Abstract

Neuronal nicotinic acetylcholine receptors

Receptor structure

Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central nervous system [1]. Each receptor/channel consists of five subunits surrounding a water-filled, cation-permeable pore [2, 3] (Fig. 1). Each nAChR subunit has a similar general linear structure and transmembrane topology. The nAChR subunits have a large extracellular N-terminal domain that contributes to ligand binding, followed by three hydrophobic transmembrane regions (M1 to M3), a large intracellular loop, a fourth transmembrane region (M4), and finally a short extracellular C-terminus (Fig. 1A) [4]. The M2 transmembrane segment in each subunit provides the main lining of the ionic pore with some contribution from M1. The M1, M3 and M4 segments separate the pore-lining region from the hydrophobic membrane [5]. Subunits α2 through α10 have been cloned and are homologous to the α1 subunit identified in muscle [6–9]. The neuronal non-α subunits, β2 through β4, have also been identified [6–9]. Many neuronal nAChRs are assembled as αβ hetero-oligomers with a typical α:β stoichiometry of 2:3. In brain, the most common hetero-oligomeric nAChRs are assembled from α4 and β2 subunits (Fig. 1B) [3, 10, 11]. More complex hetero-oligomeric compositions are also possible, such as the α4α6β2 nAChRs commonly distributed within midbrain dopamine areas. Homo-oligomeric nAChRs are only formed by α7, α8 or α9 subunits, with α7 homo-oligomers being the only ones widely distributed in the brain (Fig. 1B) [10, 12, 13]. Accumulating evidence reveals that α7-containing (α7*) and β2-containing (β2*) nAChRs are expressed at preterminal, axonal, somatic and dendritic locations [10, 14–20].

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Fig. 1

Transmembrane topology and pentameric structure of nAChRs. (A) nAChRs consist of four transmembrane domains (M1 through M4) with extracellular C- and N-termini. (B) Subunits are assembled into pentamers that include a water-filled cation-permeable pore. The most common nAChRs in the brain are hetero-oligomeric α4β2 nAChRs and homo-oligomeric α7 nAChRs. The recognized ACh binding sites are indicated by filled black squares.

Neuronal nAChRs facilitate presynaptic neurotransmitter release

Neurotransmitters are released from presynaptic terminals, a process that typically requires an action potential to invade and depolarize the terminal. Depolarization activates voltage-gated Ca²⁺ channels (VGCCs), and the intra-terminal Ca²⁺ increase triggers neurotransmitter release. Vesicular release can also occur stochastically in an action potential-independent manner. Because nAChRs are expressed presynaptically and because the cationic current through nAChRs can both depolarize membranes and raise intracellular Ca²⁺ levels, nAChRs influence neurotransmitter release.

Nicotinic AChRs regulate hippocampal synaptic plasticity in vitro

The hippocampus is central to learning, memory, and attention mechanisms, and nAChRs contribute to these functions [6, 70–77]. For instance, local application of nAChR antagonists impairs working but not reference memory [76], whereas working memory deficits are reversed by nicotine following cholinergic denervation of the hippocampus [77]. The hippocampus receives cholinergic input from the septum-diagonal band complex, whose fibers project to all regions including the dentate gyrus, CA3 and CA1, and to most cell types, including pyramidal cells, granule cells, interneurons, and hilar neurons [78, 79]. Targeted neurons robustly express nAChRs, in particular α7* nAChRs, but also β2* nAChRs [10, 16, 18–20]. Small nicotinic receptor-mediated currents have been measured in pyramidal neurons and substantially greater nicotinic currents have been measured in interneurons [6, 25, 42, 44, 80–84], with volume transmission also present [47]. In fact, α7* nAChRs are commonly found at both presynaptic and postsynaptic sites in the hippocampus CA1 region as identified with immunogold labeling and electron microscopy [16]. Remarkably, the density of nAChRs is similar to the densities of both NMDARs and AMPARs at these synapses [85].

