrating examples of AMPAR mEPSCs in cortical cultures. Frequency of AMPAR mEPSCs HG I was not altered in α-BTX– and cytisine-treated cortical neurons (n =12)com- α pared with control (n =14).(B) Examples of robust increase in synaptic activity Fig. 5. 7 nAChRs are present in enlarged glutamatergic presynaptic bou- 2+ tons. (A–C) vGLUT1 immunoreactivity and α7 nAChR labeled by tetrame- upon removal of Mg due to overlapping NMDAR-mediated mEPSCs. Synaptic ﬁ α – thylrhodamine α-BTX in control axons. (D–I) Colocalization of α7 nAChR in charge mediated by NMDAR mEPSCs was signi cantly increased in -BTX or < enlarged vGLUT1-positive glutamatergic presynaptic boutons in cytisine- or cytisine-treated cortical neurons (n =16,P 0.001 vs. control) compared with α-BTX–treated cultures. (Scale bars as indicated.) control neurons (n =13).

Furthermore, we assessed whether the enhanced presynaptic served only a slight increase. This discrepancy may reflect the NMDAR function alters transmission. Surprisingly, whole-cell abundant extrasynaptic NMDARs during early synaptic devel- patch clamp of cortical neurons in α-BTX– or cytisine-treated cul- opment (22) and the possibility that bath application of exogenous tures showed no alteration in the frequency of AMPAR mEPSCs NMDA may preferentially activate extrasynaptic receptors but (Fig. 7A), In contrast, we found a significant increase in the syn- have limited access to synaptic NMDARs. Taken together, these aptic charge mediated by NMDAR mEPSCs (P < 0.001) (Fig. 7B). results suggest the selective enhancement of postsynaptically silent NMDA-mEPSCs were assessed as synaptic charge as their long synapses upon inactivation of α7 nAChR. duration and degree of overlap prevented individual detection. Combined, this lack of change in AMPAR and enhanced NMDAR Discussion mEPSCs suggests that the enlarged boutons are contained within The present studies show that, in cortical neuronal cultures, chronic postsynaptically silent synapses containing NMDARs but lacking inactivation of α7 nAChRs increases the number and size of glu- AMPARs. tamatergic presynaptic boutons containing presynaptic NMDARs. We also measured the total pool of NMDARs in cortical neurons Presynaptic NMDAR function is enhanced in these boutons. Al- by examining the whole-cell currents evoked by exogenous appli- though a variety of nicotinic agents altered NMDAR levels, the cation of distinct concentrations (5 and 200 μM) of NMDA in the effects on presynaptic NMDARs and boutons were most prom- presence of tetrodotoxin and in the absence of Mg2+ in control and inent with exposure to compounds that block or desensitize α7 treated cultures. Whole-cell NMDA current density exhibited no nicotinic receptors. The biochemical effects appear to reflect significant increase between control and treated cultures (Fig. S5). mainly structural changes in the presynaptic membrane, with ap- Because we measured the total pool of postsynaptically localized pearance of enlarged axonal boutons containing large numbers of NMDARs including extrasynaptic and synaptic receptors, we ob- NMDARs. These presynaptic NMDARs can alter glutamate release and are potentially involved in altering postsynaptic neurotransmission and development, thus demonstrating a pharmaco-

A B NMDA logical, structural, and physiological interaction among presynaptic α7 nAChR and NMDAR during early synaptic development. A XTB similar regulatory mechanism for analogous events has been ob- – a served in cultured cerebellar neurons. Presynaptic NMDARs in- ﬂ Vh-60 mV NMDA 5mM CONCYT BTX uence development of GABAergic presynaptic boutons in 0 developing cerebellar neurons (18), whereas, as we describe here, en

i -20 les axonal α7 nAChRs alter development of glutamatergic presynaptic

a -40

bfo% boutons in developing cortical neurons. Such parallel results sug- -60 *** *** gest that presynaptic events control development of axonal bouton -80 NMDA-induced decrease size and function in multiple neurotransmitter systems. These 0.1s5pA in fluorescence intensity findings implicate presynaptic α7 nAChR/NMDAR interactions in Fig. 6. Presynaptic NMDAR function is enhanced in enlarged presynaptic synaptic development and plasticity. boutons. (A) Examples of NMDA-activated channel currents recorded in As the present data identify changes in both presynaptic outside-out membrane patches excised from enlarged presynaptic boutons NMDAR levels and function induced by α7 blockade, altered in treated cultures. Similar channel currents were recorded upon application presynaptic function may be a direct consequence of α7 nAChR/ of 5 μM NMDA from five distinct terminals, and displayed a chord conduc- glutamate receptor interactions. Because our studies also reveal tance of 58 ± 4 pS. (B) Fura-2 calcium imaging using two-photon microscopy enhancement in presynaptic NMDAR-mediated glutamate release at 780-nm excitation wavelength showed that NMDA stimulation led to in these enlarged boutons, the potential consequences of α7 fl calcium entry as indicated by decreased uorescence intensity in presynaptic blockade may include a variety of downstream events, such as al- boutons of α-BTX–treated cultures. Quantification showed that NMDA stimulation produced a greater decrease in fluorescence intensity in the teration of postsynaptic transmission and synaptic strength (16). presence of tetrodotoxin and calcium channel blockers in boutons of α-BTX– Immunocytochemical studies revealed the presence of NR1, NR2 (n = 48, P < 0.001 vs. control) or cytisine-treated (n = 44, P < 0.001 vs. control) and GluR1 subunits in the enlarged glutamatergic presynaptic cultures compared with those of control cultures (n = 12). (Scale bars, 5 μm.) boutons. Considerable anatomical evidence supports the presence

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1007397107 Lin et al. Downloaded by guest on October 2, 2021 of NR1, NR2, and GluR1 in the presynaptic terminals or boutons in high affinity of nicotine as a α4β2 agonist, and is in accordance different brain regions, especially during early development (16, with previous findings in which the effects of nicotine on NMDAR 23). NR2B is preferentially accumulated in axonal growth cones expression reflect the alterations in postsynaptic NMDARs (37, and varicosities in immature neurons (24), suggesting a role of 38). Still, it is possible that nicotine could create presynaptic ab- presynaptic NMDAR in development. Physiologically, presynaptic normalities as well with longer exposure or exposure at higher NMDARs enhance the probability of spontaneous and evoked concentrations. neurotransmitter release at cortical, hippocampal, and cerebellar As the present data implicate presynaptic α7 nAChR/NMDAR synapses (16, 18–20). Although we identified enhanced presynaptic interactions in synaptic development and plasticity during early fi NMDAR levels and physiological responses, our ndings further development, abnormal presynaptic interactions due to genetic α suggest the selective association of chronic 7 nAChR blockade variations in α7 nAChR may thus produce permanent changes and presynaptic NMDAR function with increases in post- that could contribute to cognition deficits in schizophrenia and synaptically silent synapses, as AMPAR-mediated synaptic cur- Alzheimer’s disease (AD). A convergence of recent genetic evidence rents were not altered, whereas those mediated by NMDARs were fi α fi α identi es 7 nAChR as a potential modi er gene for schizophrenia increased in treated cultures. As 7 nAChRs are preferentially and AD patients (39–41). Similarly, deleting the α7 nAChR gene located at presynaptic terminals incorporated in postsynaptically leads to impairment ofworking/episodic-likememory (42) and alters silent synapses (14, 25), our data suggest that presynaptic α7 the synaptic development and cognition in AD transgenic mice (43, nAChR/NMDAR interactions may be involved in establishing the 44). α7 nAChRs thus are therapeutic targets for cognitive deficits in conversion of silent to fully functional synapses and play crucial – α roles in presynaptic and postsynaptic development. schizophrenia and AD (45 48). Moreover, both 7 nAChR agonists The present data also demonstrate specific structural and func- and antagonists have similar effects on enhancing LTP and cognition α tional changes that could influence synaptic maturation. Cortical in animals (49, 50). Desensitization of 7 nAChRs may play an im- cultures develop in a manner that models synaptic maturation in portant role in both normal information processing and in various vivo. The time course of synapse formation was delayed due to lack disease states and thus be used as a strategy for drug development of glial cells and low density of cortical neurons in our cortical cul- (51, 52). Our findings implicate desensitization of α7nAChRsin tures, and thereby enables us to examine the events occurring in synaptic development and plasticity and thereby provide important bouton formation during early synaptic development. Regulation of insights into development of therapeutic drugs for treatment of α7 nAChR could provide a physiological mechanism that mediates cognitive deficits in schizophrenia and AD.

