The effect of α7 nicotinic receptor activation on glutamatergic transmission in the hippocampus

Abstract

Nicotinic acetylcholine receptors (nAChRs) are expressed widely in the CNS, and mediate both synaptic and perisynaptic activities of endogenous cholinergic inputs and pharmacological actions of exogenous compounds (e.g. nicotine and choline). Behavioral studies indicate that nicotine improves such cognitive functions as learning and memory, however the cellular mechanism of these actions remains elusive. With help from newly developed biosensors and optogenetic tools, recent studies provide new insights on signaling mechanisms involved in the activation of nAChRs. Here we will review α7 nAChR’s action in the tri-synaptic pathway in the hippocampus. The effects of α7 nAChR activation via either exogenous compounds or endogenous cholinergic innervation are detailed for spontaneous and evoked glutamatergic synaptic transmission and synaptic plasticity, as well as the underlying signaling mechanisms. In summary, α7 nAChRs trigger intracellular calcium rise and calcium-dependent signaling pathways to enhance glutamate release and induce glutamatergic synaptic plasticity.

Graphical abstract

1. Introduction

Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of cys-loop cationic ligand-gated channels (Jones et al., 1999; Dajas-Bailador & Wonnacott, 2004; Dani & Bertrand, 2007; Albuquerque et al., 2009a). They are activated by the endogenous ligand acetylcholine (ACh) and various exogenous ligands (e.g. nicotine and choline) to exert their modulatory function on synaptic excitability and plasticity (Dani & Bertrand, 2007; Yakel, 2012; 2013). The α7 nAChR subtype is prominent in the hippocampus, and has been linked with cognitive deficits and a variety of neurological disorders and diseases, such as Alzheimer's diseases and schizophrenia (Freedman & Goldowitz, 2010; Yakel, 2013; Dineley et al., 2015; Sadigh-Eteghad et al., 2015; Sinkus et al., 2015a). Schizophrenic patients are often heavy smokers (de Leon & Diaz, 2005), which has been suggested to be a form of self-medication to mitigate their cognitive dysfunctions (Levin et al., 1996). The β-amyloid peptide (Aβ_1–42 or Aβ peptide), a pathological hallmark of Alzheimer's disease, has been shown to affect α7 nAChR’s function (Wang et al., 2000; Liu et al., 2001; Pettit et al., 2001; Lamb et al., 2005). Moreover, significant loss of α7 nAChR protein density was observed in the hippocampus of both traumatic brain injury and prenatal restraint stress animal models (Hoffmeister et al., 2011; Baier et al., 2015). Either activation of the α7 nAChR with agonists, or potentiation with positive allosteric modulators (PAMs) (e.g. NS-1738), has been shown to improve hippocampal-dependent learning and memory (Bitner et al., 2007; Timmermann et al., 2007; Yaguchi et al., 2009; Blake et al., 2014). Conversely, genetic deletion and pharmacological inhibition of the α7 nAChR in the hippocampus results in significant learning and memory impairments (Levin et al., 2002; Fernandes et al., 2006; Nott & Levin, 2006; Young et al., 2007; Levin et al., 2009), and worsens the learning and memory deficits in a mouse model of Alzheimer’s disease (Hernandez et al., 2010). Therefore, it is critical to understand the functional properties of nAChRs in regulating hippocampal circuitry and synaptic plasticity. Below, we will briefly review recent advances, with a particular focus on α7 nAChRs (although α4β2 nAChRs also play an important role), in the regulation of glutamatergic synaptic transmission in the hippocampal formation.

