Extensive deprivation of excitatory inputs to a central network like the mammalian visual cortex largely influences its growth and function (Shatz, 1990). It is, however, less clear if a more discrete, albeit long-lasting, suppression of a certain transmitter system can be compensated, through homeostatic mechanisms, by a neuronal network. This issue raises the question of whether nerve cells can establish a functionally coherent pattern of signalling despite removal of discrete inputs: to examine this aspect, chronic block of either excitatory or inhibitory synaptic transmission has been employed as an experimental model. As a consequence of such treatment, a novel form of synaptic modification which has been termed homeostatic plasticity (Turrigiano, 1999) emerges, whereby neurons respond to sustained increases or decreases in their excitatory inputs by changing the properties, distribution and/or composition of certain transmitter postsynaptic receptors (Craig, 1998; Turrigiano, 1999). In particular, block of GABA and/or glycine receptors leads to downregulation of spontaneous glutamatergic transmission, while chronic block of glutamate receptors boosts on-going postsynaptic glutamate receptor-mediated activity. Such phenomena have been recently described in cultured mammalian neurons from various brain areas (Bessho et al. 1994; Rao & Craig, 1997; Kirsch & Betz, 1998; Levi et al. 1998; Lissin et al. 1998; O'Brien et al. 1998; Turrigiano et al. 1998) and, in addition to receptor changes, may include alterations in membrane excitability and consequently in firing activity (Desai et al. 1999). These observations are compatible with a recent hypothesis (originated from studies of the chick embryo spinal cord; Chub & O'Donovan, 1998) that the electrical behaviour of a network is governed by homeostatic mechanisms, that is, any long-lasting reduction in excitatory activity would be counteracted by increasing the efficacy of distinct excitatory transmitter systems. Whether homeostatic plasticity is a paradigm for network remodelling during neuronal development in mammals remains, however, an intriguing and scarcely explored possibility. During development, qualitative and quantitative reshaping of afferent projections may normally occur, thus leading to variations in excitatory input (Crair, 1999). Furthermore, the onset of axonal sprouting and de novo synaptogenesis is influenced by synaptic activity itself (Cline & Constantine-Paton, 1990), suggesting that activity regulates the synaptic targeting of each receptor type in a complex way (Craig, 1998).
To explore the presence of homeostatic plasticity and its role in regulating network activity during development, we wished to use a culture system which generates a considerable degree of network organization. For this purpose we employed organotypic spinal cord cultures from rat embryos (Spenger et al. 1991; Streit et al. 1991; Ballerini & Galante, 1998; Ballerini et al. 1999), as this preparation maintains the basic cytoarchitecture of a spinal segment, and enables direct recording from visually identified neurons during in vitro development (Ballerini & Galante, 1998; Ballerini et al. 1999). Activity-dependent modulation of synaptic transmission was investigated by recording postsynaptic currents (PSCs) and miniature PSCs (mPSCs) from ventral horn interneurons at 14 days in vitro (DIV) in cultures incubated for the entire second week in the presence of various receptor blockers and comparing these responses with those of control cultures. Although these interneurons have not been individually characterized in terms of their excitatory or inhibitory function, it is likely that the large population sampled in the present study included excitatory as well as inhibitory interneurons since a strong, mixed input is normally recorded from nearby motoneurons (Streit, 1993).
We adopted three experimental strategies: (1) chronic block of AMPA/kainate glutamate receptors with CNQX to remove the main, spontaneous excitatory input to these cells, since NMDA receptors play a rather minor role in excitatory transmission on these cells (Ballerini & Galante 1998; Ballerini et al. 1999); (2) chronic block of Na+-dependent action potential mechanisms in an attempt to mimic a more systematic suppression of afferent inputs, like one might expect following chronic lesions; (3) chronic block of GABAA and glycine receptors with bicuculline and strychnine to evoke sustained bursting activity (Ballerini & Galante, 1998).