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.

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.

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.

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.

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.