2. α7 nAChR properties and expression in the hippocampus

The nAChRs are widely expressed throughout the brain in both neurons and non-neuronal cells, and participate in a variety of physiological functions (McQuiston & Madison, 1999; Nashmi & Lester, 2006; Shen & Yakel, 2012). In the mammalian brain, there are six α (2 – 10) and three β (2 – 4) nAChR subunits, which have been shown to form either hetero- or homopentameric nAChRs (Wada et al., 1989; Sargent, 1993; Jones & Yakel, 1999; Alkondon & Albuquerque, 2004; Albuquerque et al., 2009b). The most prevalent nAChRs in the hippocampus are comprised of α7 and α4β2 subtypes (Jones et al., 1999; Alkondon & Albuquerque, 2004; Albuquerque et al., 2009a). While α7 subunits were initially thought to form mainly homopentameric α7 receptors, they have been shown to co-assemble with other subunits to form functionally distinct native α7-containing receptors (Anand et al., 1993; Yu & Role, 1998; Palma et al., 1999; Shao & Yakel, 2000; Liu et al., 2009; Murray et al., 2012; Mowrey et al., 2013). Initially, we found that α7 and β2 subunits co-assembled in vitro (Khiroug et al., 2002); subsequently it was found that basal forebrain cholinergic neurons express functional native α7β2 receptors with an enhanced sensitivity to the amyloid-β (Aβ) peptide associated with AD (Liu et al., 2009). Physiological studies have shown that α7 nAChRs have lower affinity for acetylcholine, faster desensitization kinetics, and higher calcium permeability than α4β2 receptors (Bertrand et al., 1993; Fayuk & Yakel, 2005; Albuquerque et al., 2009a). Because of its high calcium permeability, α7 nAChRs can function similar to NMDA receptors in the modulation of synaptic plasticity.

In the hippocampal formation, distribution of mRNA for α7 nAChRs, as well as α-bungarotoxin (α-BTX) binding sites (which label α7 nAChRs), are widespread throughout the dentate gyrus (DG), CA3 and CA1 regions (Sudweeks & Yakel, 2000; Fabian-Fine et al., 2001; Adams et al., 2002b). Functional α7 nAChR-mediated currents have also been reported in CA3 and CA1 pyramidal neurons (Ji et al., 2001; Grybko et al., 2011; Gu et al., 2012), mouse dentate granule cells (John et al., 2015) and GABAergic interneurons (Jones & Yakel, 1999). Besides neuronal expression, α7 nAChRs are expressed on non-neuronal cells such as astrocytes in the hippocampal CA1 region (Shen & Yakel, 2012), which could play a role in neuroprotection and inflammation (Shen & Yakel, 2009). Ultrastructural studies in the CA1 region revealed immuno-gold particle labeling of α7 nAChRs at both pre- and postsynaptic compartments of both GABAergic and glutamatergic synapses (Fabian-Fine et al., 2001). α7 nAChRs located on presynaptic terminals can regulate release of other neurotransmitters (Jones et al. 1999; Dajas-Bailador & Wonnacott, 2004; Dani & Bertrand, 2007), while α7 nAChRs located on postsynaptic membranes mediate cholinergic synaptic transmission. Taking advantage of their high calcium permeability, functional α7 nAChRs can be mapped with sensitive calcium dyes and genetically-encoded calcium indicators, which provide complimentary information as to the locations of functional receptors beyond the reach of traditional patch-clamp studies. The calcium rise mediated by functional α7 nAChRs has been shown in dendrites of CA1 (Fayuk & Yakel, 2005; 2007a) and CA3 pyramidal neurons (Grybko et al., 2011), and dentate granule cells at their mossy fiber terminals (Cheng & Yakel, 2014). Optical recordings revealed that the amplitude of the α7 nAChR-mediated calcium response was significantly larger in the dendrites (than in the soma) despite smaller α7 nAChR current responses (Fayuk & Yakel, 2007a), suggesting that functional α7 nAChRs cluster differentially around neuronal compartments (Kawai et al., 2002). With the aid of newly developed α7 PAMs, we observed α7 nAChR-mediated calcium rise via GCaMP3 imaging in giant mossy fiber terminals (Cheng & Yakel, 2014), which is consistent with the study using the calcium dye Fura-2 (Gray et al., 1996). In addition, α7 nAChRs also distribute perisynaptically to mediate modulating effects of non-synaptically released ACh (Kasa et al., 1995; Dani & Bertrand, 2007; Albuquerque et al., 2009a; Shen & Yakel, 2009). The density and distribution of α7 nAChRs changes markedly during early development (Adams et al., 2002a; Adams, 2003), and differs among different mouse strains and species (Rubboli et al., 1994; Adams et al., 2006). Moreover, genetic polymorphism of the α7 nAChR gene (CHRNA7) linked to schizophrenia generated a partially duplicated α7 nAChR (CHRFAMA7A, dupα7) subunit either with or without a 2-bp deletion (CHRFAMA7A-Δ2bp, dupΔα7) (Gault et al., 2003; Sinkus et al., 2009), which could change the properties of the α7 nAChR and modify neuronal connections (Sinkus et al., 2015b). An in vitro study showed that both dupα7 and dupΔα7 co-assemble with the α7 subunit to form functional receptors with normal sensitivity to ACh and lower sensitivity to choline, though the calcium permeability of these mutated α7 receptors remains unclear (Wang et al., 2014).