Timing of nAChR activation regulates hippocampal synaptic plasticity

In one set of experiments, STP was induced at Schaffer collateral–CA1 synapses in acutely prepared hippocampal slices while varying the timing of nicotine application relative to afferent stimulation (Fig. 2A–F) [92]. STP was induced by 1 sec of 100 Hz stimulation paired with 100 pA depolarization of CA1 pyramidal neurons and persisted for ~20 min. Brief (0.5 to 1 sec) puffs of ACh (1 mM, applied in the presence of atropine to block muscarinic AChRs) were delivered to the pyramidal cells and evoked action potential (AP) discharge. One of three outcomes was obtained that depended on the timing of the last ACh-induced AP relative to afferent stimulation [92]. First, if the last ACh-induced AP preceded HFS by more than 5 sec (Fig. 2B, F), or trailed HFS (Fig. 2E, F), then STP was unaltered. Second, if the last ACh-induced AP preceded HFS by 1–5 sec, LTP was produced that persisted for at least one hour (Fig. 2C, F). Third, if the last ACh-induced AP occurred <1 sec prior to HFS, then LTD resulted (Fig. 2D, F). Mimicking ACh’s postsynaptic depolarization with direct current injection failed to boost STP to LTP, revealing an obligatory requirement for nAChR activation.

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Fig. 2

Temporal-dependence of nAChR activation for hippocampal synaptic plasticity. (A) Illustration of the experimental setup for experiments shown in panels (B) through (E). Whole-cell patch recordings were obtained from CA1 pyramidal somata, with activation of Schaffer collateral afferents in stratum radiatum. A puffer pipette delivered brief pulses of ACh (in the presence of atropine) to the pyramidal neuron’s dendrites. All postsynaptic potentials (PSPs) were normalized to baseline. (B) ACh-induced APs that preceded HFS of the Schaffer collaterals by ≥ 10 sec did not affect STP. (C) In contrast, ACh-induced APs that terminated 1–5 sec prior to HFS boosted STP to LTP. (D) If the APs terminated < 1 sec before HFS, then LTD resulted. (E) If the ACh-evoked APs followed HFS then there was no effect on STP. (F) Summary of the findings in (B) through (E). The normalized PSPs from the last 15 min of post-HFS recording were averaged and then plotted against the time between the end of ACh application and the onset of HFS. Negative time values correspond to the interval between the last ACh-induced AP and the onset of HFS. Positive time values correspond to the interval between the end of HFS and the onset of the first ACh-induced AP. The curve drawn through the data points indicates the general trend of the data. Arrows indicate timing of HFS for all panels. Adapted with permission from [92] (copyright 2005 by the Society for Neuroscience).

The temporal profile revealed in Figure 2F differs substantially from reports of spike timing dependent plasticity (STDP), which have identified intervals on the order of a few msec over which single EPSPs paired with single APs can change synaptic weights [94, 95]. Cholinergic regulation of plasticity, which emphasizes pairing intervals on the order of seconds, provides a broad window over which the outcome of presynaptic information can be transmitted and influence synaptic strength.

Boosting STP to LTP requires the activation of mainly postsynaptic α7* nAChRs (with some β2* nAChRs) in the rat hippocampus [81, 89, 90, 92, 93, 96]. Despite the fast desensitization of α7 nAChRs, brief activation of these receptors in rapid succession (e.g., <200 msec inter-stimulus intervals) actually potentiates their currents [97]. During this imposed protocols, activation of α7* nAChRs is only required for the induction of LTP, and not its expression or maintenance, as blocking α7 nAChRs has no effect once LTP is established [98].

Location of nAChR activation regulates hippocampal synaptic plasticity

In another set of experiments, STP was induced at Schaffer collateral–CA1 synapses, while varying the location of agonist application (Fig. 3A–C) [81]. For these experiments a more intense stimulation protocol (3 × 1 sec, 100Hz stimulus trains) was used to produce robust LTP [81]. When nAChRs were activated by puffing ACh onto neighboring GABAergic interneurons, which have been shown to inhibit CA1 pyramidal neurons directly [81, 84, 99, 100], this protocol produced only STP. Thus nicotinic effects in the hippocampus on synaptic transmission are mediated to a significant extent by the direct activation of inhibitory GABAergic interneurons, which serves to decrease the net output of pyramidal cells, or negate plasticity mechanisms in pyramidal cells. Interestingly, for the cellular combination of interneuron-interneuron-pyramidal cell, nicotinic activation of the first GABAergic neuron can inhibit the second interneuron, which then disinhibits the pyramidal cell, thereby potentially increasing pyramidal cell output [84]. In situ the nicotinic influences via GABAergic networks are generally stronger than direct action at glutamatergic pyramidal neurons because nAChRs are more densely expressed on the GABAergic interneurons [84, 99, 101, 102].