the development or plasticity of glutamatergic presynaptic boutons. NEUROSCIENCE Structural plasticity of presynaptic boutons has crucial roles in de- Materials and Methods velopmental circuit assembly processes and neural circuit remod- Neuronal Cultures. Primary cortical cultures from E17–E19 rats were prepared, eling in adult cortex (26–29). Axonal varicosities or boutons are maintained, and treated as described in detail in SI Text. Neurobasal medium a series of swellings along the axons, and are typically the sites that for cultures contains choline chloride, a selective agonist at α7 nAChR. In fi release transmitters and form synapses. They are usually divided addition, cholinergic neurons are present in cortical cultures as identi ed by into two subtypes: en passant and terminaux. En passant varicosities choline acetyltransferase (Abcam) immunostaining as described in the manufacturer’s instructions. are swellings in the axonal shaft, whereas terminaux varicosities are swellings at the terminals that connect to the axonal shaft by a short α Two-Photon Calcium Imaging. Cortical neurons were loaded with the calcium neck. Here, chronic inactivation of axonal 7 nAChRs increased the indicator dye 5 μM fura-2-acetoxymethyl ester and subjected to two-photon number and size of glutamatergic presynaptic boutons, particularly imaging as described in detail in SI Text. NMDAR-mediated calcium images those with the appearance of en passant boutons along gluta- were captured and analyzed off-line using ImageJ software (Fiji, an Open matergic axons. Studies in Caenorhabditis elegans and in rodent Source image processing package based on ImageJ), and data were pre- cortical cultures suggest that initial formation of presynaptic ter- sented as relative changes in fluorescence with respect to background minals can occur preferentially at predefined sites within axons in fluorescence (Δf/f). the absence of postsynaptic targets such as neuronal or glial con- tacts, suggesting that many en passant synapses form specifically and Electrophysiology. Whole-cell patch-clamp and outside-out membrane patch autonomously at predefined sites in developing axons (30–32). As recordings were performed as described in detail in SI Text. The frequency of α7 nAChRs are present within these enlarged boutons, and as en AMPAR-mediated mEPSCs was measured, and NMDAR-mediated mEPSCs passant boutons are increased and enlarged upon inactivation of α7 were assessed as synaptic charge from total current integrated during nAChR, axonal α7 nAChRs may be an intrinsic factor within glu- a 1-min time interval, as their long duration and degree of overlap prevented individual detection. Outside-out membrane patches were excised tamatergic axons that modulates the formation of presynaptic fi boutons. Considerable anatomical and functional evidence supports from visually identi ed enlarged boutons. α thepresenceof 7 nAChRs in glutamatergic axonal terminals in ± – α Statistical Analysis. Data are shown as mean SEM. Experiments were analyzed different cortical regions (14, 25, 33 36). Thus, 7maybeastruc- using Student’s t test to compare two conditions or by ANOVA followed by tural determinant for presynaptic development in glutamatergic planned comparisons of multiple conditions. Significance was set at P < 0.05. synapses throughout the brain. fi Although our data focus on modi cations of the presynaptic ACKNOWLEDGMENTS. We thank Margaret Maronski (University of Penn- membrane, there may be postsynaptic interactions of NMDAR sylvania) for cortical neuronal cultures, Dr. Robert Kalb (The Children’s Hos- and nicotinic receptors as well. In our studies, nicotine itself in- pital of Philadelphia) for confocal microscopy, Dr. Guoxiang Xiong (The Children’s Hospital of Philadelphia) for technical suggestions on immunocy- creased NMDAR levels but did not produce notable presynaptic tochemistry, and Dr. Jon Lindstrom (University of Pennsylvania) for α7 cell changes, suggesting that nicotine exposure may increase post- lines. The studies were supported by National Institutes of Health Grant synaptic NMDAR levels. Pharmacologically, this could reflect the NS45986 (to D.R.L) and the CHOP Trisomy 21 Program (to D.R.L.).

