Neonatal Nicotine Exposure Primes Midbrain Neurons to a Dopaminergic Phenotype and Increases Adult Drug Consumption.

BACKGROUND: Nicotine intake induces addiction through neuroplasticity of the reward circuitry, altering the activity of dopaminergic neurons of the ventral tegmental area. Prior work demonstrated that altered circuit activity can change neurotransmitter expression in the developing and adult brain. Here we investigated the effects of neonatal nicotine exposure on the dopaminergic system and nicotine consumption in adulthood. METHODS: Male and female mice were used for two-bottle-choice test, progressive ratio breakpoint test, immunohistochemistry, RNAscope, quantitative polymerase chain reaction, calcium imaging, and DREADD (designer receptor exclusively activated by designer drugs)-mediated chemogenic activation/inhibition experiments. RESULTS: Neonatal nicotine exposure potentiates drug preference in adult mice, induces alterations in calcium spike activity of midbrain neurons, and increases the number of dopamine-expressing neurons in the ventral tegmental area. Specifically, glutamatergic neurons are first primed to express transcription factor Nurr1, then acquire the dopaminergic phenotype following nicotine re-exposure in adulthood. Enhanced neuronal activity combined with Nurr1 expression is both necessary and sufficient for the nicotine-mediated neurotransmitter plasticity to occur. CONCLUSIONS: Our findings illuminate a new mechanism of neuroplasticity by which early nicotine exposure primes the reward system to display increased susceptibility to drug consumption in adulthood.

Loss of MeCP2 in cholinergic neurons causes part of RTT-like phenotypes via α7 receptor in hippocampus.

Mutations in the X-linked MECP2 gene cause Rett syndrome (RTT), an autism spectrum disorder characterized by impaired social interactions, motor abnormalities, cognitive defects and a high risk of epilepsy. Here, we showed that conditional deletion of Mecp2 in cholinergic neurons caused part of RTT-like phenotypes, which could be rescued by re-expressing Mecp2 in the basal forebrain (BF) cholinergic neurons rather than in the caudate putamen of conditional knockout (Chat-Mecp2(-/y)) mice. We found that choline acetyltransferase expression was decreased in the BF and that α7 nicotine acetylcholine receptor signaling was strongly impaired in the hippocampus of Chat-Mecp2(-/y) mice, which is sufficient to produce neuronal hyperexcitation and increase seizure susceptibility. Application of PNU282987 or nicotine in the hippocampus rescued these phenotypes in Chat-Mecp2(-/y) mice. Taken together, our findings suggest that MeCP2 is critical for normal function of cholinergic neurons and dysfunction of cholinergic neurons can contribute to numerous neuropsychiatric phenotypes.

Nicotine recruits glutamate receptors to postsynaptic sites.

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

A novel mechanism for nicotinic potentiation of glutamatergic synapses.

Selective strengthening of specific glutamatergic synapses in the mammalian hippocampus is critical for encoding new memories. This is most commonly achieved by input-specific Hebbian-type plasticity involving glutamate-dependent coincident presynaptic and postsynaptic depolarization. Our results demonstrate a novel mechanism by which nicotinic signaling, independently of coincident fast glutamatergic transmission, increases synaptic strength in the hippocampus. Electrophysiological recordings from rat hippocampal neurons in culture revealed that 1-3 h of exposure to 1 µm nicotine, even with action potentials being blocked, produced increases in both the frequency and amplitude of miniature EPSCs. Possible mechanisms were analyzed both in mouse organotypic slice culture and in rat cell culture by inducing the cells to express super-ecliptic pHluorin-tagged GluA1-containing AMPA receptors, which fluoresce only on the cell surface. Pharmacological and genetic manipulation of the cells, in combination with fluorescence-recovery-after-photobleaching experiments, revealed that nicotine, acting through α7-containing nicotinic acetylcholine receptors on the postsynaptic neuron, induces the stabilization and accumulation of GluA1-containing AMPA receptors on dendritic spines. The process relies on intracellular calcium signaling, PDZ [postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] interactions with members of the PSD-95 family, and lateral diffusion of the GluA1 receptors on the cell surface. These findings define a new avenue by which nicotinic signaling modulates synaptic mechanisms thought to subserve learning and memory.

Activation of α7-containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamatergic synapses.

