Darwin K. Berg

Nicotinic Receptors in the Development and Modulation of CNS Synapses

Work from the laboratory of L. W. R. has been supported by grants from the National Institutes of Health, the National Institute on Drug Abuse, the Council for Tobacco Research, and the McKnight Foundation. Work from the laboratory of D. K. B. has been supported by grants from the National Institutes of Health, the Muscular Dystrophy Association, and the Council for Tobacco Research.

beta -Amyloid peptide blocks the response of alpha 7-containing nicotinic receptors on hippocampal neurons.

Alzheimers disease produces a devastating decline in mental function, with profound effects on learning and memory. Early consequences of the disease include the specific loss of cholinergic neurons in brain, diminished cholinergic signaling, and the accumulation of β-amyloid peptide in neuritic plaques. Of the nicotinic acetylcholine receptors at risk, the most critical may be those containing the α7 gene product (α7-nAChRs), because they are widespread, have a high relative permeability to calcium, and regulate numerous cellular events in the nervous system. With the use of whole-cell patch–clamp recording we show here that nanomolar concentrations of β-amyloid peptides specifically and reversibly block α7-nAChRs on rat hippocampal neurons in culture. The block is noncompetitive, voltage-independent, and use-independent and is mediated through the N-terminal extracellular domain of the receptor. It does not appear to require either calcium influx or G protein activation. β-Amyloid blockade is likely to be a common feature of α7-nAChRs because it applies to the receptors at both somato-dendritic and presynaptic locations on rat hippocampal neurons and extends to homologous receptors on chick ciliary ganglion neurons as well. Because α7-nAChRs in the central nervous system are thought to have numerous functions and recently have been implicated in learning and memory, impaired receptor function in this case may contribute to cognitive deficits associated with Alzheimers disease.

Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes

A family of genes encoding neuronal acetylcholine receptor (AChR) subunits has been identified and cloned from vertebrates. Expression studies have implied that as few as one or two kinds of subunits may be sufficient to construct neuronal AChRs and that multiple pair-wise combinations of the gene products are capable of generating functional receptors. We show here that a class of AChRs with a predominantly synaptic location on neurons contains receptors having at least three types of subunits and that the subunits are encoded by the alpha 3, beta 4, and alpha 5 AChR genes. In addition, we show that a class of extrasynaptic AChRs on the same neurons contains the alpha 7 subunits but lacks the alpha 3, beta 4, and alpha 5 subunits. The results demonstrate that native AChRs on neurons are more complex in composition than previously appreciated and suggest that constraints on subunit interactions limit the kinds of receptor species produced.

Nicotinic receptors that bind α-bungarotoxin on neurons raise intracellular free ca2+

Abstract Many populations of vertebrate neurons have a membrane component that binds α-bungarotoxin and cholinergic ligands. Despite the abundance of this component and its similarities to nicotinic receptors, its function has remained controversial. Using a fluorescence assay, we show here that activation of the component elevates the intracellular concentration of free Ca 2+ demonstrating a receptor function for the toxin-binding component. Whole-cell voltage-clamp and intracellular recordings did not detect a significant current resulting from receptor activation, possibly because the currents were small or the receptors rapidly desensitized. The rise in intracellular free Ca 2+ caused by the receptor was prevented by Ca 2+ channel blockers. This suggests a signaling cascade likely to have important regulatory consequences for the neuron.

Neuronal acetylcholine receptors that bind α-bungarotoxin with high affinity function as ligand-gated ion channels

Neuronal membrane components that bind alpha-bungarotoxin with high affinity can increase intracellular levels of free calcium, demonstrating the components function as nicotinic receptors. Though such receptors often contain the alpha 7 gene product, which by itself can produce ionotropic receptors in Xenopus oocytes, numerous attempts have failed to demonstrate an ion channel function for the native receptors on neurons. Using rapid application of agonist, we show here that the native receptors are ligand-gated ion channels which are cation selective, prefer nicotine over acetylcholine, and rapidly desensitize. Much of the calcium increase caused in neurons by the receptors under physiological conditions appears to result from their depolarizing the membrane sufficiently to trigger calcium influx through voltage-gated channels.