1. Waxman EA, Lynch DR (2005) N-methyl-D-aspartate receptor subtypes: Multiple roles 5. Zhang L, Warren RA (2002) Muscarinic and nicotinic presynaptic modulation of EPSCs in excitotoxicity and neurological disease. Neuroscientist 11:37–49. in the nucleus accumbens during postnatal development. J Neurophysiol 88: 2. Dingledine R, Borges K, Bowie D, Traynelis SF (1999) The glutamate receptor ion 3315–3330. channels. Pharmacol Rev 51:7–61. 6. Liechti ME, Markou A (2008) Role of the glutamatergic system in nicotine 3. Placzek AN, Zhang TA, Dani JA (2009) Age dependent nicotinic inﬂuences over dependence: Implications for the discovery and development of new pharmacological dopamine neuron synaptic plasticity. Biochem Pharmacol 78:686–692. smoking cessation therapies. CNS Drugs 22:705–724. 4. Nashmi R, Lester HA (2006) CNS localization of neuronal nicotinic receptors. J Mol 7. Dani JA, Bertrand D (2007) Nicotinic acetylcholine receptors and nicotinic cholinergic Neurosci 30:181–184. mechanisms of the central nervous system. Annu Rev Pharmacol Toxicol 47:699–729.

Lin et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 8. O’Leary KT, Leslie FM (2003) Developmental regulation of nicotinic acetylcholine 32. Shen K, Fetter RD, Bargmann CI (2004) Synaptic specificity is generated by the receptor-mediated [3H]norepinephrine release from rat cerebellum. J Neurochem 84: synaptic guidepost protein SYG-2 and its receptor, SYG-1. Cell 116:869–881. 952–959. 33. Ge S, Dani JA (2005) Nicotinic acetylcholine receptors at glutamate synapses facilitate 9. Dajas-Bailador FA, Lima PA, Wonnacott S (2000) The alpha7 nicotinic acetylcholine long-term depression or potentiation. J Neurosci 25:6084–6091. receptor subtype mediates nicotine protection against NMDA excitotoxicity in primary 34. Jones IW, Wonnacott S (2004) Precise localization of α7 nicotinic acetylcholine hippocampal cultures through a Ca(2+) dependent mechanism. Neuropharmacology receptors on glutamatergic axon terminals in the rat ventral tegmental area. 39:2799–2807. J Neurosci 24:11244–11252. 10. Jonnala RR, Buccafusco JJ (2001) Relationship between the increased cell surface 35. Zhong C, et al. (2008) Presynaptic type III neuregulin 1 is required for sustained alpha7 nicotinic receptor expression and neuroprotection induced by several nicotinic enhancement of hippocampal transmission by nicotine and for axonal targeting of receptor agonists. J Neurosci Res 66:565–572. alpha7 nicotinic acetylcholine receptors. J Neurosci 28:9111–9116. 11. Huang YY, Kandel ER, Levine A (2008) Chronic nicotine exposure induces a long- 36. Hancock ML, Canetta SE, Role LW, Talmage DA (2008) Presynaptic type III lasting and pathway-specific facilitation of LTP in the amygdala. Learn Mem 15: neuregulin1-ErbB signaling targets alpha7 nicotinic acetylcholine receptors to axons. 603–610. J Cell Biol 181:511–521. 