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.

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.

Capabilities of neurexins in the chick ciliary ganglion.

Transcellular interactions between neuroligins (NL) and beta-neurexin have been widely documented to promote maturation and function of both glutamatergic and GABAergic synapses. Recently it has been shown that neuroligin-1 plays a similar role at nicotinic synapses on chick ciliary ganglion neurons in culture, acting from the postsynaptic side to enhance transmitter release from adjacent cholinergic terminals and boost nicotinic input to the cells. We show here that the ciliary ganglion expresses three forms of neuroligin as well as two beta-neurexins and an alpha-neurexin. Overexpression of the beta-neurexins, but not the alpha-neurexin, can induce clustering of endogenous PSD-95 in adjacent neurons, presumably engaging neuroligin in the postsynaptic cell. The trans effects of beta-neurexins are selective; though both alpha3- and alpha7-containing nicotinic receptors are available on opposing cells, beta-neurexins induce coclustering of alpha3- but not alpha7-containing nicotinic receptors. Overexpression of other putative synaptogenic molecules, including SynCAM and L1, are ineffective at trans-clustering of PSD-95 on adjacent neurons. The beta-neurexins also exert a cis effect, coclustering presynaptic markers along with beta-neurexin in neurites juxtaposed to postsynaptic proteins, consistent with organizing presynaptic components as well. Striated muscle, the synaptic target of ciliary neurons in vivo, also expresses neuroligin. The results demonstrate that NL and neurexins are present at multiple sites in nicotinic cholinergic pathways and suggest the possibility of both cis- and trans-interactions to influence 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.

PDZ-containing proteins provide a functional postsynaptic scaffold for nicotinic receptors in neurons.

Protein scaffolds are essential for specific and efficient downstream signaling at synapses. Though nicotinic receptors are widely expressed in the nervous system and influence numerous cellular events due in part to their calcium permeability, no scaffolds have yet been identified for the receptors in neurons. Here we show that specific members of the PSD-95 family of PDZ-containing proteins are associated with specific nicotinic receptor subtypes. At postsynaptic sites, the PDZ scaffolds are essential for maturation of functional nicotinic synapses on neurons. They also help mediate downstream signaling as exemplified by activation of transcription factors. By tethering components to postsynaptic nicotinic receptors, PDZ scaffolds can organize synaptic structure and determine which calcium-dependent processes will be subject to nicotinic modulation.

Potentiation of alpha7-containing nicotinic acetylcholine receptors by select albumins.

Nicotinic receptors containing alpha7 subunits are ligand-gated ion channels widely distributed in the nervous system; they influence a diverse array of events because of their high relative calcium permeability. We show here that nicotine-induced whole-cell responses generated by such receptors can be dramatically potentiated in a rapidly reversible manner by some but not all albumins. The potentiation involves increases both in potency and efficacy with no obvious differences in rise and fall times of the response. The potentiation is not reduced by removing absorbed components; it is abolished by proteolysis, suggesting that the albumin protein backbone is essential. The fact that some albumins are ineffective indicates that minor differences in amino acid sequence may be critical. Experiments with open channel blockers indicate that the potentiation involves increased responses from active receptors rather than recruitment of receptors from a previously silent pool. Single channel recordings reveal that the potentiation correlates with increased single channel opening probability, reflected in increased frequency of channel opening and increased mean channel open time. The potentiation can be exploited to overcome blockade by noncompetitive inhibitors such as beta-amyloid peptide. The results raise the possibility that endogenous compounds use the site to modulate receptor function in vivo, and suggest that the receptors may represent useful targets for therapeutic intervention in cases where they have been implicated in neuropathologies such as Alzheimer's disease.

Developmental expression of nicotinic receptors in the chick and human spinal cord.

Naturally occurring programmed cell death of lumbar motor neurons in the chick spinal cord occurs between embryonic day (E) 6 and E12; whereas, a peak of motor neuron degeneration in the human spinal cord occurs between 12 and 16 weeks gestation. One of the major neurotransmitters, acetylcholine, is released from the embryonic motor neuron early in development and is thought to be responsible for early muscle activity that serves as a signal for regulating motor neuron survival. The effects of acetylcholine are mediated by two functionally distinct classes of receptors; namely, muscarinic and nicotinic with nicotinic receptors being used at the neuromuscular synapse. In this study, we determined the developmental expression profile of nicotinic acetylcholine receptor subunits in the chick and human lumbar motor neurons and skeletal muscle using reverse transcription polymerase chain reaction, immunoblots, and immunocytochemistry. Our results show that, in the chick, nicotinic receptor subunits alpha1, alpha4, alpha7, alpha8, and beta2 appear to be regulated during the process of naturally occurring motor neuron cell death in the spinal cord. A new finding was the expression of alpha8 mRNA and protein from E4.5 through E7 in chick motor neurons. Interestingly, we also found that, at E14, alpha8 protein was localized only in sensory dorsal horn neurons. In the developing human spinal cord, we determined that nicotinic receptor subunits alpha1, alpha2, alpha3, alpha4, alpha7, beta2, and beta3 were expressed before the programmed cell death period, and alpha2, alpha4, alpha7, beta2, beta3, and beta4 were expressed during the programmed cell death period. Our data demonstrate that neuronal and muscle nicotinic receptor mRNAs and proteins are expressed during important embryonic periods. This finding raises the possibility that nicotinic receptors play an important role in the spinal cord and skeletal muscle during early development.

Nicotinic alpha 7 receptors: synaptic options and downstream signaling in neurons.

Nicotinic receptors are cation-ion selective ligand-gated ion channels that are expressed throughout the nervous system. Most have significant calcium permeabilities, enabling them to regulate calcium-dependent events. One of the most abundant is a species composed of the alpha 7 gene product and having a relative calcium permeability equivalent to that of NMDA receptors. The alpha 7-containing receptors can be found presynaptically where they modulate transmitter release, and postsynaptically where they generate excitatory responses. They can also be found in perisynaptic locations where they modulate other inputs to the neuron and can activate a variety of downstream signaling pathways. The effects the receptors produce depend critically on the sites at which they are clustered. Instructive preparations for examining alpha 7-containing receptors are the rat hippocampus, where they are thought to play a modulatory role, and the chick ciliary ganglion, where they participate in throughput transmission as well as regulatory signaling. Relatively high levels of alpha 7-containing receptors are found in the two preparations, and the receptors display a variety of synaptic options and functions in the two cases. Progress is starting to be made in understanding the mechanisms responsible for localizing the receptors at specific sites and in identifying components tethered in the vicinity of the receptors that may facilitate signal transduction and downstream signaling.