Synaptogenesis in hippocampal cultures: evidence indicating that axons and dendrites become competent to form synapses at different stages of neuronal development

Abstract

Hippocampal neurons in culture develop extensive axonal and dendritic arbors and form numerous synapses. Presynaptic specializations occur at sites of contact between axons and somata or dendrites but they do not appear until day 3 in culture, even though numerous contacts between cells develop within the first 24 hr (Fletcher et al., 1991). To determine whether this delay in the appearance of presynaptic specializations could be related to maturational events in the presynaptic axon or in the postsynaptic target, “heterochronic” cocultures were prepared by adding newly dissociated neurons to cultures containing mature neurons. The competence of axons to form presynaptic vesicle clusters in response to contact with the somata or dendrites of mature or immature neurons was determined by immunofluorescent staining for synapsin I or synaptophysin. After only 1 d of coculture, there was a fivefold increase in the number of synapses along the somata and dendrites of the mature neurons, compared to mature neurons cultured alone. If newly dissociated neurons were labeled with a fluorescent dye before coculture, dye-labeled axons frequently were colocalized with presynaptic specializations on mature cells. In contrast, when the axons of mature neurons contacted immature neurons, synapses were first observed only after coculture for 3 d. These results suggest that the axons of hippocampal neurons have the capacity to form presynaptic specializations soon after they emerge, provided they encounter appropriate targets, but that the cell bodies and dendrites of hippocampal neurons are not capable of inducing the formation of presynaptic specializations until they reach a critical stage of maturation.

Cultured rat hippocampal neural progenitors generate spontaneously active neural networks

“…These E18 networks formed functional synapses that fired spontaneously within 7 days, consistent with previous studies (36)(37)(38). The onset of spontaneous activity in progenitorderived networks was delayed by 10-14 days relative to the E18 networks.…”

Calcium Channel Density and Hippocampal Cell Death with Age in Long-Term Culture

“…Conceivably, the rapid phase of cell death occurring during the first 3-6 DIV is more analogous to apoptotic cell death, which occurs during early development (Oppenheim, 1991). During this early phase, hippocampal neurons in culture are relatively immature and show little evoked synaptic activity (Basarsky et al, 1994;Fletcher et al, 1994); however, we also consistently observed evidence of necrosis, particularly for neurons 15-28 DIV, as indicated by the remnants of swollen cell bodies. It seems feasible that the initial increase in C a 2ϩ current density may contribute to both the early growth phase and developmentally programmed apoptosis, whereas the later gradual phase of C a 2ϩ current density increase may eventually result in heightened vulnerability of neurons to degenerative or necrotic death.…”

“…Accompanying the alterations in Ca 2ϩ channel density are ongoing morphological changes in neurons, including the extension of processes and the growth of the soma, which are quite rapid in the first week (Banker and Cowan, 1979;Basarsky et al, 1994;Fletcher et al, 1994) (Figs. 1, 3A).…”

SynCAMs Organize Synapses through Heterophilic Adhesion

“…In the absence of antibodies applicable for immunostaining, we expressed epitope-tagged constructs in dissociated hippocampal neurons at 7 d.i.v., when neurons in culture can rapidly form synapses (Fletcher et al, 1994). In these constructs, one extracellular flag epitope was inserted C-terminal of the third Ig-like domain to minimally interfere with putative intracellular sorting signals.…”