Astrocytes, an abundant form of glia, are known to promote and modulate synaptic signaling between neurons. They also express α7-containing nicotinic acetylcholine receptors (α7-nAChRs), but the functional relevance of these receptors is unknown. We show here that stimulation of α7-nAChRs on astrocytes releases components that induce hippocampal neurons to acquire more α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors post-synaptically at glutamatergic synapses. The increase is specific in that no change is seen in synaptic NMDA receptor clusters or other markers for glutamatergic synapses, or in markers for GABAergic synapses. Moreover, the increases in AMPA receptors on the neuron surface are accompanied by increases in the frequency of spontaneous miniature synaptic currents mediated by the receptors and increases in the ratio of evoked synaptic currents mediated by AMPA versus NMDA receptors. This suggests that stimulating α7-nAChRs on astrocytes can convert 'silent' glutamatergic synapses to functional status. Astrocyte-derived thrombospondin is necessary but not sufficient for the effect, while tumor necrosis factor-α is sufficient but not necessary. The results identify astrocyte α7-nAChRs as a novel pathway through which nicotinic cholinergic signaling can promote the development of glutamatergic networks, recruiting AMPA receptors to post-synaptic sites and rendering the synapses more functional. We find that activation of nicotinic receptors on astrocytes releases a component that specifically recruits AMPA receptors to glutamatergic synapses. The recruitment appears to occur preferentially at what may be 'silent synapses', that is, synapses that have all the components required for glutamatergic transmission (including NMDA receptors) but lack sufficient AMPA receptors to generate a response. The results are unexpected and open up new possibilities for mechanisms underlying network formation and synaptic plasticity.

Induction of dendritic spines by ß2-containing nicotinic receptors.

Glutamatergic synapses are located mostly on dendritic spines in the adult nervous system. The spines serve as postsynaptic compartments, containing components that mediate and control the synaptic signal. Early in development, when glutamatergic synapses are initially forming, waves of excitatory activity pass through many parts of the nervous system and are driven in part by a class of heteropentameric ß2-containing nicotinic acetylcholine receptors (ß2*-nAChRs). These ß2*-nAChRs are widely distributed and, when activated, can depolarize the membrane and elevate intracellular calcium levels in neurons. We show here that ß2*-nAChRs are essential for acquisition of normal numbers of dendritic spines during development. Mice constitutively lacking the ß2-nAChR gene have fewer dendritic spines than do age-matched wild-type mice at all times examined. Activation of ß2*-nAChRs by nicotine either in vivo or in organotypic slice culture quickly elevates the number of spines. RNA interference studies both in vivo and in organotypic culture demonstrate that the ß2*-nAChRs act in a cell-autonomous manner to increase the number of spines. The increase depends on intracellular calcium and activation of calcium, calmodulin-dependent protein kinase II. Absence of ß2*-nAChRs in vivo causes a disproportionate number of glutamatergic synapses to be localized on dendritic shafts, rather than on spines as occurs in wild type. This shift in synapse location is found both in the hippocampus and cortex, indicating the breadth of the effect. Because spine synapses differ from shaft synapses in their signaling capabilities, the shift observed is likely to have significant consequences for network function.

Glutamatergic synapse formation is promoted by α7-containing nicotinic acetylcholine receptors.

Glutamate is the primary excitatory transmitter in adult brain, acting through synapses on dendritic spines and shafts. Early in development, however, when glutamatergic synapses are only beginning to form, nicotinic cholinergic excitation is already widespread; it is mediated by acetylcholine activating nicotinic acetylcholine receptors (nAChRs) that generate waves of activity across brain regions. A major class of nAChRs contributing at this time is a species containing α7 subunits (α7-nAChRs). These receptors are highly permeable to calcium, influence a variety of calcium-dependent events, and are diversely distributed throughout the developing CNS. Here we show that α7-nAChRs unexpectedly promote formation of glutamatergic synapses during development. The dependence on α7-nAChRs becomes clear when comparing wild-type (WT) mice with mice constitutively lacking the α7-nAChR gene. Ultrastructural analysis, immunostaining, and patch-clamp recording all reveal synaptic deficits when α7-nAChR input is absent. Similarly, nicotinic activation of α7-nAChRs in WT organotypic culture, as well as cell culture, increases the number of glutamatergic synapses. RNA interference demonstrates that the α7-nAChRs must be expressed in the neuron being innervated for normal innervation to occur. Moreover, the deficits persist throughout the developmental period of major de novo synapse formation and are still fully apparent in the adult. GABAergic synapses, in contrast, are undiminished in number under such conditions. As a result, mice lacking α7-nAChRs have an altered balance in the excitatory/inhibitory input they receive. This ratio represents a fundamental feature of neural networks and shows for the first time that endogenous nicotinic cholinergic signaling plays a key role in network construction.

