Activity-independent segregation of excitatory and inhibitory synaptic terminals in cultured hippocampal neurons

Abstract

Cultured hippocampal neurons were used as a model system to address experimentally the spatial and temporal sequence leading to the appropriate sorting of excitatory and inhibitory synaptic terminals to different cellular target domains and the role of neural activity in this process. By using antibodies against glutamic acid decarboxylase 65 (GAD65) and synaptophysin, we examined the development and segregation of GABAergic and non-GABAergic synaptic terminals on single neurons. Electron microscopy confirmed that GAD65-labeled swellings observed using light microscopy corresponded to synaptic boutons. From the time at which GABAergic terminals first appeared, they developed at a more rapid rate on neuronal somata than non-GABAergic terminals did, such that by 18 d in culture, 60% of the total boutons on somata were GABAergic. By contrast, the majority (70%) of boutons on dendrites were non-GABAergic. These data suggest that inhibitory synaptic terminals are targeted preferentially to or maintained on cell somata at the expense of excitatory terminals. Interestingly, non-GABAergic terminals were not inhibited from forming synapses on cell somata, because in the absence of GABAergic terminals they attained the same total somatic terminal density seen in the presence of GABAergic terminals. Chronic blockade of neuronal activity did not affect the differential targeting of GABAergic and non-GABAergic axons; however, it did reduce the extent of dendritic arborization. Our findings support a two-step model for synaptic segregation whereby the majority of terminals is initially targeted in an activity-independent manner to the appropriate cellular domains, but an additional developmental mechanism serves to further restrict and refine the original synaptic distribution.

Fig. 1.

Diagram of a neuron soma and dendrites illustrating modified Scholl analysis. For each of the five primary dendrites in the neuron illustrated, a ray measures the distance from the soma to the farthest extent of the dendritic branches. The rays are averaged and divided by 3, and the resultant length (x) is used for the radius of the innermost circle. For the outer circle,r = 2x. In this way the neuron is divided into four regions: soma, proximal, middle, and distal. See Materials and Methods for details.

Fig. 2.

GAD65 labels synaptic boutons. Electron photomicrographs showing GAD65 immunolabeling of synaptic terminals in 18-d-old cultured hippocampal neurons. In both A andB, DAB reaction product is localized to vesicle-filled synaptic boutons contacting unlabeled dendrites (filled arrows). Open arrows indicate unlabeled axo-dendritic synaptic terminals. Magnification: 20,000× (A); 30,400× (B).

Fig. 3.

Fluorescence (A, B, D, E) and phase-contrast (C, F) photomicrographs showing the distribution of GAD65 (A, D) and synaptophysin (B, E) immunoreactivity in cultured hippocampal neurons. In 4-d-old neurons (A–C), GAD65 immunoreactivity is restricted to cell somata with a slightly greater concentration in what is probably the Golgi apparatus (A, arrow). Synaptophysin immunoreactivity is concentrated in the same area of the soma (B, arrow), but vesicle-like particles are also distributed in growing neurites (arrowheads) and occasional presynaptic clusters are observed (open arrowhead). At 18 d in culture (D–F), GAD65 immunoreactivity is found throughout axons and brightly labeled presynaptic boutons (small arrows), the latter of which correspond precisely to a subpopulation of the synaptophysin-labeled boutons shown inE (small arrows). Virtually all of the synaptophysin label in E is localized to brightly labeled terminals that have been demonstrated to be synaptic boutons (see introductory remarks). Scale bars, 10 μm.

Fig. 4.

Graphs showing increase in synaptic terminal number (A) and dendritic length (B) during development in culture. Synaptic terminal number (A) was determined by counting the total number of synaptophysin-labeled boutons on individual neurons grown in culture. Dendritic length was determined by measuring all dendritic branches of MAP2-labeled neurons.n = 30 except for 18-d-old cells in A, wheren = 20. Error bars represent SEM.

Fig. 5.

Percentage and distribution of terminals that were GABAergic at 11 and 18 d in culture. Bar graph shows the percentage of total (synaptophysin-immunoreactive) terminals that contained GAD65 immunoreactivity within soma, proximal, middle, and distal regions (as defined in Materials and Methods). At 11 d, the percentage of terminals that was GABAergic is significantly greater on somata than elsewhere on the neuron. By 18 d in culture, an even greater proportion of somatic terminals contained GAD65 label.Asterisks indicate columns that are significantly different from all other columns (A, p < 0.02; B, p < 0.001). Error bars represent SEM.

Fig. 6.

Rates of synaptic terminal development on somata (A) and dendritic trees (B). On somata (A), the rate at which GAD-positive terminals form (GAD +; dashed line) increases more rapidly than the rate for GAD-negative terminals (GAD −; solid thin line). The rate at which all terminals develop is indicated by the solid thick line. Differences in slopes (GAD+ vs GAD−) are statistically significant by paired t test: between 4 and 11 d, p < 0.002, and between 11 and 18 d, p < 0.003. On dendrites (B), the rate at which GAD+ terminals form flattens beyond 11 d in culture, whereas GAD− terminals continue to proliferate at a steady rate (p < 0.0002).