BMP-mediated inhibition of FGF signaling promotes cardiomyocyte differentiation of anterior heart field progenitors.

The anterior heart field (AHF) encompasses a niche in which mesoderm-derived cardiac progenitors maintain their multipotent and undifferentiated nature in response to signals from surrounding tissues. Here, we investigate the signaling mechanism that promotes the shift from proliferating cardiac progenitors to differentiating cardiomyocytes in chick embryos. Genomic and systems biology approaches, as well as perturbations of signaling molecules, in vitro and in vivo, reveal tight crosstalk between the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signaling pathways within the AHF niche: BMP4 promotes myofibrillar gene expression and cardiomyocyte contraction by blocking FGF signaling. Furthermore, inhibition of the FGF-ERK pathway is both sufficient and necessary for these processes, suggesting that FGF signaling blocks premature differentiation of cardiac progenitors in the AHF. We further revealed that BMP4 induced a set of neural crest-related genes, including MSX1. Overexpression of Msx1 was sufficient to repress FGF gene expression and cell proliferation, thereby promoting cardiomyocyte differentiation. Finally, we show that BMP-induced cardiomyocyte differentiation is diminished following cranial neural crest ablation, underscoring the key roles of these cells in the regulation of AHF cell differentiation. Hence, BMP and FGF signaling pathways act via inter- and intra-regulatory loops in multiple tissues, to coordinate the balance between proliferation and differentiation of cardiac progenitors.

Neuronal density determines network connectivity and spontaneous activity in cultured hippocampus.

The effects of neuronal density on morphological and functional attributes of the evolving networks were studied in cultured dissociated hippocampal neurons. Plating at different densities affected connectivity among the neurons, such that sparse networks exhibited stronger synaptic connections between pairs of recorded neurons. This was associated with different patterns of spontaneous network activity with enhanced burst size but reduced burst frequency in the sparse cultures. Neuronal density also affected the morphology of the dendrites and spines of these neurons, such that sparse neurons had a simpler dendritic tree and fewer dendritic spines. Additionally, analysis of neurons transfected with PSD95 revealed that in sparse cultures the synapses are formed on the dendritic shaft, whereas in dense cultures the synapses are formed primarily on spine heads. These experiments provide important clues on the role of neuronal density in population activity and should yield new insights into the rules governing neuronal network connectivity.

Determinants of spontaneous activity in networks of cultured hippocampus.

The brain generates extensive spontaneous network activity patterns, even in the absence of extrinsic afferents. While the cognitive correlates of these complex activities are being unraveled, the rules that govern the generation, synchronization and spread of different patterns of intrinsic network activity in the brain are still enigmatic. Using hippocampal neurons grown in dissociated cultures, we are able to study these rules. Network activity emerges at 3-7 days in-vitro (DIV) independent of either ongoing excitatory or inhibitory synaptic activity. Network activity matures over the following several weeks in culture, when it becomes sensitive to chronic drug treatment. The size of the network determines its properties, such that dense networks have higher rates of less synchronized activity than that of sparse networks, which are more synchronized but rhythm at lower rates. Large networks cannot be triggered to fire by activating a single neuron. Small networks, on the other hand, do not burst spontaneously, but can be made to discharge a network burst by stimulating a single neuron. Thus, the strength of connectivity is inversely correlated with spontaneous activity and synchronicity. In the absence of confirmed 'leader' neurons, synchronous bursting network activity appears to be triggered by at least several local subthreshold synaptic events. We conclude that networks of neurons in culture can produce spontaneous synchronized activity and serve as a viable model system for the analysis of the rules that govern network activity in the brain.

Simultaneous NMDA-dependent long-term potentiation of EPSCs and long-term depression of IPSCs in cultured rat hippocampal neurons.

A fundamental issue in understanding activity-dependent long-term plasticity of neuronal networks is the interplay between excitatory and inhibitory synaptic drives in the network. Using dual whole-cell recordings in cultured hippocampal neurons, we examined synaptic changes occurring as a result of a transient activation of NMDA receptors in the network. This enhanced transient activation led to a long-lasting increase in synchrony of spontaneous activity of neurons in the network. Simultaneous long-term potentiation of excitatory synaptic strength and a pronounced long-term depression of inhibitory synaptic currents (LTDi) were produced, which were independent of changes in postsynaptic potential and Ca2+ concentrations. Surprisingly, miniature inhibitory synaptic currents were not changed by the conditioning, whereas both frequency and amplitudes of miniature EPSCs were enhanced. LTDi was mediated by activation of a presynaptic GABAB receptor, because it was blocked by saclofen and CGP55845 [(2S)-3-{[(15)-1-(3, 4-dichlorophenyl)ethyl]amino-2-hydroxypropyl)(phenylmethyl)phosphinic acid]. The cAMP antagonist Rp-adenosine 3 ', 5 ' -cyclic monophosphothioate abolished all measured effects of NMDA-dependent conditioning, whereas a nitric oxide synthase inhibitor was ineffective. Finally, network-induced plasticity was not occluded by a previous spike-timing-induced plasticity, indicating that the two types of plasticity may not share the same mechanism. These results demonstrate that network plasticity involves opposite affects on inhibitory and excitatory neurotransmission.

Age-dependent glutamate induction of synaptic plasticity in cultured hippocampal neurons.

A common denominator for the induction of morphological and functional plasticity in cultured hippocampal neurons involves the activation of excitatory synapses. We now demonstrate massive morphological plasticity in mature cultured hippocampal neurons caused by a brief exposure to glutamate. This plasticity involves a slow, 70%-80% increase in spine cross-section area associated with a significant reduction in the width of dendrites. These changes are age dependent and expressed only in cells >18 d in vitro (DIV). Activation of both NMDARs and AMPARs as well as a sustained rise of internal calcium levels are necessary for induction of this plasticity. On the other hand, blockade of network activity or mGluRs does not abolish the observed morphological plasticity. Electrophysiologically, a brief exposure to glutamate induces an increase in the magnitude of EPSCs evoked between pairs of neurons, as well as in mEPSC frequency and amplitude, in mature but not young cultures. These results demonstrate an age-dependent, rapid and robust morphological and functional change in cultured central neurons that may contribute to their ability to express long term synaptic plasticity.