PMCA2 via PSD-95 controls calcium signaling by α7-containing nicotinic acetylcholine receptors on aspiny interneurons.

Local control of calcium concentration within neurons is critical for signaling and regulation of synaptic communication in neural circuits. How local control can be achieved in the absence of physical compartmentalization is poorly understood. Challenging examples are provided by nicotinic acetylcholine receptors that contain α7 nicotinic receptor subunits (α7-nAChRs). These receptors are highly permeable to calcium and are concentrated on aspiny dendrites of interneurons, which lack obvious physical compartments for constraining calcium diffusion. Using functional proteomics on rat brain, we show that α7-nAChRs are associated with plasma membrane calcium-ATPase pump isoform 2 (PMCA2). Analysis of α7-nAChR function in hippocampal interneurons in culture shows that PMCA2 activity limits the duration of calcium elevations produced by the receptors. Unexpectedly, PMCA2 inhibition triggers rapid calcium-dependent loss of α7-nAChR clusters. This extreme regulatory response is mediated by CaMKII, involves proteasome activity, depends on the second intracellular loop of α7-nAChR subunits, and is specific in that it does not alter two other classes of calcium-permeable ionotropic receptors on the same neurons. A critical link is provided by the scaffold protein PSD-95 (postsynaptic density-95), which is associated with α7-nAChRs and constrains their mobility as revealed by single-particle tracking on neurons. The PSD-95 link is required for PMCA2-mediated removal of α7-nAChR clusters. This three-component combination of PMCA2, PSD-95, and α7-nAChR offers a novel mechanism for tight control of calcium dynamics in neurons.

Nicotinic control of adult-born neuron fate.

The hippocampus is one of only two regions in the adult brain where neurons are generated in significant numbers throughout the lifetime of the animal. Numerous studies have demonstrated that these adult-born neurons are essential for optimal cognitive function with unimpaired memory formation and retrieval. The extent to which adult-born neurons survive through an early "critical period" and become integrated into functional networks has been shown to depend on the richness of stimulation they receive during these formative stages. The dentate gyrus in the hippocampus - home of the adult-born neurons - receives extensive cholinergic innervation, and newly generated neurons in the adult hippocampus express substantial numbers of both major types of neuronal nicotinic acetylcholine receptors. Early studies indicated that nicotinic signaling may be important for the development of adult-born neurons: repeated exposure to nicotine impaired their long-term survival. Recent studies with mutant mice lacking either one of the two major nicotinic receptor subtypes demonstrate that receptor loss results in fewer adult-born neurons surviving the critical period and becoming integrated into neural networks. The key nicotinic receptor mediating the largest effects is one that has a high relative permeability to calcium. In view of this feature, it may not be surprising that excessive exposure to nicotine can have detrimental effects on survival and maturation of adult-born neurons in the dentate; these same receptors appear to be key. The results pose serious challenges for therapeutic strategies targeting an individual class of nicotinic receptors for global treatment in the recipient.

EphB receptors co-distribute with a nicotinic receptor subtype and regulate nicotinic downstream signaling in neurons.

Activation of nicotinic acetylcholine receptors (nAChRs) on neurons engages calcium-dependent signaling pathways regulating numerous events. Receptors containing alpha7 subunits (alpha7-nAChRs) are prominent in this because of their abundance and high relative calcium permeability. We show here that EphB2 receptors are co-localized with postsynaptic alpha7-nAChRs on chick ciliary ganglion neurons and that treatment of the cells with an ephrinB1 construct to activate the EphB receptors exerts physical restraints on both classes of receptors, diminishing their dispersal after spine retraction or lipid raft disruption. Moreover, the ephrinB1/EphB receptor complex specifically enhances the ability of alpha7-nAChRs to activate the transcription factor CREB, acting through a pathway including a receptor tyrosine kinase, a Src family member, PI3 kinase, and protein kinase A most distally. The enhancement does not appear to result from a change in the alpha7-nAChR current amplitude, suggesting a downstream target. The results demonstrate a role for ephrin/EphB action in nicotinic signaling.