3. The effect of α7 nAChR’s activation by exogenous agonists

Many groups have studied how α7 nAChR activation modulates glutamatergic synaptic transmission with nicotine or selective agonists to activate α7 nAChRs in the hippocampus. We will focus mainly on the direct α7 nAChR-mediated effect on glutamatergic synapses, and not the indirect impact derived from α7 nAChR activation on GABAergic neurons, which also can modulate synaptic plasticity in pyramidal cells.

For spontaneous glutamate release, acute nicotine application increased the frequency of spontaneous miniature EPSCs from dissociated hippocampal neurons (Gray et al., 1996; Radcliffe & Dani, 1998), and CA1 (Le Magueresse et al., 2006) and CA3 pyramidal neurons (Sharma & Vijayaraghavan, 2003); this suggests a presynaptic location of nicotine’s action. The nicotine-induced frequency increase of mEPSCs was blocked by MLA (an α7-selective antagonist) (Gray et al., 1996), and absent in α7 knockout mice (Le Magueresse et al., 2006), suggesting that α7 nAChRs mediated this enhancement. Moreover, a brief application of nicotine converted presynaptically silent synapses into conductive ones at immature Schaffer collateral-CA1 connections (Maggi et al., 2003). Besides its modulatory effect on mEPSC frequency, activation of α7 nAChRs could directly trigger glutamate release from mossy fiber terminals (Sharma et al., 2008). The direct measurement of glutamate release from hippocampal synaptosomes showed significant release upon choline application (Zappettini et al., 2010), which is consistent with electrophysiological findings. In vivo extracellular recordings from CA3 pyramidal neurons showed robust increases in firing induced by nicotine and blocked by MLA; this was found to be due to enhanced glutamate release (Huang et al., 2010). The α7 nAChR’s effect on spontaneous glutamate release occurred within seconds after agonist application and lasted for several minutes after agonist removal, suggesting that both immediate calcium rise through α7 nAChR activation and longer-term calcium-dependent signaling cascades are involved.

For the evoked release of glutamate, α7 nAChR activation can increase the amplitude of evoked EPSCs in cultured hippocampal neurons (Radcliffe & Dani, 1998), for CA3 to CA1 pyramidal neuron synapses, DG to CA3 synapses, and perforant path to DG glutamate synapses (Sola et al., 2006; Ondrejcak et al., 2012; Cheng & Yakel, 2014). The increase in amplitude was associated with a significant increase in the probability of release along with a significant decrease of the PPR (paired-pulse ratio) (Sola et al., 2006). Our study (Cheng & Yakel, 2014) of CA3 pyramidal neurons showed that α7 nAChR activation enhanced eEPSC amplitude, which persisted for 5-10 minutes after the removal of agonist. Similar long-lasting effects of nAChR agonists on neurotransmitter release were observed by others as well (Lena & Changeux, 1997; Zhong et al., 2008). Studies from both spontaneous and evoked glutamatergic synaptic transmission showed that presynaptic α7 nAChRs mediated enhancement of glutamate release, suggesting that α7 nAChRs could be activating the same calcium-dependent signaling cascades to sustain the nicotinic enhancement of glutamate release. In the prefrontal cortex, nicotine induced an increase in frequency and amplitude of spontaneous EPSCs, but did not affect the amplitude of evoked glutamatergic transmission from layer II/III to layer V pyramidal neurons (Couey et al., 2007), suggesting that multiple calcium-dependent pathways could be involved in its action.

