Rapid modulation of local neural activity by controlled drug release from polymer-coated recording microelectrodes.

We demonstrate targeted perturbation of neuronal activity with controlled release of neurochemicals from conducting polymer-coated microelectrodes. Polymer coating and chemical incorporation are achieved through individually addressable electrodeposition, a process that does not compromise the recording capabilities of the electrodes. Release is realized by the application of brief voltage pulses that electrochemically reduce the polymer and dissociate incorporated neurochemicals; whereby they can diffuse away and achieve locally effective concentrations. Inhibition of evoked synaptic currents in neurons within 200 µm of a 6-cyano-7-nitroquinoxaline-2,3-dione releasing electrode lasts for several seconds. Spiking activity of neurons in local circuits recorded extracellularly near the releasing electrode is silenced for a similar duration following release. This methodology is compatible with many neuromodulatory chemicals and various recording electrodes, including in vitro and implantable neural electrode arrays, thus providing an inexpensive and accessible technique capable of achieving sophisticated patterned chemical modulation of neuronal circuits.

Axon formation: a molecular model for the generation of neuronal polarity.

Neurons have unique structural and functional polarity. In general, information flows from the short dendrites to the long axon, and each neuron has multiple dendrites but only one axon. A detailed description of the cellular events leading to the establishment of axonal-dendritic polarity has been given from an in vitro hippocampal culture model system. Little is known, however, about the nature of the underlying molecular events. New data strongly suggest that actin depolymerization at a growth cone is crucial for axon fate determination. We hypothesize that an autocatalytic positive feedback loop at all growth cones locally regulates actin dynamics and other cellular events required for axon formation. Meanwhile, a negative feedback signal, produced by the positive feedback loop, propagates from all growth cones throughout the neuron and counteracts the positive feedback loops. Such feedback regulation provides a robust mechanism for spontaneous symmetry breaking and the formation of only one axon, even in a symmetric in vitro environment. Based on data from studies of cell migration, axon guidance, vesicle exocytosis, and the regulation of actin and microtubule polymerization, we propose a molecular scheme for the positive feedback loop and discuss possible negative feedback signals. BioEssays 22:172-179, 2000.

Stable Hebbian learning from spike timing-dependent plasticity.

We explore a synaptic plasticity model that incorporates recent findings that potentiation and depression can be induced by precisely timed pairs of synaptic events and postsynaptic spikes. In addition we include the observation that strong synapses undergo relatively less potentiation than weak synapses, whereas depression is independent of synaptic strength. After random stimulation, the synaptic weights reach an equilibrium distribution which is stable, unimodal, and has positive skew. This weight distribution compares favorably to the distributions of quantal amplitudes and of receptor number observed experimentally in central neurons and contrasts to the distribution found in plasticity models without size-dependent potentiation. Also in contrast to those models, which show strong competition between the synapses, stable plasticity is achieved with little competition. Instead, competition can be introduced by including a separate mechanism that scales synaptic strengths multiplicatively as a function of postsynaptic activity. In this model, synaptic weights change in proportion to how correlated they are with other inputs onto the same postsynaptic neuron. These results indicate that stable correlation-based plasticity can be achieved without introducing competition, suggesting that plasticity and competition need not coexist in all circuits or at all developmental stages.

The mechanism of facilitated cell membrane resealing.

