The Polarized Sorting of Membrane Proteins Expressed in Cultured Hippocampal Neurons Using Viral Vectors

One model of neuronal polarity (Dotti and Simons, 1990) proposes that neurons and polarized epithelia use similar mechanisms to sort membrane proteins. To explore this hypothesis, we used viral vectors to express proteins in cultured neurons and assessed their distribution using quantitative immunofluorescence microscopy. Basolateral epithelial proteins were polarized to dendrites; more significantly, mutations of sequences required for their basolateral targeting in epithelia also disrupted dendritic targeting. Unexpectedly, apical proteins were not polarized to axons but were expressed at roughly equal amounts in dendrites and axons. These data provide strong evidence that targeting of basolateral and dendritic proteins depends on common mechanisms. In contrast, the sorting of proteins to the axon requires signals that are not present in apical proteins

Assembly of α4β2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal Mammalian Cells and in Cultured Midbrain Neurons

Fura-2 recording of Ca^(2+) influx was used to show that incubation in 1 μM nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHβE-sensitive component that presumably corresponds to α4β2 receptors. To study changes in α4β2 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the α4 or β2 M3-M4 intracellular loops, and these subunits were coexpressed in human embryonic kidney (HEK) 293T cells and cultured ventral midbrain neurons. The fluorescent receptors resembled wild-type receptors in maximal responses to ACh, dose-response relations, ACh-induced Ca^(2+) influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent α4 and β2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of α4β2 receptors, coexpression of the α4-YFP and β2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic α4β2 nAChRs displayed greater FRET than receptors inside the soma, and in HEK293T cells, a similar increase was noted for receptors that were translocated to the surface during PKC stimulation. When cultured transfected midbrain neurons were incubated in 1 μMnicotine, there was increased FRET in the cell body, denoting increased assembly of α4β2 receptors. Thus, changes in α4β2 receptor assembly play a role in the regulation of α4β2 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca^(2+)-stimulated pathways

Inhibition of axonal growth by brefeldin A in hippocampal neurons in culture

The outgrowth of neuronal processes involves a great increase in the surface area of the cell. The supply of membrane material necessarily must be coordinated with the demands for neurite growth. The selective growth of only one or two neurites at any given time during the development of polarity raises the possibility that the production of materials by the soma is limiting for growth (Dotti and Banker, 1987; Dotti et al., 1988; Goslin and Banker, 1990). To examine the role of the availability of membrane components during the development of polarity and axonal elongation, we treated neurons with brefeldin A, an antibiotic that disrupts the trafficking of vesicles from the Golgi complex to the plasma membrane. Treatment with brefeldin A (1 �g/ml) inhibited axonal growth within 0.5 hr; in unpolarized cells it prevented the formation of an axon. These results indicate that the availability of membrane components of Golgiderived vesicles is required for axonal growth and hence the development of polarity. Inhibitors of protein and RNA synthesis also blocked axonal growth and the development of polarity, but over a much slower time course. This suggests that the full complement of proteins and mRNAs required for the initial development of polarity is present for several hours before polarity is actually established. Key words: polarity; neurite outgrowth; hippocampal neurons; brefeldin A; Golgi complex; axonal transpor

Mapping of Presynaptic Nicotinic Acetylcholine Receptors Using Fluorescence Imaging of Neuritic Calcium

Neuronal nicotinic receptors (nAChRs) appear to function at both pre- and postsynaptic sites, to modulate the release of neurotransmitter, and to mediate synaptic transmission, respectively. Localization of functional nAChRs at presynaptic structures has only been possible under the best of circumstances where the presynaptic structure is very large allowing direct nAChR channel recording. We report here a novel and simple method that allows the visualization of stimulus-evoked changes in Fura-2 fluorescence in the presynaptic structures of essentially any neuron type in vitro. Following ‘loading’ of all neurons by incubation with the calcium-sensitive dye, Fura-2-AM, we selectively reduced the fluorescent signal in the postsynaptic neuron by injecting the Fura-2 quenching agent, Mn2+, into the postsynaptic neuron. After quenching, nicotine treatment elicits calcium transients that can be observed in spatially distinct regions of neurite bundles contacting the Mn2+-infused neuron. Thus, the approach described allows one to readily map the distribution of activated nAChRs on presynaptic inputs in vitro