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Fig. 3

Spatial-dependence of nAChR activation for hippocampal synaptic plasticity. (A) Illustration of the experimental setup for experiments shown in panels (B) and (C). The puffer pipette applies ACh to a GABAergic interneuron neighboring the pyramidal neuron. All PSPs were normalized to baseline. (B, C) HFS-evoked LTP (B) was prevented by activating inhibitory interneurons with ACh (C). Arrows indicate timing of HFS for all panels. Adapted with permission from [81] (copyright 2001 by Cell Press).

Nicotinic AChRs regulate hippocampal synaptic plasticity in vivo

Our understanding of nAChR regulation of synaptic transmission and plasticity has for the most part been gleaned from in vitro studies, which offer a high level of experimental control to probe mechanistic questions. However, potential drawbacks, such as the severing or remodeling of network connections and changes in cellular physiology that may impact plasticity [116], suggest the need for systems-level approaches with in vivo strategies.

Systemic administration of very high doses of nicotine has been shown to evoke LTP at perforant path–dentate gyrus synapses in anesthetized mice [34, 117]. In the dentate gyrus, chemical LTP evoked by 3 mg/kg nicotine gradually develops over the first ~10 min post-injection and reaches a stable >2-fold increase over baseline that persists for at least 2 hours [117]. In contrast to in vitro cases, where nicotine boosts STP to LTP, in the intact anesthetized animal very high doses of nicotine actually induce LTP in the absence of the tetanization of afferent inputs. Interestingly, nicotine-induced LTP and perforant path HFS-induced LTP are similar in time course and amplitude, and both are blocked by pre-administration of mecamylamine, suggesting that tetanus-induced LTP requires the endogenous activation of cholinergic inputs and nAChRs. Neither nicotine-induced nor HFS-induced LTP is blocked by mecamylamine delivered after nicotine injection or following HFS. As seen in vitro, this result indicates that the induction, but not the expression or maintenance of LTP, is dependent on nAChRs. This same study also showed that the α7* nAChR agonist choline elicited a dose-dependent LTP in vivo, and that the maximum LTP evoked by choline could be further increased by nicotine [117]. In a complementary experiment, these authors showed that epibatidine, a somewhat specific β2* agonist, also produced chemical LTP [118]. Like choline-induced LTP, epibatidine-induced LTP was sub-maximal and was increased to maximum values by nicotine. The amount of potentiation evoked by choline or epibatidine individually reveals that α7* and β2* receptors, respectively, each contribute about 50% to the LTP evoked by nicotine [117, 118]. At Schaffer collateral–CA1 synapses in anesthetized rats, α7* nAChRs contribute approximately one-third of the HFS-induced LTP [119].

Although these results are intriguing, there are a number of issues to consider. The antagonist mecamylamine in wake rodents also weakly inhibits NMDARs [120] and inhibits ACh synthesis [121]. Thus, work with more specific blockers that exclude alternative conclusions will be necessary. In addition, the rodents were anesthetized and LTP was evoked by very high concentrations of agonists: concentrations of nicotine that would induce seizures in wake mice [122]. The use of anesthesia further confounds the nicotinic role because some anesthetics inhibit nAChRs [123]. In addition, nAChRs often influence circuit events by a predominant action upon GABAergic inhibitory interneurons [84, 101], and GABAergic signaling is important during the induction of nicotine-evoked plasticity [81, 124]. Given the propensity for anesthetics to modulate GABAergic activity, GABARs, and nAChRs [123, 125], additional studies using freely-moving animals and physiological concentrations of nicotine are going to be extremely important to understand the biological implications of nicotine influences over plasticity in vivo.

Nicotinic AChRs contribute to hippocampal plasticity during early postnatal development

During the first few weeks of postnatal life, the rodent brain undergoes extraordinary development. As noted for adult animals, nAChR-mediated plasticity appears to play important roles during these times as well. In early (<1 week) postnatal rats, α7* nAChRs regulate the frequency of GABA-mediated giant depolarizing potentials (GDPs) in CA3 [126]. As pairing GDPs with mossy fiber input is known to strengthen mossy fiber–CA3 synapses [127], early nicotinic regulation may contribute to the maturation of these neuronal connections.

