MicroRNA-218 instructs proper assembly of hippocampal networks.

The assembly of the mammalian brain is orchestrated by temporally coordinated waves of gene expression. Post-transcriptional regulation by microRNAs (miRNAs) is a key aspect of this program. Indeed, deletion of neuron-enriched miRNAs induces strong developmental phenotypes, and miRNA levels are altered in patients with neurodevelopmental disorders. However, the mechanisms used by miRNAs to instruct brain development remain largely unexplored. Here, we identified miR-218 as a critical regulator of hippocampal assembly. MiR-218 is highly expressed in the hippocampus and enriched in both excitatory principal neurons (PNs) and GABAergic inhibitory interneurons (INs). Early life inhibition of miR-218 results in an adult brain with a predisposition to seizures. Changes in gene expression in the absence of miR-218 suggest that network assembly is impaired. Indeed, we find that miR-218 inhibition results in the disruption of early depolarizing GABAergic signaling, structural defects in dendritic spines, and altered intrinsic membrane excitability. Conditional knockout of Mir218-2 in INs, but not PNs, is sufficient to recapitulate long-term instability. Finally, de-repressing Kif21b and Syt13, two miR-218 targets, phenocopies the effects on early synchronous network activity induced by miR-218 inhibition. Taken together, the data suggest that miR-218 orchestrates formative events in PNs and INs to produce stable networks.

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.

Neurotransmitter Switching Regulated by miRNAs Controls Changes in Social Preference.

Changes in social preference of amphibian larvae result from sustained exposure to kinship odorants. To understand the molecular and cellular mechanisms of this neuroplasticity, we investigated the effects of olfactory system activation on neurotransmitter (NT) expression in accessory olfactory bulb (AOB) interneurons during development. We show that protracted exposure to kin or non-kin odorants changes the number of dopamine (DA)- or gamma aminobutyric acid (GABA)-expressing neurons, with corresponding changes in attraction/aversion behavior. Changing the relative number of dopaminergic and GABAergic AOB interneurons or locally introducing DA or GABA receptor antagonists alters kinship preference. We then isolate AOB microRNAs (miRs) differentially regulated across these conditions. Inhibition of miR-375 and miR-200b reveals that they target Pax6 and Bcl11b to regulate the dopaminergic and GABAergic phenotypes. The results illuminate the role of NT switching governing experience-dependent social preference. VIDEO ABSTRACT.

Evidence for opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation.

The signaling mechanisms that choreograph the assembly of the highly asymmetric pre- and postsynaptic structures are still poorly defined. Using synaptosome fractionation, immunostaining, and coimmunoprecipitation, we found that Celsr3 and Vangl2, core components of the planar cell polarity (PCP) pathway, are localized at developing glutamatergic synapses and interact with key synaptic proteins. Pyramidal neurons from the hippocampus of Celsr3 knockout mice exhibit loss of ∼50% of glutamatergic synapses, but not inhibitory synapses, in culture. Wnts are known regulators of synapse formation, and our data reveal that Wnt5a inhibits glutamatergic synapses formed via Celsr3. To avoid affecting earlier developmental processes, such as axon guidance, we conditionally knocked out Celsr3 in the hippocampus 1 week after birth. The CA1 neurons that lost Celsr3 also showed a loss of ∼50% of glutamatergic synapses in vivo without affecting the inhibitory synapses assessed by miniature excitatory postsynaptic current (mEPSC) and electron microscopy. These animals displayed deficits in hippocampus-dependent behaviors in adulthood, including spatial learning and memory and fear conditioning. In contrast to Celsr3 conditional knockouts, we found that the conditional knockout of Vangl2 in the hippocampus 1 week after birth led to a large increase in synaptic density, as evaluated by mEPSC frequency and spine density. PCP signaling is mediated by multiple core components with antagonizing functions. Our results document the opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation.

MicroRNA-101 Regulates Multiple Developmental Programs to Constrain Excitation in Adult Neural Networks.

A critical feature of neural networks is that they balance excitation and inhibition to prevent pathological dysfunction. How this is achieved is largely unknown, although deficits in the balance contribute to many neurological disorders. We show here that a microRNA (miR-101) is a key orchestrator of this essential feature, shaping the developing network to constrain excitation in the adult. Transient early blockade of miR-101 induces long-lasting hyper-excitability and persistent memory deficits. Using target site blockers in vivo, we identify multiple developmental programs regulated in parallel by miR-101 to achieve balanced networks. Repression of one target, NKCC1, initiates the switch in γ-aminobutyric acid (GABA) signaling, limits early spontaneous activity, and constrains dendritic growth. Kif1a and Ank2 are targeted to prevent excessive synapse formation. Simultaneous de-repression of these three targets completely phenocopies major dysfunctions produced by miR-101 blockade. Our results provide new mechanistic insight into brain development and suggest novel candidates for therapeutic intervention.

