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.

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We have analyzed the polarity orientation of microtubules in the axons and dendrites of cultured rat hippocampal neurons. As previously reported of axons from other neurons, microtubules in these axons are uniform with respect to polarity; (+)-ends are directed away from the cell body toward the growth cone. In sharp contrast, microtubules in the mid-region of the dendrite, -75 Jtm from the cell body, are not of uniform polarity orientation. Roughly equal proportions of these microtubules are oriented with (+)-ends directed toward the growth cone and ( + )-ends directed .toward the cell body. At distances within 15 ,um of the growth cone, however, microtubule polarity orientation in dendrites is similar to that in axons; (+)-ends are uniformly directed toward the growth cone. These findings indicate a clear difference between axons and dendrites with respect to microtubule organization, a difference that may underlie the differential distribution of organelles within the neuron.Vertebrate neurons generate and maintain two morphologically and functionally distinct types of neurites, axons and dendrites (1-6). It has long been recognized that axons and dendrites differ in their complements of cytoplasmic organelles (1, 6). Most notable in this regard, ribosomes and Golgi elements are present in dendrites but are absent from axons. What is the basis for the nonuniform distribution of organelles in neurons? Several lines of evidence indicate that the distribution of organelles in a cell reflects active transport processes that selectively convey organelles from their sites of synthesis and assembly to other locations in the cell (7, 8).These observations raise the possibility that many of the differences between the organelle composition of axons and dendrites are produced by differences in the organization of the transport systems that convey materials from the cell body into the axon or dendrite.The transport of organelles is a microtubule-based process; microtubules provide the substrate for organelle translocation and, by virtue of their intrinsic polarity, influence the directionality of transport (7-9). The intrinsic polarity of microtubules is based on the asymmetry of the tubulin subunit and its self-assembly characteristics; the (+)-end is preferred for subunit addition over the (-)-end (10, 11). Microtubule-based translocators convey organelles specifically toward either the ( + )-or the ( -)-end ofthe microtubule (7-9). In the axon, microtubules are uniform with respect to polarity, with the (+ )-ends directed away from the cell body (12-15). Thus, only those organelles that translocate toward (+ )-ends of microtubules will be conveyed from the cell body into the axon.Do microtubules in dendrites have the same polarity orientation as those in axons? To date, information concerning the polarity orientation of dendritic microtubules derives from a few atypical cell types. In the dendrite-like processes of teleost retinal cone cells (16) and frog primary olfactory neurons (17), microtubules are unifo...

As a first step toward elucidating mechanisms involved in the sorting of synaptic vesicle proteins in neurons, we have used immunofluorescence microscopy to determine the distribution of two synaptic vesicle proteins, synapsin I and synaptophysin, in hippocampal neurons developing in culture. In mature cultures, synapsin I and synaptophysin immunoreactivity was concentrated in puncta that were restricted to sites where axons contacted neuronal cell bodies or dendrites. Electron-microscopic immunocytochemistry demonstrated that these puncta corresponded to vesicle-filled axonal varicosities that were exclusively presynaptic. At early stages of development, before cell-cell contact, both synapsin I and synaptophysin were preferentially localized in axons, where they were particularly concentrated in the distal axon and growth cone. In axons that did not contact other cells, immunostaining for these two proteins had a granular appearance, which persisted for at least 7 d, but focal accumulations of vesicles comparable to those seen at sites of synaptic contact were not observed. When neurons contacted one another, numerous puncta of synapsin I and synaptophysin formed within the first week in culture. Double-label immunofluorescence demonstrated that the two vesicle antigens were closely codistributed throughout these stages of development. These observations demonstrate that synaptic vesicle proteins assume a polarized distribution within nerve cells beginning early in development, as soon as the axon can be identified. In contrast, differences in microtubule polarity orientation that distinguish mature axons and dendrites, and that have been proposed to account for the selective sorting of some materials in nerve cells, first appear at a subsequent stage of development. The selective distribution of synaptic vesicle proteins to the axon occurs in isolated cells, independent of interactions with other cells. In contrast, the formation of large clusters of vesicles typical of presynaptic specializations requires contact with an appropriate postsynaptic target. Thus, in cultured hippocampal neurons, the localization of synaptic vesicles in presynaptic specializations is the result of sorting mechanisms intrinsic to individual neurons as well as to mechanisms mediated by cell-cell contact.