Hippocampal LTP is a well-accepted cellular model for learning and memory. The effect of α7 nAChR’s activation on synaptic plasticity is dictated by the timing of its activation and electrical stimulation on glutamatergic synaptic currents. When α7 nAChR agonists were bath perfused, the activation of α7 nAChRs facilitated the induction of LTP (Hunter et al., 1994; Fujii et al., 1999), and enhanced both early and late LTP (Kroker et al., 2011) in the CA1 hippocampal region. When α7 nAChRs were activated via brief application of ACh, which coincided or preceded (by 1-5 s) high frequency stimulation (HFS), this produced LTP under mild presynaptic stimulation conditions at CA1 pyramidal neurons (Ji, 2001). Outside of these time frames, the mismatch of nAChR activity and stimulation led to short-term potentiation (STP) (Ge & Dani, 2005). The α7 nAChR-mediated enhancement of LTP was due to its ability to increase presynaptic release of glutamate and postsynaptic depolarization. Activation of α7 nAChRs has also been shown to suppress LTD induction at CA3-CA1 synapses (Fujii & Sumikawa, 2001; Nakauchi & Sumikawa, 2014). In the DG, acute α7 nAChR activation enhanced HFS-induced LTP, and α7 nAChR PAMs reduced the threshold for LTP induction, an effect which required ERK and PKA activity (Welsby et al., 2009). For mossy fiber LTP, α7 nAChR agonists rescued LTP that was deficient in BACE1 knock-out mice, but had very little effect on LTP in wildtype mice (Wang et al., 2010). The rescue by α7 nAChR agonists was abolished by blockers of ryanodine-sensitive calcium stores, suggesting that calcium-induced calcium release played a key role in this regulation. However, the effect of α7 nAChR activation on STP needs to be tested on mossy fiber transmission. These studies also pointed to a calcium rise induced by α7 nAChR activation as the central mechanism for these actions, and α7 nAChR activation also triggers differential signaling molecules to produce various influences in the hippocampus.

4. α7 activation via cholinergic synapses in the hippocampus

Under physiological conditions, α7 nAChRs are activated by endogenous ACh released from cholinergic neurons. The primary cholinergic input to the hippocampus arises from the medial septum and diagonal band of Broca (Amaral & Kurz, 1985; Dutar et al., 1995), although a small population of cholinergic interneurons do exist in the hippocampus as recently reconfirmed from ChAT-tauGFP and ChAT-CRE/Rosa-YFP transgenic mice (Yi et al., 2015). Initial studies using electrical cholinergic stimulation to dissect the function of endogenous ACh showed prominent muscarinic receptor’s effect of Schaffer collateral input to CA1 pyramidal neurons (Fernandez de Sevilla & Buno, 2003). The α7 nAChR-dependent modulation of synaptic plasticity of CA3-CA1 glutamatergic synapses was uncovered with localized application of ACh, which is sensitive to the location and timing of α7 nAChR activation (Ji et al., 2001; Ge & Dani, 2005). The development of optogenetic techniques allows both cell-type specific and temporally precise stimulation of cholinergic inputs to the hippocampus. Taking advantage of this approach, our lab investigated how activation of endogenous cholinergic inputs may regulate hippocampal synaptic plasticity (Gu & Yakel, 2011). We found that α7 nAChRs mediated two forms of hippocampal synaptic plasticity with a timing precision in the millisecond range. When the cholinergic input to the CA1 hippocampal region was activated 100 ms prior to activation of the Schaffer collateral (SC) pathway, this induced an α7 nAChR-dependent form of LTP. When the cholinergic input was activated only 10 ms prior to the SC pathway, this induced an α7 nAChR-dependent short-term depression (STD) that was mediated primarily through the presynaptic inhibition of glutamate release. Furthermore, we observed α7 nAChR-dependent enhancement of calcium responses during this LTP both pre- and post synaptically, and short-term depression of calcium responses during STD (Gu et al., 2012). In aged α7-nAChR KO mice, both evoked field synaptic potentials and LTP were significantly reduced in hippocampal CA3-CA1 synapses (Ma et al., 2014), suggesting that cholinergic modulation of LTP required the activation of α7 nAChRs.