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.

Selectively driving cholinergic fibers optically in the thalamic reticular nucleus promotes sleep.

Cholinergic projections from the basal forebrain and brainstem are thought to play important roles in rapid eye movement (REM) sleep and arousal. Using transgenic mice in which channelrhdopsin-2 is selectively expressed in cholinergic neurons, we show that optical stimulation of cholinergic inputs to the thalamic reticular nucleus (TRN) activates local GABAergic neurons to promote sleep and protect non-rapid eye movement (NREM) sleep. It does not affect REM sleep. Instead, direct activation of cholinergic input to the TRN shortens the time to sleep onset and generates spindle oscillations that correlate with NREM sleep. It does so by evoking excitatory postsynaptic currents via α7-containing nicotinic acetylcholine receptors and inducing bursts of action potentials in local GABAergic neurons. These findings stand in sharp contrast to previous reports of cholinergic activity driving arousal. Our results provide new insight into the mechanisms controlling sleep.

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.

Long-lasting changes in neural networks to compensate for altered nicotinic input.

The nervous system must balance excitatory and inhibitory input to constrain network activity levels within a proper dynamic range. This is a demanding requirement during development, when networks form and throughout adulthood as networks respond to constantly changing environments. Defects in the ability to sustain a proper balance of excitatory and inhibitory activity are characteristic of numerous neurological disorders such as schizophrenia, Alzheimer's disease, and autism. A variety of homeostatic mechanisms appear to be critical for balancing excitatory and inhibitory activity in a network. These are operative at the level of individual neurons, regulating their excitability by adjusting the numbers and types of ion channels, and at the level of synaptic connections, determining the relative numbers of excitatory versus inhibitory connections a neuron receives. Nicotinic cholinergic signaling is well positioned to contribute at both levels because it appears early in development, extends across much of the nervous system, and modulates transmission at many kinds of synapses. Further, it is known to influence the ratio of excitatory-to-inhibitory synapses formed on neurons during development. GABAergic inhibitory neurons are likely to be key for maintaining network homeostasis (limiting excitatory output), and nicotinic signaling is known to prominently regulate the activity of several GABAergic neuronal subtypes. But how nicotinic signaling achieves this and how networks may compensate for the loss of such input are important questions remaining unanswered. These issues are reviewed.

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.

Lateral mobility of presynaptic α7-containing nicotinic receptors and its relevance for glutamate release.

Surface diffusion of postsynaptic receptors shapes synaptic transmission. Presynaptic receptors also influence transmission, but the relevance of their mobility for synaptic function is unknown. Using single-particle tracking with quantum dots, we show that calcium-permeable α7-containing nicotinic acetylcholine receptors (α7-nAChRs), capable of promoting transmitter release, are mobile on presynaptic terminals but constrained in synaptic space on rat hippocampal neurons in culture. Additional immobilization of presynaptic α7-nAChRs by antibody crosslinking increases glutamate release capacity as seen in the frequency of spontaneous miniature postsynaptic currents and the size of the readily releasable pool of transmitter. Conversely, blocking glutamate release by targeting tetanus toxin to individual synapses increases α7-nAChR dwell time at presynaptic sites. The effects on release require functional α7-nAChRs and may to depend on CAST/ELKS (calpastatin/glutamine, leucine, lysine, and serine-rich protein), which an unbiased proteomic screen yielded. The results support a new homeostatic regulatory mechanism in which α7-nAChR restrain may be adjusted as needed at presynaptic sites via active zone proteins to maintain transmitter release capability.

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.

Timing is everything, even for cholinergic control.

Synaptic plasticity is widely considered to be a cellular mechanism underlying learning and memory. In this issue of Neuron, Gu and Yakel show that the precise timing of a single cholinergic pulse of activity can determine whether plasticity will occur at a glutamatergic synapse and confer long-term potentiation versus depression.

Endogenous signaling through alpha7-containing nicotinic receptors promotes maturation and integration of adult-born neurons in the hippocampus.

