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.

Normal gonadal development is dependent upon stimulation by the gonadotropic hormones, and it is likely that control of ovarian development is regulated by expression of gonadotropin receptors. In this study, quantitative changes in LH- and FSH-receptor mRNA levels were measured in the ovary during development in normal mice and in hypogonadal (hpg/hpg) mice, which lack circulating gonadotropins. The relative abundance of alternate transcripts encoding LH and FSH receptors was also determined. Results show that shortened transcripts of the receptors were abundant at all ages. Full-length transcripts of the LH receptor were not detectable until postnatal Day 5 although shortened transcripts encoding the extracellular domain of the receptor were present from birth. Between Days 5 and 7, LH-receptor transcript levels showed a marked increase in normal animals but no change in hpg/hpg animals. FSH-receptor transcripts encoding all domains of the receptor were detectable at low levels at birth, increased in concentration between Days 3 and 5, and peaked at Day 10. In hpg/hpg animals, FSH-receptor mRNA levels were normal up to Day 7 but failed to increase thereafter. These results show that early development of both LH- and FSH-receptor mRNA levels in the ovary is gonadotropin-independent. This coincides with early folliculogenesis to the primary stage. Further development of LH-receptor mRNA levels is gonadotropin-dependent although FSH-receptor mRNA levels continue to increase independently until early secondary follicles are present.

Several theories have been advanced to explain how cross-frequency coupling, the interaction of neuronal oscillations at different frequencies, could enable item multiplexing in neural systems. The communication-through-coherence theory proposes that phase-matching of gamma oscillations between areas enables selective processing of a single item at a time, and a later refinement of the theory includes a theta-frequency oscillation that provides a periodic reset of the system. Alternatively, the theta-gamma neural code theory proposes that a sequence of items is processed, one per gamma cycle, and that this sequence is repeated or updated across theta cycles. In short, both theories serve to segregate representations via the temporal domain, but differ on the number of objects concurrently represented. In this study, we set out to test whether each of these theories is actually physiologically plausible, by implementing them within a single model inspired by physiological data. Using a spiking network model of visual processing, we show that each of these theories is physiologically plausible and computationally useful. Both theories were implemented within a single network architecture, with two areas connected in a feedforward manner, and gamma oscillations generated by feedback inhibition within areas. Simply increasing the amplitude of global inhibition in the lower area, equivalent to an increase in the spatial scope of the gamma oscillation, yielded a switch from one mode to the other. Thus, these different processing modes may co-exist in the brain, enabling dynamic switching between exploratory and selective modes of attention.

Our constant eye movements mean that updating processes, such as saccadic remapping, are essential for the maintenance of a stable spatial representation of the world around us. It has been proposed that, rather than continually update a full spatiotopic map, only the location of a few key objects is updated, suggesting that the process is linked to attention. At the same time, mounting evidence links attention to oscillatory neuronal processes. We therefore hypothesized that updating processes should themselves show oscillatory characteristics, inherited from underlying attentional processes. To test this, we carried out a combined psychophysics and EEG experiment in human participants, using a saccadic mislocalization task as a behaviourally measureable proxy for spatial updating, and simultaneously recording 64-channel EEG. We then used a time-frequency analysis to test for a correlation between oscillation phase and perceptual outcome. We found a significant phase-dependence of mislocalization in a time-frequency region from around 400 ms prior to saccade initiation and peaking at around 7 Hz, principally apparent over occipital electrodes. Thus the degree of perceived mislocalization is correlated with the phase of a theta-frequency oscillation prior to saccade onset. We conclude that spatial updating processes are indeed linked to rhythmic processes in the brain.

Visual area V5/MT in the rhesus macaque has a distinct functional organization, where neurons with specific preferences for direction of motion and binocular disparity are co-organized in columns or clusters. Here, we analyze the pattern of intrinsic connectivity within cortical area V5/MT in both parasagittal sections of the intact brain and tangential sections from flatmounted cortex using small injections of the retrograde tracer cholera toxin subunit b. Labeled cells were predominantly found in cortical layers 2, 3, and 6. Going along the cortical layers, labeled cells were concentrated in regularly spaced clusters. The clusters nearest to the injection site were approximately 2 mm from its center. In flatmounted cortex, along the dorsoventral axis of V5/MT, we identified further clusters of labeled cells up to 10 mm from the injection site. Quantitative analysis of parasagittal sections estimated average cluster spacing at 2.2 mm; in cortical flatmounts, spacing was 2.3 mm measured radially from the injection site. The results suggest a regular pattern of intrinsic connectivity within V5/MT, which is consistent with connectivity between sites with a common preference for both direction of motion and binocular depth. The long-range connections can potentially account for the large suppressive surrounds of V5/MT neurons.

Adaptation to static scenes is a familiar and fundamental aspect of visual perception that causes negative afterimages, fading, and many other visual illusions. To establish a foundation for understanding the neuronal bases of such phenomena and to constrain the contributions of retinal versus cortical processing, we studied the responses of neurons in the dorsal lateral geniculate nucleus during and after the presentation of prolonged static visual stimuli. We found that parvocellular (P) cells (the more numerous and color-sensitive pathway) showed response adaptation with a time constant on the order of tens of seconds and that their response after the removal of a visual stimulus lasting 1 min was similar in amplitude and time course to the response evoked by the photographic negative stimulus. Magnocellular (M) cells (the faster-conducting and achromatic pathway) had after responses that were substantially weaker than responses evoked by patterned visual stimuli. This difference points to the existence of an adaptive mechanism in the P-pathway that is absent or impaired in the M-pathway and is inconsistent with full adaptation of photoreceptors, which feed both pathways. Cells in both pathways often maintained a substantial tonic response throughout 1 min stimuli, suggesting that these major feedforward inputs to cortex adapt too slowly to account for visual fading. Our findings suggest that faster-adapting mechanisms in cortex are likely to be required to account for the dynamics of perception during and after the viewing of prolonged static images.