Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-induced versus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK.Bone morphogenetic proteins (BMPs) 1 are members of the transforming growth factor-␤ (TGF-␤) superfamily that play important roles in most morphogenetic processes during development (1). BMPs are able to induce the formation of bone at nonbony sites in the adult animal by influencing the differentiation of mesenchymal progenitor cells along the cartilage lineage pathway. BMPs act on osteoblasts as well as chondrocytes but also on many other cell types such as neuronal cells (2, 3). Signaling by BMP-2 involves two types of transmembrane serine/threonine kinases, termed type I (BRI) and type II (BRII) receptors (4 -8). Receptors of both types are needed to form a functional complex to initiate further signaling events. Activated BMP type I receptors phosphorylate Smad1, Smad5, and Smad8 (R-Smads), which then assemble into heteromeric complexes with Smad4 (Co-Smad) and translocate into the nucleus to regulate transcription of target genes (9, 10). In addition, BMP receptors initiate other signaling pathways, distinct from the Smad pathway, resulting in the activation of p38 MAPK and JNK (11-13).We have shown recently that the oligomerization pattern of ...

Cortical dynamics can be imaged at high spatiotemporal resolution with voltage-sensitive dyes (VSDs) and calcium-sensitive dyes (CaSDs). We combined these two imaging techniques using epifluorescence optics together with whole cell recordings to measure the spatiotemporal dynamics of activity in the mouse somatosensory barrel cortex in vitro and in the supragranular layers in vivo. The two optical signals reported distinct aspects of cortical function. VSD fluorescence varied linearly with membrane potential and was dominated by subthreshold postsynaptic potentials, whereas the CaSD signal predominantly reflected local action potential firing. Combining VSDs and CaSDs allowed us to monitor the synaptic drive and the spiking activity of a given area at the same time in the same preparation. The spatial extent of the two dye signals was different, with VSD signals spreading further than CaSD signals, reflecting broad subthreshold and narrow suprathreshold receptive fields. Importantly, the signals from the dyes were differentially affected by pharmacological manipulations, stimulation strength, and depth of isoflurane anesthesia. Combined VSD and CaSD measurements can therefore be used to specify the temporal and spatial relationships between subthreshold and suprathreshold activity of the neocortex.

GABA (gamma-aminobutyric acid) can mediate inhibition via pre- and post/extrasynaptic GABA receptors. In this paper we demonstrate potentially post/extrasynaptic GABA(B) receptor-dependent tonic inhibition in L2/3 pyramidal cells of rat medial prefrontal cortex (mPFC) in vitro. First, we show via voltage-clamp experiments the presence of a tonic GABA(B) receptor-dependent outward current in these neurons. This GABA(B)ergic current could be induced by ambient GABA when present at sufficient concentrations. To increase ambient GABA levels in the usually silent slice preparation, we amplified network activity and hence synaptic GABA release with a modified artificial cerebrospinal fluid. The amplitude of tonic GABA(B) current was similar at different temperatures. In addition to the tonic GABA(B) current, we found presynaptic GABA(B) effects, GABA(B)-mediated inhibitory postsynaptic currents and tonic GABA(A) currents. Second, we performed current-clamp experiments to evaluate the functional impact of GABA(B) receptor-mediated inhibition in the mPFC. Activating or inactivating GABA(B) receptors led to rightward (reduction of excitability) or leftward (increase of excitability) shifts, respectively, of the input-output function of mPFC L2/3 pyramidal cells without effects on the slope. Finally, we showed in electrophysiological recordings and epifluorescence Ca(2+)-imaging that GABA(B) receptor-mediated tonic inhibition is capable of regulating network activity. Blocking GABA(B) receptors increased the frequency of excitatory postsynaptic currents impinging on a neuron and prolonged network upstates. These results show that ambient GABA via GABA(B) receptors is powerful enough to modulate neuronal excitability and the activity of neural networks.

Population dynamics of patterned neuronal firing are fundamental to information processing in the brain. Multiphoton microscopy in combination with calcium indicator dyes allows circuit dynamics to be imaged with single-neuron resolution. However, the temporal resolution of fluorescent measures is constrained by the imaging frequency imposed by standard raster scanning techniques. As a result, traditional raster scans limit the ability to detect the relative timing of action potentials in the imaged neuronal population. To maximize the speed of fluorescence measures from large populations of neurons using a standard multiphoton laser scanning microscope (MPLSM) setup, we have developed heuristically optimal path scanning (HOPS). HOPS optimizes the laser travel path length, and thus the temporal resolution of neuronal fluorescent measures, using standard galvanometer scan mirrors. Minimizing the scan path alone is insufficient for prolonged high-speed imaging of neuronal populations. Path stability and the signal-to-noise ratio become increasingly important factors as scan rates increase. HOPS addresses this by characterizing the scan mirror galvanometers to achieve prolonged path stability. In addition, the neuronal dwell time is optimized to sharpen the detection of action potentials while maximizing scan rate. The combination of shortest path calculation and minimization of mirror positioning time allows us to optically monitor a population of neurons in a field of view at high rates with single-spike resolution, ∼ 125 Hz for 50 neurons and ∼ 8.5 Hz for 1,000 neurons. Our approach introduces an accessible method for rapid imaging of large neuronal populations using traditional MPLSMs, facilitating new insights into neuronal circuit dynamics.

Integrative properties of single neurons have been extensively studied in acute brain slices. However, these preparations are characterized by extremely low levels of synaptic and action potential activity. In comparison to in vivo, reduced intracortical input and lack of subcortical modulation increase the effective difference between mean membrane potential and spiking threshold, preventing selfsustained network activity in vitro. To elicit an increased and stable network activity (INA) in vitro comparable to that found in awake animals, we mimicked subcortical cholinergic and serotoninergic inputs using carbachol or barium alone or in combination with serotonin in layer 5 pyramidal cells in slices of mouse somatosensory cortex. INA is primarily induced by a modulation of intrinsic conductances resulting in a depolarization of the membrane potential. We studied the impact of INA on synaptic and somatodendritic integration using extracellular stimulation and dendritic calcium imaging. Synaptic inhibition is strengthened due to an increased driving force for chloride. The critical frequency at which somatic action potentials induce a dendritic calcium action potential is lowered. Simultaneous inhibitory synaptic input is powerful enough to suppress dendritic calcium action potential generation. Pharmacologically induced INA enables the study of neuronal integration in well-accessible cortical slices within an active network.