Afferent innervation influences the development of dendritic branches and spines via both activity-dependent and non-activity-dependent mechanisms

Abstract

The present investigation uses an in vitro co-culture system to study the role of afferent innervation in early development and differentiation of hippocampal neurons. Our experiments indicate that the formation of two key morphological features, dendritic branches and dendritic spines, is induced by afferent innervation. Hippocampal neurons develop multiple dendritic branches and spines only when extensively innervated by living axonal afferents. No morphological changes occurred when hippocampal neurons were plated on other cell surfaces such as fixed axons or astrocytes. Furthermore, afferents exerted their effect locally on individual dendrites that they contacted. When one portion of the dendritic arbor of a neuron was contacted by afferents and the other portion was not, morphological effects were restricted to the innervated dendrites. Innervation of some of the dendrites on a neuron did not produce global effects throughout the neuron. Afferent-induced dendritic branching is independent of activity, since branch induction was unaffected by chronic application of TTX or glutamate receptor blockers. In contrast, the formation of dendritic spines is influenced by activity. The number of developing spines was reduced when TTX or a cocktail of three glutamate receptor blockers was applied. Blockade of individual AMPA, NMDA, or metabotropic glutamate receptors did not affect the number of spines. These results, taken together, demonstrate that afferents can have a prominent influence on the development of postsynaptic target cells via both activity-dependent and non-activity-dependent mechanisms, indicating the presence of multiple signals. Accordingly, this suggests an important interplay between pre- and postsynaptic elements early in development.

Fig. 1.

Innervation of neurons plated on a network of entorhinal axons and on polylysine. A, Hippocampal neurons grown on a dense net of entorhinal axons are embedded in an extensive meshwork of entorhinal axons, whereas neurons grown on polylysine (B) are contacted by only a few axons from neighboring neurons at that cell density. C, Immunofluorescent staining with an antibody against synaptophysin shows punctated staining along the dendrites of a hippocampal pyramidal neuron growing on the axonal net for 14 d, thus demonstrating the formation of synapses between the entorhinal axonal net and hippocampal neurons. The antibody, a marker for presynaptic terminals, stained only parts of the axons contacting the postsynaptic neuron.D, Electron micrograph showing the presence of numerous synapses between hippocampal neurons and the axonal net. Scale bars:A–C, 25 μm; D, 0.5 μm.

Fig. 2.

Development of dendritic branching dependent on the presence of innervation. Micrograph of hippocampal neurons growing off (A) and on (B) a net of entorhinal axons. Neurons growing on polylysine developed basal dendrites with no or only few branches (A), whereas neurons growing in contact with the entorhinal axons have a highly differentiated dendritic tree with numerous branches (B). C, Time course of development of dendritic branches for neurons growing on and off the axonal net. Neurons growing in contact with the axons started to develop highly branched dendritic trees after 6 d in vitro. After 14 d in culture these neurons had formed on average four times more branches than neurons growing off the net. Scale bar (shown inA): 25 μm. Error bars represent SEM.

Fig. 3.

Role of activity and glutamate receptors in dendritic branching. The number of dendritic branches was not affected by chronic blockade of spontaneous activity with TTX (n = 48). Also, the chronic blockade of different glutamate receptors (AMPA, NMDA, metabotropic) by DNQX (n = 32), AP-5 (n = 27), and MCPG (n = 15) alone or combined in a cocktail (n = 32) did not affect the branching. The average number of branches in treated cultures is shown as percentage of controls. Controls were performed in parallel to each experimental group to allow comparison (average number of branches in controls = 16.1 ± 1.3; n = 56). Error bars represent SEM.

Fig. 4.

Role and specificity of different cell surfaces on dendritic branching. Neurons were grown on a fixed carpet of axons (n = 18) and a monolayer of living cortical astrocytes (n = 18) for 14 d. On both surfaces hippocampal neurons remain unbranched. Only neurons contacting living axons developed stable dendritic branches (n = 48). The average number of branches is depicted. Error bars represent SEM.

Fig. 5.

Localized induction of branching by afferents.A, Schematic illustration of our culture system, where neurons were grown along a border of afferents with one part of their dendritic tree touching the afferent net while the other part was growing on polylysine. B, Micrograph illustrating how axons extending from the explants (right and left lanes) formed sharp borders along a stripe of a nongrowth-permissive substrate (middle lane).C, Micrograph illustrating the presence of the afferent border even after removal of the nonadhesive substrate (right side). D, E, Example of a DiI-labeled neuron with phase contrast growing along a border of afferent axons for 11 d, after the nonadhesive substrate had been peeled off. Only dendrites in contact with the afferents developed a rich branching pattern, as can be seen in the fluorescence picture. Dendrites from the same neuron, but growing on polylysine, remain unbranched.F, Quantification of branching in border neurons. Basal dendrites of all cell types (n = 32) as well as apical dendrites of pyramidal neurons (n = 11) developed branches only on those dendrites in contact with the afferents. This suggests that the afferents promote branching by means of a local effect on the dendrites rather than on the cell globally. Error bars represent SEM. Scale bars: B, 500 μm;C–E, 20 μm.

Fig. 6.

Development of dendritic spines in the presence of innervation. Micrograph of hippocampal neurons growing off (A) and on (B) the entorhinal axonal net. Only neurons growing in contact with the entorhinal axons develop numerous spines. C, Fluorescent micrograph showing spines of DiI-stained neurons at higher magnification. D, Electron micrograph of a spine. Scale bars: A, B, 25 μm; C, 5 μm;D, 0.5 μm.

Fig. 7.

Role of activity and glutamate receptors in the development of dendritic spines. Chronic blockade of spontaneous activity by TTX (n = 20) significantly reduced the number of spines that developed after 14 d in vitro. Chronic blockade of individual glutamate receptors with different inhibitors (DNQX, n = 26;APV, n = 23; MCPG,n = 21) alone did not significantly decrease the number of spines; however, combination of all three blockers (COCKT, n = 80) again significantly reduced the number of spines, suggesting a conjoint action of different glutamate receptors in the induction of spines. The average number of spines in treated cultures is shown as percentage of control. (Average number of spines in controls per 100 μm: 12.6 ± 0.8;n = 101.) Error bars represent SEM.