Polyethylenimine (PEI), a polycation with high ionic charge density, has recently been used as a gene therapy delivery agent. We have defined the optimal conditions for PEI-based transfection of airway epithelial cells in vitro and in vivo and used these conditions to restore Cl À channel activity in a CF mouse model. Three forms of PEI, a linear 22 kDa (ExGen 500) form and branched 25 or 50 kDa forms were evaluated. All forms of PEI significantly increased luciferase reporter gene expression compared to the liposome DCChol/DOPE in a human bronchial epithelial cell line (16HBE) irrespective of the extent of cell confluency. With subconfluent cells, gene expression was around 1000-, 200-and 25-fold higher than liposomes using linear 22, 25 and 50 kDa PEI, respectively. The transfection efficiency was reduced in confluent and polarized epithelial cells but linear 22 kDa PEI showed the smallest decrease and gave 8000-fold better transfection in polarized cells compared to liposomes. A comparison of linear 22 or 25 kDa PEI with DCChol/DOPE for airway delivery in vivo via intranasal instillation was also performed. Linear 22 kDa PEI gave significantly better luciferase reporter gene expression of 350-fold in the lung, 180-fold in the nose and 85-fold in the trachea compared to liposome. In contrast, the 25 kDa form of PEI was no better than DCChol/ DOPE. Repeat dosing with linear 22 kDa PEI failed to give reporter gene delivery comparable to the initial dose. To establish that PEI can be used to deliver a physiologically relevent gene in vivo, we used it to restore Cl À secretion by CFTR gene delivery in the airways of a CF mouse model.

Difficult search tasks are known to involve attentional resources, but the spatiotemporal behavior of attention remains unknown. Are multiple search targets processed in sequence or in parallel? We developed an innovative methodology to solve this notoriously difficult problem. Observers performed a difficult search task during which two probes were flashed at varying delays. Performance in reporting probes at each location was considered a measure of attentional deployment. By solving a second-degree equation, we determined the probability of probe report at the most and least attended probe locations on each trial. Because these values differed significantly, we conclude that attention was focused on one stimulus or subgroup of stimuli at a time, and not divided uniformly among all search stimuli. Furthermore, this deployment was modulated periodically over time at ∼7 Hz. These results provide evidence for a nonuniform spatiotemporal deployment of attention during difficult search.V isual search tasks (e.g., to find a target embedded among similar looking distracters) have long been used to investigate the deployment of attention (1-6). Certain tasks are performed "efficiently," in which case the search time and accuracy are independent of the number of distracters. Other tasks are more difficult, or "inefficient," characterized by an increase in reaction times (RTs) and/or a decrease in accuracy with the number of distracting elements, a result typically attributed to the need to allocate attention (4-7). For more than 30 y now, since the pioneering study of Treisman and Gelade in 1980 (4), two opposing theories of attention deployment during difficult search have persisted. Attention could either be allocated nonuniformly to the stimuli, such that in some cases it would switch sequentially from one stimulus (or group of stimuli) to another (4, 5), or be divided uniformly to process all of the stimuli in parallel, but with a drop in efficiency for increasing distractor numbers (2,(8)(9)(10). To date, neither of these two theories has been unequivocally disproved. Overall performance in the search task itself is not directly informative, because both theories predict an increase in RT with the number of distracters (11,12). One alternative is to use briefly flashed probes to test for the deployment of attention at a specific location and time. With two probes, it should be possible to differentiate parallel and sequential processing strategies: The strict parallel theory predicts that both probes should receive equal amounts of attention, whereas the sequential theory predicts that one of the probes will receive more attention than the other. Of course, the most attended probe may not be the same one on every trial, but a simple mathematical manipulation, the solution of a quadratic equation, allows us to access this information despite the need to average performance over trials.In recent years, a second, related, debate has arisen in the literature concerning the temporal behavior of attention. It has been p...

Theoretical and experimental studies suggest that oscillatory modes of processing play an important role in neuronal computations. One well supported idea is that the net excitatory input during oscillations will be reported in the phase of firing, a 'rate-to-phase transform', and that this transform might enable a temporal code. Here, we investigate the efficiency of this code at the level of fundamental single cell computations. We first develop a general framework for the understanding of the rate-to-phase transform as implemented by single neurons. Using whole cell patch-clamp recordings of rat hippocampal pyramidal neurons in vitro, we investigated the relationship between tonic excitation and phase of firing during simulated theta frequency (5 Hz) and gamma frequency (40 Hz) oscillations, over a range of physiological firing rates. During theta frequency oscillations, the phase of the first spike per cycle was a near-linear function of tonic excitation, advancing through a full 180 deg, from the peak to the trough of the oscillation cycle as excitation increased. In contrast, this relationship was not apparent for gamma oscillations, during which the phase of firing was virtually independent of the level of tonic excitatory input within the range of physiological firing rates. We show that a simple analytical model can substantially capture this behaviour, enabling generalization to other oscillatory states and cell types. The capacity of such a transform to encode information is limited by the temporal precision of neuronal activity. Using the data from our whole cell recordings, we calculated the information about the input available in the rate or phase of firing, and found the phase code to be significantly more efficient. Thus, temporal modes of processing can enable neuronal coding to be inherently more efficient, thereby allowing a reduction in processing time or in the number of neurons required.