Neuronal acetylcholine receptors that bind alpha-bungarotoxin mediate neurite retraction in a calcium-dependent manner

Neuronal membrane components that bind alpha-bungarotoxin with high affinity have only recently been shown unambiguously to function as nicotinic receptors. Activation of the receptors increases intracellular levels of free calcium in neurons. In the chick ciliary ganglion, where the receptors have been studied in some detail, they have been shown to have a predominantly nonsynaptic location on neurons and may be concentrated on pseudodendrites emerging from the somata. This has raised questions about the physiological significance of the receptors for the neurons. Here we show that activation of the receptors on isolated ciliary ganglion neurons in cell culture produces neurite retraction. Focal application of either nicotine or ACh at low concentrations induces the retraction, and alpha-bungarotoxin blocks the effect. The retraction requires external calcium and is confined to the individual neurite stimulated with agonist. Brief exposure to elevated concentrations of K+ also induces neurite retraction, and both the K(+)-induced and the nicotine-induced retractions can be prevented by the calcium channel blocker omega-conotoxin. The results suggest that activation of the alpha-bungarotoxin-binding nicotinic receptors on neurites triggers activation of voltage-gated calcium channels presumably by depolarizing the membrane, and that together they permit sufficient calcium to enter the neurite to prevent further outgrowth and induce retraction.

The α5 gene product assembles with multiple acetylcholine receptor subunits to form distinctive receptor subtypes in brain

The acetylcholine receptor (AChR) alpha 5 gene has been classified as a member of the AChR gene family based on sequence homology. Expression studies, however, have yet to identify a function for the alpha 5 gene product or even to demonstrate an interaction with known AChR subunits. We report here that the alpha 5 gene product is identical to the 49 kd protein previously found on immunoblots of AChRs purified from brain and ciliary ganglia. In brain the alpha 5 gene product is present both in alpha 3- and in alpha 4-based receptor subtypes, while in the ganglion it is found in an alpha 3-based receptor subtype concentrated in postsynaptic membrane. Immunoprecipitation experiments with subunit-specific monoclonal antibodies indicate that some native AChRs are likely to have at least three kinds of subunits, with two being of the alpha type. These findings support new views about the construction of AChRs in neurons.

Synaptic Currents Generated by Neuronal Acetylcholine Receptors Sensitive to α-Bungarotoxin

Abstract Nicotinic acetylcholine receptors are widely distributed throughout the nervous system, but their functions remain largely unknown. One of the most abundant is a class of receptors that contains the α7 gene product, has a high relative permeability to calcium, and binds α-bungarotoxin. Here, we report that receptors sensitive to α-bungarotoxin, though concentrated in perisynaptic clusters on neurons, can generate a large amount of the synaptic current. Residual currents through other nicotinic receptors are sufficient to elicit action potentials, but with slower rise times. This demonstrates a postsynaptic response for α-bungarotoxin-sensitive receptors on neurons and suggests that the functional domain of the postsynaptic membrane is broader than previously recognized.

Sequential interplay of nicotinic and GABAergic signaling guides neuronal development.

GABA (γ-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.

Voltage-Gated Channels Block Nicotinic Regulation of CREB Phosphorylation and Gene Expression in Neurons

Synaptic activation of the transcription factor CREB and downstream gene expression usually depend on calcium influx aided by voltage-gated calcium channels. We find that nicotinic signaling, in contrast, activates CREB and gene expression in ciliary ganglion neurons both in culture and in situ only if voltage-gated channels are silent. The nicotinic response requires calcium influx and release from internal stores and acts through CaMK and MAPK pathways to sustain activated CREB. Voltage-gated channels mobilize CaMK to activate CREB initially, but they also enable calcineurin and PP1 to terminate the activation before transcription is affected. L-type voltage-gated channels dominate the outcome and block the effects of nicotinic signaling on transcription. This demonstrates a novel aspect of activity-dependent gene regulation.