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Background: Mechanisms of antibody-mediated neutralization are of much interest. For plant and bacterial A-B toxins, A chain mediates toxicity and B chain binds target cells. It is generally accepted and taught that antibody (Ab) neutralizes by preventing toxin binding to cells. Yet for some toxins, ricin included, anti-A chain Abs afford greater protection than anti-B. The mechanism(s) whereby Abs to the A chain neutralize toxins are not understood., Methodology/principal Findings: We use quantitative confocal imaging, neutralization assays, and other techniques to study how anti-A chain Abs function to protect cells. Without Ab, ricin enters cells and penetrates to the endoplasmic reticulum within 15 min. Within 45-60 min, ricin entering and being expelled from cells reaches equilibrium. These results are consistent with previous observations, and support the validity of our novel methodology. The addition of neutralizing Ab causes ricin accumulation at the cell surface, delays internalization, and postpones retrograde transport of ricin. Ab binds ricin for >6hr as they traffic together through the cell. Ab protects cells even when administered hours after exposure. CONCLUSIONS/KEY FINDINGS: We demonstrate the dynamic nature of the interaction between the host cell and toxin, and how Ab can alter the balance in favor of the cell. Ab blocks ricin's entry into cells, hinders its intracellular routing, and can protect even after ricin is present in the target organelle, providing evidence that the major site of neutralization is intracellular. These data add toxins to the list of pathogenic agents that can be neutralized intracellularly and explain the in vivo efficacy of delayed administration of anti-toxin Abs. The results encourage the use of post-exposure passive Ab therapy, and show the importance of the A chain as a target of Abs.

The retinocollicular pathway undergoes activity-dependent refinement during postnatal development, which results in the precise retinotopic order seen in adults. This process is NMDA- and nitric oxide-dependent. Recent studies have shown that L-type Ca2+ channels may also play a role in synaptic plasticity, but such channel activity has not previously been reported in the developing superior colliculus (SC). Here we report the presence of a postsynaptic plateau potential mediated by L-type Ca2+ channels using whole-cell current clamp of the SC in an isolated brainstem preparation of rats. Seventy percent of SC neurons showed these potentials as early as postnatal day 0 (P0)-P2. The potential was blocked by nitrendipine and/or APV and facilitated by bicuculline, showing that the channel is activated by NMDA receptor-mediated EPSPs and deactivated by GABAA receptor-mediated IPSPs. Blockade of L-type Ca2+ channels also diminished long-term depression, which we could induce in the retinocollicular pathway in neonatal animals. The incidence of plateau potentials decreased to 39% of neurons by P10-P14, suggesting that L-type calcium channels may contribute to retinocollicular pathway refinement in the developing SC.

By using an in vitro isolated brain stem preparation, we recorded extracellular responses to electrical stimulation of the optic tract (OT) from 71 neurons in the superficial superior colliculus (SC) of neonatal rats (P1-13). At postnatal day 1 (P1), all tested neurons (n = 10) already received excitatory input from the retina. Sixty-nine (97%) superficial SC neurons of neonatal rats showed three response patterns to OT stimulation, which depended on stimulus intensity. A weak stimulus evoked only one spike that was caused by activation of non-N-methyl-D-aspartate (NMDA) glutamate receptors. A moderate stimulus elicited a short train (<250 ms) of spikes, which was induced by activation of both NMDA and non-NMDA receptors. A strong stimulus gave rise to a long train (>300 ms) of spikes, which was associated with additional activation of L-type high-threshold calcium channels. The long train firing pattern could also be induced either by temporal summation of retinal inputs or by blocking gamma-aminobutyric acid-A receptors. Because retinal ganglion cells show synchronous bursting activity before eye opening at P14, the retinotectal inputs appear to be sufficient to activate L-type calcium channels in the absence of pattern vision. Therefore activation of L-type calcium channels is likely to be an important source for calcium influx into SC neurons in neonatal rats.

We made intracellular recordings from 74 neurons in the optic layer of the rat superior colliculus (SC). Resting membrane potentials were -62.3 +/- 6.2 (SD) mV, and input resistances were 37.9 +/- 10.1 MOmega. Optic layer neurons had large sodium spikes (74.2 +/- 12.3 mV) with an overshoot of 12 mV and a half-amplitude duration of 0.75 +/- 0.2 ms. Each sodium spike was followed by two afterhyperpolarizations (AHPs), one of short duration and one of longer duration, which were mediated by tetraethylammonium (TEA)-sensitive (IC) or apamin-sensitive (IAHP) calcium-activated potassium currents, respectively. Sodium spikes were also followed by an afterdepolarization (ADP), which was only revealed when the AHPs were blocked by TEA or apamin. In response to hyperpolarizing current pulses, optic layer neurons showed an inward rectification mediated by H channels. At the break of the current pulse, there was a rebound low-threshold spike (LTS) with a short duration of <25 ms. The LTS usually induced two sodium spikes (doublet). Most optic layer neurons (84%) behaved as intrinsically bursting cells. They responded to suprathreshold depolarization with an initial burst (or doublet) followed by a train of regular single spikes. The remaining 16% of cells acted as chattering cells with high-frequency gamma (20-80 Hz) rhythmic burst firing within a narrow range of depolarized potentials. The interburst frequency was voltage dependent and also time dependent, i.e., showed frequency adaptation. Unmasking the ADP with either TEA or apamin converted all of the tested intrinsically bursting cells into chattering cells, indicating that the ADP played a crucial role in the generation of rhythmic burst firing. Optic layer neurons receive direct retinal excitation mediated by both N-methyl--aspartate (NMDA) and non-NMDA receptors. Optic tract (OT) stimulation also led to gamma-aminobutyric acid-A (GABAA) receptor-mediated inhibition, the main effect of which was to curtail the excitatory response to retinal inputs by shunting the excitatory postsynaptic current. Intracellular staining with biocytin showed that the optic layer neurons that we recorded from were mostly either wide-field vertical neurons or other cells with predominately superficially projecting dendrites. These cells were similar to calbindin immunoreactive cells seen in the optic layer. The characteristics of these optic layer neurons, such as prominent AHPs, strong shunting effect of inhibition, and short-lasting LTS, suggest that they respond transiently to retinal inputs. This is consistent with a function for these cells as the first relay station in the extrageniculate visual pathway.

During development, rabbits were exposed only to vertical or horizontal lines to determine if the receptive field characteristics of visual cortex cells would be altered as they are in the cat. Motion and directional selectivity were preserved, and orientation specificity remained unaffected by the restricted experience, which suggests that the rabbit may lack the neural plasticity seen in some other mammals.