Disruption of the plasma membrane evokes an exocytotic response that is required for rapid membrane resealing. We show here in Swiss 3T3 fibroblasts that a second disruption at the same site reseals more rapidly than the initial wound. This facilitated response of resealing was inhibited by both low external Ca2+ concentration and specific protein kinase C (PKC) inhibitors, bisindolylmaleimide I (BIS) and Gö-6976. In addition, activation of PKC by phorbol ester facilitated the resealing of a first wound. BIS and Gö-6976 suppressed the effect of phorbol ester on resealing rate. Fluorescent dye loss from a FM1-43 pre-labeled endocytotic compartment was used to investigate the relationship between exocytosis, resealing and the facilitation of resealing. Exocytosis of endocytotic compartments near the wounding site was correlated with successful resealing. The destaining did not occur when exocytosis and resealing were inhibited by low external Ca2+ concentration or by injected tetanus toxin. When the dye loaded cells were wounded twice, FM1-43 destaining at the second wound was less than at the first wound. Less destaining was also observed in cells pre-treated with phorbol ester, suggesting newly formed vesicles, which were FM1-43 unlabeled, were exocytosed in the resealing at repeated woundings. Facilitation was also blocked by brefeldin A (BFA), a fungal metabolite that inhibits vesicle formation at the Golgi apparatus. Lowering the temperature below 20 degrees C also blocked facilitation as expected from a block of Golgi function. BFA had no effect on the resealing rate of an initial wound. The facilitation of the resealing by phorbol ester was blocked by pre-treatment with BFA. These results suggest that at first wounding the cell used the endocytotic compartment to add membrane necessary for resealing. At a second wounding, PKC, activated by Ca2+ entry at the first wound, stimulated vesicle formation from the Golgi apparatus, resulting in more rapid resealing of the second membrane disruption. Since vesicle pools were implicated in both membrane resealing and facilitation of membrane resealing, we reasoned that artificial decreases in membrane surface tension would have the same result. Decreases in surface tension induced by the addition of a surfactant (Pluronic F68 NF) or cytochalasin D facilitated resealing at first wounding. Furthermore, Pluronic F68 NF restored resealing when exocytosis was blocked by tetanus toxin. These results suggest that membrane resealing requires a decrease in surface tension and under natural conditions this is provided by Ca2+-dependent exocytosis of new membrane near the site of disruption.

Synaptic modifications in cultured hippocampal neurons: dependence on spike timing, synaptic strength, and postsynaptic cell type.

In cultures of dissociated rat hippocampal neurons, persistent potentiation and depression of glutamatergic synapses were induced by correlated spiking of presynaptic and postsynaptic neurons. The relative timing between the presynaptic and postsynaptic spiking determined the direction and the extent of synaptic changes. Repetitive postsynaptic spiking within a time window of 20 msec after presynaptic activation resulted in long-term potentiation (LTP), whereas postsynaptic spiking within a window of 20 msec before the repetitive presynaptic activation led to long-term depression (LTD). Significant LTP occurred only at synapses with relatively low initial strength, whereas the extent of LTD did not show obvious dependence on the initial synaptic strength. Both LTP and LTD depended on the activation of NMDA receptors and were absent in cases in which the postsynaptic neurons were GABAergic in nature. Blockade of L-type calcium channels with nimodipine abolished the induction of LTD and reduced the extent of LTP. These results underscore the importance of precise spike timing, synaptic strength, and postsynaptic cell type in the activity-induced modification of central synapses and suggest that Hebb's rule may need to incorporate a quantitative consideration of spike timing that reflects the narrow and asymmetric window for the induction of synaptic modification.

Kinesin- and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis.

Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca2+-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin- dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.

Calcium-regulated exocytosis is required for cell membrane resealing.

Using confocal microscopy, we visualized exocytosis during membrane resealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of localized Ca(2+)-regulated exocytosis. The rate of exocytosis was correlated quantitatively with successfully resealing. In embryos, whose activated surfaces must first dock vesicles before fusion, exocytosis and membrane resealing were inhibited by neurotoxins that selectively cleave the SNARE complex proteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly undocked with externally applied stachyose. If cortical vesicles were undocked both exocytosis and plasma membrane resealing were completely inhibited. When cortical vesicles were transiently undocked, exposure to tetanus toxin and botulinum neurotoxin type C1 rendered them no longer competent for resealing, although botulinum neurotoxin type A was still ineffective. Cortical vesicles transiently undocked in the presence of tetanus toxin were subsequently fusion incompetent although to a large extent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exocytosis is required and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane.