Gary Banker

Culturing hippocampal neurons

We provide protocols for preparing low-density dissociated-cell cultures of hippocampal neurons from embryonic rats or mice. The neurons are cultured on polylysine-treated coverslips, which are suspended above an astrocyte feeder layer and maintained in serum-free medium. When cultured according to this protocol, hippocampal neurons become appropriately polarized, develop extensive axonal and dendritic arbors and form numerous, functional synaptic connections with one another. Hippocampal cultures have been used widely for visualizing the subcellular localization of endogenous or expressed proteins, for imaging protein trafficking and for defining the molecular mechanisms underlying the development of neuronal polarity, dendritic growth and synapse formation. Preparation of glial feeder cultures must begin 2 weeks in advance, and it takes 5 d to prepare coverslips as a substrate for neuronal growth. Dissecting the hippocampus and plating hippocampal neurons takes 2–3 h.

The distribution of glutamate receptors in cultured rat hippocampal neurons : postsynaptic clustering of AMPA-selective subunits

The distribution of several glutamate receptor subunits was investigated in cultured rat hippocampal neurons by in situ hybridization and immunocytochemistry. The AMPA/kainate-selective receptors GluR1-6 exhibited two patterns of mRNA expression: most neurons expressed GluR1, R2, and R6, whereas only about 20% expressed significant levels of GluR3, R4, and R5. By immunocytochemistry, the metabotropic glutamate receptor mGluR1 alpha was detectable only in a subpopulation of GABAergic interneurons. GluR1 and GluR2/3 segregated to the somatodendritic domain within the first week in culture, even in the absence of synaptogenesis. Glutamate receptor-enriched spines developed later and were present only on presumptive pyramidal cells, not on GABAergic interneurons. Clusters of GluR1 and GluR2/3 completely colocalized and were restricted to a subset of postsynaptic sites. Thus, glutamate receptor subunits exhibit both a cell type-specific expression and a selective subcellular localization.

Getting the message from the gene to the synapse: sorting and intracellular transport of RNA in neurons

A key question in cellular neurobiology is how neurons target molecules to cellular microdomains at a distance from the nucleus. Of special importance are the thousands of postsynaptic sites that form the basis for synaptic communication. Recent evidence suggests that an important aspect of molecular trafficking involves differential sorting, selective intracellular transport, and docking of particular mRNA molecules and associated protein synthetic machinery at postsynaptic sites. This offers the potential for local regulation of the production of key proteins in response to conditions at individual synapses. This article reviews what is known about the mechanisms of mRNA trafficking in neurons and in other cells ranging from oocytes to oligodendrocytes, and considers the possible role that mRNA trafficking and the resulting local synthesis of particular proteins may play in cellular function.

Two Distinct Mechanisms Target Membrane Proteins to the Axonal Surface

We have investigated the trafficking of two endogenous axonal membrane proteins, VAMP2 and NgCAM, in order to elucidate the cellular events that underlie their polarization. We found that VAMP2 is delivered to the surface of both axons and dendrites, but preferentially endocytosed from the dendritic membrane. A mutation in the cytoplasmic domain of VAMP2 that inhibits endocytosis abolished its axonal polarization. In contrast, the targeting of NgCAM depends on sequences in its ectodomain, which mediate its sorting into carriers that preferentially deliver their cargo proteins to the axonal membrane. These observations show that neurons use two distinct mechanisms to polarize proteins to the axonal domain: selective retention in the case of VAMP2, selective delivery in the case of NgCAM.

A Change in the Selective Translocation of the Kinesin-1 Motor Domain Marks the Initial Specification of the Axon

We used the accumulation of constitutively active kinesin motor domains as a measure of where kinesins translocate in developing neurons. Throughout development, truncated Kinesin-3 accumulates at the tips of all neurites. In contrast, Kinesin-1 selectively accumulates in only a subset of neurites. Before neurons become polarized, truncated Kinesin-1 accumulates transiently in a single neurite. Coincident with axon specification, truncated Kinesin-1 accumulates only in the emerging axon and no longer appears in any other neurite. The translocation of Kinesin-1 along a biochemically distinct track leading to the nascent axon could ensure the selective delivery of Kinesin-1 cargoes to the axon and hence contribute to its molecular specification. Imaging YFP-tagged truncated Kinesin-1 provides the most precise definition to date of when neuronal polarity first emerges and allows visualization of the molecular differentiation of the axon in real time.

Differential subcellular localization of particular mRNAs in hippocampal neurons in culture.

In situ hybridization was used to assess the subcellular distribution of mRNAs encoding several important neuronal proteins in hippocampal neurons in culture. mRNA encoding GAP-43, a protein that is largely excluded from dendrites, was restricted to nerve cell bodies, as were mRNAs encoding neurofilament-68 and beta-tubulin, which are prominent constituents of dendrites and of axons. In contrast, mRNA encoding MAP-2, a protein that is selectively distributed in dendrites and cell bodies, was present in both dendrites and cell bodies. These results demonstrate that different mRNAs are differentially distributed within individual hippocampal neurons. Taken together with previous findings from other laboratories, our results suggest that only a limited set of mRNAs are available for local translation within dendrites.

The economics of neurite outgrowth — the addition of new membrane to growing axons

Recent studies have shown that axonal growth is disrupted by treatments that block the synthesis of membrane components or their delivery by microtubule-based transport. This implies that a continuous supply of newly synthesized membrane components is necessary to sustain growth. In contrast, no clear consensus has yet been achieved about the site of insertion of new membrane components in the membrane of the growing axon, despite the application of new and refined biophysical and molecular techniques to the study of this issue. Until the site of insertion of new membrane components is resolved, little progress can be made in defining the feedback mechanisms by which the supply of new membrane components is co-ordinated with the demands of growth, particularly in cases where the dynamics of neurite growth change from minute to minute.

Pathogenic huntingtin inhibits fast axonal transport by activating JNK3 and phosphorylating kinesin

Selected vulnerability of neurons in Huntingtons disease suggests that alterations occur in a cellular process that is particularly critical for neuronal function. Supporting this idea, pathogenic Htt (polyQ-Htt) inhibits fast axonal transport (FAT) in various cellular and animal models of Huntingtons disease (mouse and squid), but the molecular basis of this effect remains unknown. We found that polyQ-Htt inhibited FAT through a mechanism involving activation of axonal cJun N-terminal kinase (JNK). Accordingly, we observed increased activation of JNK in vivo in cellular and mouse models of Huntingtons disease. Additional experiments indicated that the effects of polyQ-Htt on FAT were mediated by neuron-specific JNK3 and not by ubiquitously expressed JNK1, providing a molecular basis for neuron-specific pathology in Huntingtons disease. Mass spectrometry identified a residue in the kinesin-1 motor domain that was phosphorylated by JNK3 and this modification reduced kinesin-1 binding to microtubules. These data identify JNK3 as a critical mediator of polyQ-Htt toxicity and provide a molecular basis for polyQ-Htt–induced inhibition of FAT.

Delayed localization of synelfin (synuclein, NACP) to presynaptic terminals in cultured rat hippocampal neurons.

Synelfin is a presynaptic protein of unknown function that is differentially regulated in the avian song control circuit during the critical period for song learning; in humans, it gives rise to an amyloidogenic peptide found in senile plaques of Alzheimers disease. To gain insight into the potential involvement of synelfin in synapse development, we investigated its expression in neurons cultured from the embryonic rat hippocampus. These neurons express a variety of defined synaptic proteins, and form numerous synaptic connections after several days in culture. Synapsin I, a synaptic vesicle-associated protein, was detected within one day after the neurons were put in culture, but significant immunoreactivity for synelfin was not detected until approximately 5 days in vitro (DIV). By 3 DIV, synapsin-positive puncta (previously shown to correspond to presynaptic specializations) were detected surrounding the soma and proximal dendritic processes, whereas comparable aggregations of synelfin did not appear until several days later. By 14 DIV the punctate concentrations of synelfin and synapsin overlapped completely. Thus synelfin is expressed in these cultured neurons and eventually becomes localized to presynaptic terminals, but it is absent from these specializations when they first form. We conclude that presynaptic terminals can change in molecular composition, and that synelfin is associated with later stages in synaptic development or modulation.

Aligned microcontact printing of micrometer-scale poly-L-Lysine structures for controlled growth of cultured neurons on planar microelectrode arrays

We describe a method for producing high-resolution chemical patterns on surfaces to control the attachment and growth of cultured neurons. Microcontact printing has been extended to allow the printing of /spl mu/m-scale protein lines aligned to an underlying pattern of planar microelectrodes. Poly-L-lysine (PL) lines have been printed on the electrode array for electrical studies on cultured neural networks. Rat hippocampal neurons showed degree of attachment selectivity to the PL and produced neurites that faithfully grew onto the electrode recording sites.