Cell cultures were used to analyze the role of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the development of synaptic transmission. Neurons obtained from embryonic day 18 (E18) rat hippocampus and cultured for 2 weeks exhibited extensive spontaneous synaptic activity. By comparison, neurons obtained from E16 hippocampus expressed very low levels of spontaneous or evoked synaptic activity. Neurotrophin treatment produced a sevenfold increase in the number of functional synaptic connections in the E16 cultures. BDNF induced formation of both excitatory and inhibitory synapses, whereas NT-3 induced formation of only excitatory synapses. These effects were independent of serum or the age of the glia bed used for the culture. They were not accompanied by significant changes in synaptic-vesicle-associated proteins or glutamate receptors. Treatment of the cultures with the neurotrophins for 3 d was sufficient to establish the maximal level of functional synapses. During this period, neurotrophins did not affect the viability or the morphology of the excitatory neurons, although they did produce an increase in the number and length of dendrites of the GABAergic neurons. Remarkably, only BDNF caused an increase in the number of axonal branches and in the total length of the axons of the GABAergic neurons. These results support a unique and differential role for neurotrophins in the formation of excitatory and inhibitory synapses in the developing hippocampus.

Astrocytes are present in large numbers in the nervous system, are associated with synapses, and propagate ionic signals. Astrocytes influence neuronal physiology by responding to and releasing neurotransmitters, but the mechanisms that establish the close interaction between these cells are not defined. Here we use hippocampal neurons in culture to demonstrate that vasoactive intestinal polypeptide (VIP) promotes neuronal differentiation through activity-dependent neurotrophic factor (ADNF), a protein secreted by VIP-stimulated astroglia. ADNF is produced by glial cells and acts directly on neurons to promote glutamate responses and morphological development. ADNF causes secretion of neurotrophin 3 (NT-3), and both proteins regulate NMDA receptor subunit 2A (NR2A) and NR2B. These data suggest that the VIP-ADNF-NT-3 neuronal-glial pathway regulates glutamate responses from an early stage in the synaptic development of excitatory neurons and may also contribute to the known effects of VIP on learning and behavior in the adult nervous system. Key words: glia; synapse; hippocampus; ADNF; NT-3; VIPAn important role for glial cells in synapse function has long been suggested by the intimate relationship that exists between astrocytes and synaptic terminals in vivo (Peters et al., 1991;Ventura and Harris, 1999). Growing evidence indicates that astrocytes play active roles in the CNS (Dani et al., 1992;Porter and McCarthy, 1996). Astrocytes can signal to neurons by releasing soluble factors such as glutamate (Nedergaard, 1994;Parpura et al., 1994) or ␤-chemokines (Brenneman et al., 1999a,b) that regulate neuronal activity (Araque et al., 1998a,b;Meucci et al., 1998). In fact, astrocytes are now viewed as active partners of the presynaptic and postsynaptic terminals in the elaboration of tripartite synaptic structures (Araque et al., 1999). The temporal correlation between glial development and synaptogenesis also suggests an involvement of astrocytes in the formation of the first synapses. In the rat CNS, neurons form most of their synapses during the third postnatal week (Aghajanian and Bloom, 1967), after the differentiation of astrocytes has already been completed (Parnavelas et al., 1983). It has been proposed that glia-derived, soluble factors are necessary for the maturation of developing synapses in vitro (Pfrieger and Barres, 1997). However, the molecular mechanisms that regulate the interactions between neurons and glia are not well defined.Secreted factors such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been shown to regulate synaptogenesis in the developing hippocampus (Vicario-Abejón et al., 1998), but these factors are neuron-derived proteins that do not require the presence of glia to act on neurons (Song et al., 1997). Among the known regulatory peptides found in the CNS, only vasoactive intestinal polypeptide (VIP) is thought to stimulate glia to generate neurotrophic factors (for review, see Brenneman et al., 1999b). The most potent of these glial signals is the activit...

Transverse hippocampal slices were cut from 8- to 9-day-old rats and maintained in an interface chamber for periods of 1-4 wk, in tissue culture conditions. Neurons in the slice preserved their spatial organization and connectivity. Dendritic spine density in CA1 neurons was very low at 1 wk in culture, and long, filopodia-like structures were abundant. Spine density increased in these neurons nearly threefold during the course of 3 wk in vitro, to approach values of those of the normal, in vivo hippocampus. The magnitude of long-term potentiation (LTP) of reactivity of Ca1 to stimulation of CA3 neurons also increased during weeks in culture in parallel with the change in spine density. Chronic exposure of slices to drugs that interact with synaptic activity caused changes in their dendritic spine density. Blockade of the N-methyl-D-aspartate (NMDA) receptors with the receptor antagonist 2-aminophosphonovalerate (D-APV) or blockade of action potential discharges with tetrodotoxin (TTX) prevented dendritic spine development in immature cultures. Enhancing synaptic activity by blockade of GABAergic inhibition with picrotoxin did not affect spine density to a significant degree. D-APV-treated slices expressed larger LTP than controls. TTX-treated slices expressed smaller LTP than controls. Picrotoxin treated slices did not express LTP. It is proposed that LTP and dendritic spine density are correlated strongly during development, whereas they are not correlated in the more mature slice/culture of the hippocampus where spine density can be modulated by chronic exposure to blockers of synaptic activity, which will not affect LTP in a similar manner.