5. Second messenger signaling upon activation of α7 nAChRs-

The α7 nAChRs have a high calcium permeability, comparable to that of NMDA receptors (Seguela et al., 1993; Uteshev, 2012); this confers the ability of α7 nAChRs to produce long-lasting modulations of synaptic transmission (Fagen et al., 2003; McKay et al., 2007). The intracellular calcium rise alters the amount of neurotransmitter release directly (Neher & Sakaba, 2008), and triggers multiple signaling cascades to modify the efficacy of synaptic transmission. Indeed, we observed a prominent calcium rise upon activation of α7 nAChRs at mossy fiber terminals (using the genetically-encoded calcium sensor GCaMP3), and found that the peak of the action potential (AP)-evoked calcium transient was enhanced by the α7 nAChR agonist PNU-282987 (Cheng & Yakel, 2014; 2015). Similar actions of presynaptic α7 nAChRs on AP-evoked calcium transients were also reported in CA1 pyramidal neurons (Szabo et al., 2008). Changes in the AP-induced calcium transients would lead to changes in the amount of neurotransmitter released at presynaptic terminals (Jackson et al., 1991). The source of the calcium increase would be mainly comprised of the calcium influx directly through the α7 nAChR itself (Fayuk & Yakel, 2007a; Gilbert et al., 2009), and any calcium-induced calcium release from intracellular calcium stores (Weisskopf & Nicoll, 1995; Sharma & Vijayaraghavan, 2003; Sharma et al., 2008). Although calcium influx through voltage-gated calcium channels (VGCC) previously was shown not to contribute significantly to the α7 nAChR-mediated presynaptic effect (Dickinson et al., 2008), other studies reported that calcium influx through VGCCs could contribute to some degree (Barrantes et al., 1995; Fayuk & Yakel, 2007b; del Barrio et al., 2011). In addition, recent studies showed that α7 nAChR activation enhanced NMDAR-mediated whole-cell currents and glutamate release from hippocampal synaptosomes (Zappettini et al., 2010; Li et al., 2013). Lastly, membrane depolarization induced by α7 nAChR activation could release the magnesium blockade of NMDARs to reduce the threshold for NMDAR-dependent synaptic plasticity.

