Spatial patterning of proteins is a valuable technique for many biological applications and is the prevailing tool for defining microenvironments for cells in culture, a required procedure in developmental biology and tissue engineering research. However, it is still challenging to achieve protein patterns that closely mimic native microenvironments, such as gradient protein distributions with desirable mechanical properties. By combining projection dynamic mask lithography and protein engineering with non-canonical photosensitive amino acids, we demonstrate a simple, scalable strategy to fabricate any userdefined 2D or 3D stable gradient pattern with complex geometries from an artificial extracellular matrix (aECM) protein. We show that the elastic modulus and chemical nature of the gradient profile are biocompatible and allow useful applications in cell biological research.

GABAergic amacrine cells, cultured from embryonic chick retina, display spontaneous mini frequencies ranging from 0-4.6 Hz as a result of the release of quanta of transmitter from both synapses and autapses. We show here that at least part of this variation originates from differences in the degree to which endocannabinoids, endogenously generated within the culture, are present at terminals presynaptic to individual cells. Though all cells examined scored positive for cannabinoid receptor type I (CB1R), only those showing a low initial rate of spontaneous minis responded to CB1R agonists with an increase in mini frequency, caused by a Gi/o-mediated reduction in [cAMP]. Cells displaying a high initial rate of spontaneous minis, on the other hand, were unaffected by CB1R agonists, but they did show a rate decrease with CB1R antagonists. Such a regulation of spontaneous transmitter release by endocannabinoids might be important in network maintenance in amacrine cells and other inhibitory interneurons.

The Ca(2+) that promotes transmitter release is generally thought to enter presynaptic terminals through voltage-gated Ca(2+)channels. Using electrophysiology and Ca(2+) imaging, we show that, in amacrine cell dendrites, at least some of the Ca(2+) that triggers transmitter release comes from endoplasmic reticulum Ca(2+) stores. We show that both inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) are present in these dendrites and both participate in the elevation of cytoplasmic [Ca(2+)] during the brief depolarization of a dendrite. Only the Ca(2+) released through IP(3)Rs, however, seems to promote the release of transmitter. Antagonists for the IP(3)R reduced transmitter release, whereas RyR blockers had no effect. Application of an agonist for metabotropic glutamate receptor, known to liberate Ca(2+) from internal stores, enhanced both spontaneous and evoked transmitter release.

While plasticity is typically associated with persistent modifications of synaptic strengths, recent studies indicated that modulations of dendritic excitability may form the other part of the engram and dynamically affect computational processing and output of neuronal circuits. However it remains unknown whether modulation of dendritic excitability is controlled by synaptic changes or whether it can be distinct from them. Here we report the first observation of the induction of a persistent plastic decrease in dendritic excitability decoupled from synaptic stimulation, which is localized and purely activity-based. In rats this local plasticity decrease is conferred by CamKII mediated phosphorylation of A-type potassium channels upon interaction of a back propagating action potential (bAP) with dendritic depolarization.

The depletion of ER Ca2+ stores, following the release of Ca 2+ during intracellular signalling, triggers the Ca 2+ entry across the plasma membrane known as store-operated calcium entry (SOCE). We show here that brief, local [Ca 2+ ] i increases (motes) in the thin dendrites of cultured retinal amacrine cells derived from chick embryos represent the Ca 2+ entry events of SOCE and are initiated by sphingosine-1-phosphate (S1P), a sphingolipid with multiple cellular signalling roles. Externally applied S1P elicits motes but not through a G protein-coupled membrane receptor. The endogenous precursor to S1P, sphingosine, also elicits motes but its action is suppressed by dimethylsphingosine (DMS), an inhibitor of sphingosine phosphorylation. DMS also suppresses motes induced by store depletion and retards the refilling of depleted stores. These effects are reversed by exogenously applied S1P. In these neurons formation of S1P is a step in the SOCE pathway that promotes Ca 2+ entry in the form of motes.