12. De Filippi G, Baldwinson T, Sher E (2005) Nicotinic receptor modulation of 37. Yamazaki Y, Jia Y, Niu R, Sumikawa K (2006) Nicotine exposure in vivo induces long- – neurotransmitter release in the cerebellum. Prog Brain Res 148:307 320. lasting enhancement of NMDA receptor-mediated currents in the hippocampus. Eur 13. Girod R, Barazangi N, McGehee D, Role LW (2000) Facilitation of glutamatergic J Neurosci 23:1819–1828. neurotransmission by presynaptic nicotinic acetylcholine receptors. Neuropharmacology 38. Yamazaki Y, Jia Y, Wong JK, Sumikawa K (2006) Chronic nicotine-induced switch in – 39:2715 2725. Src-family kinase signaling for long-term potentiation induction in hippocampal CA1 14. Aramakis VB, Metherate R (1998) Nicotine selectively enhances NMDA receptor- pyramidal cells. Eur J Neurosci 24:3271–3284. mediated synaptic transmission during postnatal development in sensory neocortex. 39. Heinzen EL, et al. (2010) Genome-wide scan of copy number variation in late-onset – J Neurosci 18:8485 8495. Alzheimer’s disease. J Alzheimers Dis 19:69–77. 15. Rezvani K, Teng Y, Shim D, De Biasi M (2007) Nicotine regulates multiple synaptic 40. Sinkus ML, et al. (2009) A 2-base pair deletion polymorphism in the partial duplication – proteins by inhibiting proteasomal activity. J Neurosci 27:10508 10519. of the alpha7 nicotinic acetylcholine gene (CHRFAM7A) on chromosome 15q14 is 16. Corlew R, Brasier DJ, Feldman DE, Philpot BD (2008) Presynaptic NMDA receptors: associated with schizophrenia. Brain Res 1291:1–11. Newly appreciated roles in cortical synaptic function and plasticity. Neuroscientist 14: 41. Carson R, et al. (2008) Genetic variation in the alpha 7 nicotinic acetylcholine receptor 609–625. is associated with delusional symptoms in Alzheimer’s disease. Neuromolecular Med 17. Corlew R, Wang Y, Ghermazien H, Erisir A, Philpot BD (2007) Developmental switch in 10:377–384. the contribution of presynaptic and postsynaptic NMDA receptors to long-term 42. Fernandes C, Hoyle E, Dempster E, Schalkwyk LC, Collier DA (2006) Performance depression. J Neurosci 27:9835–9845. deficit of alpha7 nicotinic receptor knockout mice in a delayed matching-to-place 18. Fiszman ML, et al. (2005) NMDA receptors increase the size of GABAergic terminals task suggests a mild impairment of working/episodic-like memory. Genes Brain and enhance GABA release. J Neurosci 25:2024–2031. Behav 5:433–440. 19. Madara JC, Levine ES (2008) Presynaptic and postsynaptic NMDA receptors mediate 43. Dziewczapolski G, Glogowski CM, Masliah E, Heinemann SF (2009) Deletion of the α7 distinct effects of brain-derived neurotrophic factor on synaptic transmission. nicotinic acetylcholine receptor gene improves cognitive deficits and synaptic J Neurophysiol 100:3175–3184. pathology in a mouse model of Alzheimer’s disease. J Neurosci 29:8805–8815. 20. Grilli M, et al. (2009) Exposure to an enriched environment selectively increases the 44. Hernandez CM, Kayed R, Zheng H, Sweatt JD, Dineley KT (2010) Loss of alpha7 functional response of the pre-synaptic NMDA receptors which modulate nor- nicotinic receptors enhances beta-amyloid oligomer accumulation, exacerbating adrenaline release in mouse hippocampus. J Neurochem 110:1598–1606. early-stage cognitive decline and septohippocampal pathology in a mouse model of 21. Varas R, et al. (2006) Electrophysiological characterization of nicotinic acetylcholine ’ – receptors in cat petrosal ganglion neurons in culture: Effects of cytisine and its bromo Alzheimer s disease. J Neurosci 30:2442 2453. derivatives. Brain Res 1072:72–78. 