In the developing hippocampus, nAChRs provide a powerful control over glutamate release probability, converting low probability synapses to high probability synapses [128] and vice versa [129]. The effect is sufficiently dramatic that nicotine can convert ‘silent’ synapses to functional synapses [128]. In the adult, ‘silent’ synapses do not express AMPARs in the postsynaptic membrane and, thus, cannot detect glutamate release at hyperpolarized potentials [130]. In contrast, in the early postnatal brain, ‘silent’ synapses express postsynaptic glutamate receptors, but the probability of presynaptic neurotransmitter release is near zero. As noted in adult neurons, nicotine application to juvenile neurons markedly increases the probability of release from Schaffer collateral fibers onto CA1 pyramidal neurons in an α7* nAChR-dependent manner. Presynaptic nAChR activity increases the frequency, but not the amplitude, of spontaneous synaptic currents. Importantly, stimulating cholinergic fibers within juvenile slices mimics the effect of nicotine, suggesting that the switch from low to high probability synapse is mediated by endogenous ACh. For those rare cases when synapses with a high probability of release were identified, nicotine application decreased release probability, an effect that required both α7* and β2* nAChRs [129]. Taken together, these results suggest that activation of nAChRs may function as a bidirectional switch on release probability in developing hippocampal neurons.

Interestingly, the transition of GABA as an excitatory neurotransmitter in the juvenile brain to an inhibitory neurotransmitter in the adult brain is also regulated by nAChR signaling [131]. In juvenile neurons, expression of the Cl⁻ transporter NKCC1 maintains a high intracellular Cl⁻ concentration, and thus GABA-mediated channel openings favor an outward flow of negative current that is depolarizing. In adult neurons the Cl⁻ transporter KCC2 is more robustly expressed, and it maintains a high extracellular Cl⁻ concentration that favors hyperpolarization when the Cl− channels are opened [132, 133]. During the second postnatal week NKCC1 expression is reduced and KCC2 expression is increased, rendering GABA inhibitory. These events were recently shown to involve α7* nAChR-mediated signaling, albeit through an unidentified transduction mechanism [131].

Nicotinic AChRs regulate synaptic plasticity in midbrain dopaminergic neurons

Nicotinic receptors are known to regulate synaptic plasticity in areas that play a critical role in reward and addiction, such as the neurons of the ventral midbrain. Of particular significance are the dopaminergic neurons of the ventral tegmental area (VTA), which respond to rewarding stimuli or reward-predicting stimuli [134]. VTA neurons broadcast reward and salience information to areas of the brain underlying emotion and decision making, including the prefrontal cortex, amygdala, striatum, and nucleus accumbens [41]. The VTA dopaminergic neurons receive excitatory input from numerous sources, including the prefrontal cortex, and inhibitory inputs from afferent projections and from local GABAergic interneurons. Cholinergic inputs arrive from the laterodorsal and the pedunculopontine tegmental nuclei and project to both dopaminergic and GABAergic cells in this region. The ventral midbrain neurons express many nAChR subunits, with α7* and β2* nAChRs predominating [135, 136]. Nicotine robustly activates this system, resulting in the acquisition of behaviors that are reinforced by drug use [137–139]. In fact, a single exposure to nicotine elevates dopamine levels in the nucleus accumbens for more than two hours in rats (Fig. 4A) [140, 141]. The question of how a drug such as nicotine triggers long-term changes in the midbrain dopamine system has been explored from the hypothesis that nicotine, acting through nAChRs, modulates short- and long-term plasticity.

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Fig. 4

Synaptic plasticity in the VTA. (A) Dopamine levels are elevated in the nucleus accumbens of rats for more than two hours following a single i.p. injection of 0.6 mg/kg nicotine. (B) Bath applied nicotine increases the frequency of AP discharge in dopaminergic neurons mainly via β2* nAChRs. (C) Bath applied nicotine increases the frequency of spontaneous EPSCs (sEPSCs) mainly via presynaptic α7* nAChRs. The coincidence of postsynaptic and presynaptic activation by nicotine is sufficient to induce LTP of the glutamatergic synapses. (D) The frequency of spontaneous IPSCs (sIPSCs) first increases, and then decreases below baseline following desensitization of β2* nAChRs on the somata of GABAergic neurons. The decrease in IPSC frequency indirectly enhances the glutamatergic excitation of dopamine neurons. Adapted with permission from [140, 144] (copyright 1997 by Nature Publishing Group [144] and copyright 2004 by Cold Spring Harbor Laboratory Press [140]).