Although activation of the α7 nAChRs induces transient calcium rises, the kinetics of this do not explain the long-lasting effects caused by α7 nAChR agonists on synaptic plasticity (Lena & Changeux, 1997; Zhong et al., 2008; Zhong et al., 2013). Many calcium signaling cascades have been linked to α7 nAChRs, including the CaMKII (Sharma et al., 2008) and cAMP-PKA pathways (Dajas-Bailador et al., 2002; Welsby et al., 2009). Recently, we reported that the action of α7 nAChRs at mossy fiber terminals depended on PKA activity (Cheng & Yakel, 2014). Since it was shown that the α7 nAChR and adenylyl cyclase 1 (AC1) are associated physically and functionally in epithelium (Maouche et al., 2013), we investigated any connection between the activation of the α7 nAChR and cAMP signaling in hippocampal neurons. Using a FRET-based cAMP sensor, we found that activation of α7 nAChRs significantly increased intracellular cAMP levels. The cAMP rise was abolished by the α7 nAChR antagonist MLA, the calcium chelator BAPTA, the selective AC1 inhibitor CB-6673567, and siRNA-mediated AC1 knockdown; this suggested that the calcium-dependent AC1 is required for linking the activation of α7 nAChRs to a cAMP rise (Cheng & Yakel, 2015). We also found that the activation of α7 nAChRs leads to the widespread increase of phosphorylated synapsin I (via its PKA site), which is a prominent presynaptic PKA substrate (Huttner & Greengard, 1979) and can regulate neurotransmitter release and calcium-dependent synaptic plasticity (Hilfiker et al., 1999; Hilfiker et al., 2005; Menegon et al., 2006; Fiumara et al., 2007; Cousin & Evans, 2011). The PKA pathway was also shown to be required for the α7 nAChR-mediated enhancement of LTP in dentate granule cells (Welsby et al., 2009). Moreover, α7 nAChR activation was shown to interact with G proteins and G protein-regulated inducer of neurite outgrowth 1 to reduce axonal growth (Nordman & Kabbani, 2014; Nordman et al., 2014). With the advancement of new technologies, novel signaling pathways might be linked to α7 nAChRs to interpret its complex actions in the brain.

6.Significance and conclusion

The changes in α7 nAChR expression and density in the hippocampus have been observed in many cognitive impairment disorders, such as schizophrenia (Freedman & Goldowitz, 2010) and Alzheimer’s disease (Dani & Bertrand, 2007; Dineley et al., 2015; Sadigh-Eteghad et al., 2015). However, the physiological mechanism and consequences of these changes are still unclear, in terms of how these receptors regulate neuronal excitability, synaptic plasticity and brain circuit function. Furthermore, the outcome of α7 nAChR activation on the hippocampal circuit is complicated by the extensive distribution of α7 nAChRs on both glutamatergic and GABAergic neurons. With the newly-generated floxed α7 nAChR transgenic mouse (Hernandez et al., 2014) combining with Cre and Cre-ER dependent systems, selective deletion of α7 nAChRs from distinct cell populations at specific developmental stages will allow us to unravel systematically the impact of α7 nAChR activation on hippocampal network both in vitro and in vivo. This will also help us to understand how the alteration of α7 nAChR function will impact cognitive function.

The agonists and modulators of α7 nAChRs have been attractive candidates for developing therapeutic treatments for these neuronal disorders (Deutsch et al., 2013; Deutsch et al., 2014). Indeed, several drugs targeting α7 nAChRs demonstrated their potential in treating schizophrenic patients (Freedman et al., 2008; Preskorn et al., 2014; Keefe et al., 2015). With the development of improved biosensors and emerging tools such as optogenetics (combined with electrophysiology), we will continue to probe cellular and molecular mechanisms of α7 nAChR’s functions in neural circuits. The insights gained from these studies will lead to new treatments to mitigate cognitive impairments.

Figure 1.

Mechanisms of α7 nAChR-mediated presynaptic enhancement of glutamate release. The intracellular calcium level at presynaptic terminals is increased by calcium influx through α7 nAChRs, VGCCs, and NMDARs from the outside, and calcium release from intracellular calcium stores. The raised calcium levels could increase vesicular glutamate release directly, and trigger cAMP-PKA-dependent pathways for prolonged enhancement of glutamate release. Phosphorylation of synapsins by PKA leads to synaptic vesicle mobilization from the reserve pool to the readily releasable pool, hence increasing glutamate release. (Purple trapeziods depict unphosphorylated synapsins, while red trapezoids depict phosphorylated synapsins.)

Abbreviations

ACh

acetylcholine

DG

dentate gyrus

HFS

high frequency stimulation

LTD

long-term depression

LTP

long-term potentiation

nAChR

nicotinic ACh receptor

STP

short-term potentiation

PAM

positive allosteric modulator