Postsynaptic neuroligin enhances presynaptic inputs at neuronal nicotinic synapses.

Neuroligins are cell adhesion molecules that interact with neurexins on adjacent cells to promote glutamatergic and GABAergic synapse formation in culture. We show here that neuroligin enhances nicotinic synapses on neurons in culture, increasing synaptic input. When neuroligin is overexpressed in neurons, the extracellular domain induces presynaptic specializations in adjacent cholinergic neurons as visualized by SV2 puncta. The intracellular domain is required to translate the SV2 puncta into synaptic input as reflected by increases in the frequency of spontaneous mini-synaptic currents. The PDZ-binding motif of neuroligin is not needed for these effects. Together, the extracellular and proximal intracellular domains of neuroligin are sufficient to induce presynaptic specializations, align them over postsynaptic receptor clusters, and increase synaptic function. Manipulation of endogenous neuroligin with beta-neurexin-expressing cells confirms its presence; repressing function with dominant negative constructs and inhibitory shRNA shows that endogenous neuroligin helps confer functionality on existing nicotinic synaptic contacts. Endogenous neuroligin does not appear to be required, however, for initial formation of the contacts, suggesting that other components under these conditions can also initiate synapse formation. The results indicate that postsynaptic neuroligin is important for functional nicotinic synapses on neurons and that the effects achieved will likely depend on neuroligin levels.

Sequential interplay of nicotinic and GABAergic signaling guides neuronal development.

GABA (gamma-aminobutyric acid), the major inhibitory transmitter in the brain, goes through a transitory phase of excitation during development. The excitatory phase promotes neuronal growth and integration into circuits. We show here that spontaneous nicotinic cholinergic activity is responsible for terminating GABAergic excitation and initiating inhibition. It does so by changing chloride transporter levels, shifting the driving force on GABA-induced currents. The timing of the transition is critical, because the two phases of GABAergic signaling provide contrasting developmental instructions. Synergistic with nicotinic excitation, GABAergic inhibition constrains neuronal morphology and innervation. The results reveal a multitiered activity-dependent strategy controlling neuronal development.

Rapid activity-driven SNARE-dependent trafficking of nicotinic receptors on somatic spines.

Rapid trafficking of glutamate receptors contributes importantly to synaptic plasticity, but whether similar trafficking extends to other ionotropic receptors is unknown. Nicotinic acetylcholine receptors containing alpha7 subunits are widely expressed in the nervous system and allow calcium influx. Because of this, alpha7-containing receptors regulate diverse events, depending on the signaling pathways available. We find that the receptors codistribute with target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) postsynaptically and that nicotinic stimulation rapidly induces SNARE-dependent vesicular endocytosis accompanied by receptor internalization. At the same time, a SNARE-dependent process recruits receptors to the cell surface from internal pools. Overall, the trafficking does not markedly change the number of surface receptors or their combined whole-cell response to nicotine. SNARE-dependent trafficking is needed, however, for the receptors to remain capable of activating the transcription factor cAMP response element-binding protein and attendant gene expression when repeatedly challenged. Thus, trafficking appears to be essential for maintaining functional coupling between alpha7-receptor responses and downstream signaling.

Nicotinic regulation of CREB activation in hippocampal neurons by glutamatergic and nonglutamatergic pathways.

Activity-dependent gene expression is essential for form and function in the nervous system. Best understood is the role of glutamatergic signaling in controlling such events, but nicotinic signaling can also regulate transcription. We show here that nicotine can alter gene expression in rat hippocampal neurons, as reflected by activation of the transcription factor CREB and appearance of the immediate early gene product c-Fos. The process depends on both CaM and MAP kinases and on calcium release from internal stores. Part of the nicotinic effect is mediated via glutamatergic transmission, even in the absence of action potentials. Voltage-gated calcium channels are not necessary for nicotine-induced activation of CREB in hippocampal neurons. The low levels of sustained nicotinic stimulation required for transcriptional effects are consistent with those likely to be achievable either by the normal septal cholinergic innervation of the hippocampus or by repeated tobacco usage.