Comparison of nAChR LTP mechanisms between hippocampus and VTA

The contributions of nAChRs to synaptic plasticity in the hippocampus can be summarized as follows: 1) nAChRs increase presynaptic glutamate release, 2) nAChRs contribute a small postsynaptic membrane depolarization, which presumably increases the open probability of NMDARs via the voltage-dependent relief of Mg²⁺ blockade, 3) nAChRs directly and indirectly contribute an intracellular Ca²⁺ signal in the postsynaptic cell that may activate plasticity-evoking Ca²⁺-dependent signaling and gene transcription, 4) presynaptic activity and nAChR activation may be temporally coordinated to influence synaptic transmission and plasticity, 5) nAChR activation may be spatially coordinated to enhance GABAergic interneuron signaling to block LTP induction on pyramidal neurons, and 6) nAChR signaling can awaken or silence synapses during early postnatal development and control the transition of GABA from an excitatory to an inhibitory neurotransmitter. Taken together, the data support the view that nAChRs exert a temporally- and spatially-dependent bidirectional control over hippocampal synaptic plasticity, both in vitro and in vivo.

In contrast to the facilitory (modulatory) role for nAChRs during LTP induction in the in vitro hippocampal slice, nAChR activity induces LTP in the midbrain slice containing the VTA. Within the VTA, nicotine contributes to LTP of glutamatergic synapses onto dopamine neurons via three major mechanisms: 1) transient activation of postsynaptic β2* nAChRs to depolarize the dopaminergic neurons, 2) sustained activation of presynaptic α7* nAChRs to augment glutamate transmission onto dopaminergic neurons, and 3) activation and desensitization of β2* receptors on GABAergic interneurons to decrease inhibition onto dopaminergic neurons. Within the VTA, nicotinic cholinergic input could conceivably act as a coincidence mechanism, simultaneously recruiting both presynaptic and postsynaptic elements. The fact that nAChR activation in the hippocampus in vivo produces de novo LTP in the absence of afferent tetanization suggests that the hippocampal slice preparation may lack all the long-range connections and the network properties of the in vivo situation. Thus, nAChRs modulate, regulate and likely induce synaptic plasticity in both the hippocampus and the VTA.

Potential therapeutic roles for nAChRs in pathological states

The importance of nAChRs to synaptic transmission and synaptic plasticity throughout the brain is evident from pharmacological studies and investigations with nAChR-subunit knockout mice [152, 154–57]}. Any changes to the expression levels of these receptors, or changes to the afferents that supply these receptors with ACh, could influence the physiology and behavior. A corollary is that some forms of pathology that include compromised synaptic transmission or plasticity could potentially benefit from exogenous stimulation of nAChRs to boost transmission or plasticity capabilities closer to normal levels.

Cholinergic signaling and nAChRs are implicated in many pathological changes to the brain, including Alzheimer’s disease, Parkinson’s disease, schizophrenia, and addiction [137, 158–160]. For instance, in Alzheimer’s disease, the numbers of nAChRs decrease, particularly in the hippocampus and cortex [158, 161]. Evidence indicates that augmenting nAChR activity allosterically or inhibiting acetylcholinesterase may improve attention and rates of learning [75, 162–164], and these effects also may slow neurodegeneration in models of Alzheimer’s disease [165, 166]. Other evidence has shown that, in rats and nonhuman primates, nicotine is neuroprotective against loss of nigrostriatal dopamine neurons [167]. Given the deficit of dopaminergic neurotransmission in Parkinson’s disease, therapeutic activation of nAChRs to facilitate dopamine release onto its target cells may benefit those patients as well. For example, Alzheimer’s drugs, such as donepezil and galantamine, were found to enhance nicotinic function and indirectly influence dopamine release in the striatum [168].

Improved treatments will require the development of new nAChR agonists and antagonists that target nAChRs locally in some cases and broadly in other cases [169, 170]. In addition, the development of new allosteric modulators [171] – those compounds that do not directly activate the receptor but augment function of the receptor when the ligand is present –will also be valuable. Because allosteric modulators do not themselves activate receptors, they will operate with the same spatial and temporal profile as endogenous cholinergic signals. The development of novel compounds, as well as new molecular tools like siRNA and conditional/inducible knock-out or knock-in mice, will also be a benefit to basic research. Presently the nicotinic field has not extensively benefited from the combination of molecular tools and powerful, high-resolution in vivo recordings from freely-moving animals. Those techniques will open up the black-box observations of behavioral studies. The application of these powerful approaches will provide new kinds of data and open additional avenues for research and provide new targets for drug development. These combinations of tools and approaches could be used to determine exactly how and when specific nAChR subunits contribute to processes such as synaptic transmission, synaptic plasticity, normal development, and dysfunction as well as higher order functions such as cognition and consciousness.

Acknowledgments

Abbreviations

Footnotes

References