45. Chen L, Yamada K, Nabeshima T, Sokabe M (2006) alpha7 Nicotinic acetylcholine fi 22. Petralia RS, et al. (2010) Organization of NMDA receptors at extrasynaptic locations. receptor as a target to rescue de cit in hippocampal LTP induction in beta-amyloid – Neuroscience 167:68–87. infused rats. Neuropharmacology 50:254 268. 23. Schenk U, Matteoli M (2004) Presynaptic AMPA receptors: More than just ion 46. Thomsen MS, Christensen DZ, Hansen HH, Redrobe JP, Mikkelsen JD (2009) alpha(7) channels? Biol Cell 96:257–260. Nicotinic acetylcholine receptor activation prevents behavioral and molecular 24. Herkert M, Röttger S, Becker CM (1998) The NMDA receptor subunit NR2B of changes induced by repeated phencyclidine treatment. Neuropharmacology 56: – neonatal rat brain: Complex formation and enrichment in axonal growth cones. Eur 1001 1009. J Neurosci 10:1553–1562. 47. Leiser SC, Bowlby MR, Comery TA, Dunlop J (2009) A cog in cognition: How the alpha 25. Metherate R, Hsieh CY (2003) Regulation of glutamate synapses by nicotinic 7 nicotinic acetylcholine receptor is geared towards improving cognitive deficits. acetylcholine receptors in auditory cortex. Neurobiol Learn Mem 80:285–290. Pharmacol Ther 122:302–311. 26. Majewska AK, Sur M (2006) Plasticity and specificity of cortical processing networks. 48. Hajós M, Rogers BN (2010) Targeting alpha7 nicotinic acetylcholine receptors in the Trends Neurosci 29:323–329. treatment of schizophrenia. Curr Pharm Des 16:538–554. 27. Gogolla N, Galimberti I, Caroni P (2007) Structural plasticity of axon terminals in the 49. Arendash GW, Sengstock GJ, Sanberg PR, Kem WR (1995) Improved learning and adult. Curr Opin Neurobiol 17:516–524. memory in aged rats with chronic administration of the nicotinic receptor agonist 28. De Paola V, et al. (2006) Cell type-specific structural plasticity of axonal branches and GTS-21. Brain Res 674:252–259. boutons in the adult neocortex. Neuron 49:861–875. 50. Fujii S, Ji Z, Sumikawa K (2000) Inactivation of alpha7 ACh receptors and activation of 29. Stettler DD, Yamahachi H, Li W, Denk W, Gilbert CD (2006) Axons and synaptic non-alpha7 ACh receptors both contribute to long term potentiation induction in the boutons are highly dynamic in adult visual cortex. Neuron 49:877–887. hippocampal CA1 region. Neurosci Lett 286:134–138. 30. Sabo SL, Gomes RA, McAllister AK (2006) Formation of presynaptic terminals at 51. Quick MW, Lester RA (2002) Desensitization of neuronal nicotinic receptors. predefined sites along axons. J Neurosci 26:10813–10825. J Neurobiol 53:457–478. 31. Martín-Peña A, et al. (2006) Age-independent synaptogenesis by phosphoinositide 52. Buccafusco JJ, Beach JW, Terry AV, Jr (2009) Desensitization of nicotinic acetylcholine 3 kinase. J Neurosci 26:10199–10208. receptors as a strategy for drug development. J Pharmacol Exp Ther 328:364–370.

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