Neurogenesis in the dentate gyrus occurs throughout adult mammalian life and is essential for proper hippocampal function. Early in their development, adult-born neurons express homomeric alpha7-containing nicotinic acetylcholine receptors (alpha7-nAChRs) and receive direct cholinergic innervation. We show here that functional alpha7-nAChRs are necessary for normal survival, maturation, and integration of adult-born neurons in the dentate gyrus. Stereotaxic retroviral injection into the dentate gyrus of wild-type and alpha7-knock-out (alpha7KO) male and female mice was used to label and birthdate adult-born neurons for morphological and electrophysiological measures; BrdU (5-bromo-2-deoxyuridine) injections were used to quantify cell survival. In alpha7KO mice, we find that adult-born neurons develop with truncated, less complex dendritic arbors and display GABAergic postsynaptic currents with immature kinetics. The neurons also have a prolonged period of GABAergic depolarization characteristic of an immature state. In this condition, they receive fewer spontaneous synaptic currents and are more prone to die during the critical period when adult-born neurons are normally integrated into behaviorally relevant networks. Even those adult-born neurons that survive the critical period retain long-term dendritic abnormalities in alpha7KO mice. Interestingly, local infection with retroviral constructs to knockdown alpha7-mRNA mimics the alpha7KO phenotype, demonstrating that the relevant alpha7-nAChR signaling is cell autonomous. The results indicate a profound role for alpha7-nAChRs in adult neurogenesis and predict that alpha7-nAChR loss will cause progressive impairment in hippocampal circuitry and function over time as fewer neurons are added to the dentate gyrus and those that are added integrate less well.

Lateral mobility of nicotinic acetylcholine receptors on neurons is determined by receptor composition, local domain, and cell type.

The lateral mobility of surface receptors can define the signaling properties of a synapse and rapidly change synaptic function. Here we use single-particle tracking with Quantum Dots to follow nicotinic acetylcholine receptors (nAChRs) on the surface of chick ciliary ganglion neurons in culture. We find that both heteropentameric alpha3-containing receptors (alpha3*-nAChRs) and homopentameric alpha7-containing receptors (alpha7-nAChRs) access synaptic domains by lateral diffusion. They have comparable mobilities and display Brownian motion in extrasynaptic space but are constrained and move more slowly in synaptic space. The two receptor types differ in the nature of their synaptic restraints. Disruption of lipid rafts, PDZ-containing scaffolds, and actin filaments each increase the mobility of alpha7-nAChRs in synaptic space while collapse of microtubules has no effect. The opposite is seen for alpha3*-nAChRs where synaptic mobility is increased only by microtubule collapse and not the other manipulations. Other differences are found for regulation of alpha3*-nAChR and alpha7-nAChR mobilities in extrasynaptic space. Most striking are effects on the immobile populations of alpha7-nAChRs and alpha3*-nAChRs. Disruption of either lipid rafts or PDZ scaffolds renders half of the immobile alpha3*-nAChRs mobile without changing the proportion of immobile alpha7-nAChRs. Similar results were obtained with chick sympathetic ganglion neurons, though regulation of receptor mobility differed in at least one respect from that seen with ciliary ganglion neurons. Control of nAChR lateral mobility, therefore, is determined by mechanisms that are domain specific, receptor subtype dependent, and cell-type constrained. The outcome is a system that could tailor nicotinic signaling capabilities to specific needs of individual locations.

Synchronous and asynchronous transmitter release at nicotinic synapses are differentially regulated by postsynaptic PSD-95 proteins.

The rate and timing of information transfer at neuronal synapses are critical for determining synaptic efficacy and higher network function. Both synchronous and asynchronous neurotransmitter release shape the pattern of synaptic influences on a neuron. The PSD-95 family of postsynaptic scaffolding proteins, in addition to organizing postsynaptic components at glutamate synapses, acts transcellularly to regulate synchronous glutamate release. Here we show that PSD-95 family members at nicotinic synapses on chick ciliary ganglion neurons in culture execute multiple functions to enhance transmission. Together, endogenous PSD-95 and SAP102 in the postsynaptic cell appear to regulate transcellularly the synchronous release of transmitter from presynaptic terminals onto the neuron while stabilizing postsynaptic nicotinic receptor clusters under the release sites. Endogenous SAP97, in contrast, has no effect on receptor clusters but acts transcellularly from the postsynaptic cell through N-cadherin to enhance asynchronous release. These separate and parallel regulatory pathways allow postsynaptic scaffold proteins to dictate the pattern of cholinergic input a neuron receives; they also require balancing of PSD-95 protein levels to avoid disruptive competition that can occur through common binding domains.