” (Daily Telegraph, December 11, 2008) In summary, prescribing ac

” (Daily Telegraph, December 11, 2008) In summary, prescribing actions for optimizing brain performance was a salient theme around which Galunisertib cell line media coverage of neuroscience assembled. It communicated a view of brain health as a resource that required constant attention and calculated effort and was drawn into discussion about

childrearing practices. The second theme captured the use of neuroscientific findings to underline differences between categories of people in ways that were symbolically layered and socially loaded. This theme was most evident in articles within the categories psychopathology, sexuality, morality (particularly antisocial behavior), and bodily conditions (particularly obesity). Articles devoted considerable space to demonstrating male-female neurobiological differences and also to evidence that substance abusers, criminals, homosexuals, obese people, and people with mental health conditions had distinctive brain types. The content of media coverage of such groups tended to correspond with the content of existing stereotypes: for example, articles regularly linked obesity to low intelligence,

adolescence to disagreeableness, and women to irrationality. “Under stress or pressure, a woman sees spending time talking with her man as a reward, but a man sees it as an interference in his problem-solving process. She wants to talk and cuddle, and all he wants to do is watch football. To a woman, he seems uncaring and disinterested and a man sees her as annoying or pedantic.

These perceptions are a reflection of the different organisation and priorities of their brains.” (Daily Mail, January Levetiracetam Baf-A1 price 16, 2008) There was little room for ambiguity in media portrayal of group-related brain differences. It was common to encounter the phrase “the [adjective] brain,” with the brackets filled by categories like “male,” “teenage,” “criminal,” “addicted,” or “gay.” This implied the existence of a single brain type common across all members of the category and distinctly different from the brains of the categorical alternatives. Social groups were essentialized and portrayed as wholly internally homogeneous. “Addiction is viewed as a mental disorder, and gays are known to be at higher risk of anxiety, depression, self-harm, suicide and drug abuse. Most studies suggest that these problems are brought on by years of discrimination and bullying. But there is another controversial thesis—that gays lead inherently riskier lives. Gambling stimulates the dopamine system in the brain; illicit drugs pep up the same system. Are gays dopamine junkies?” (Times, December 18, 2006) The emphasis on group differences had particularly important implications for laying boundaries between the normal and the pathological. The brains typical of certain pathological categories were repeatedly contrasted with the brains of “normal” or “healthy” people. Detail about what exactly constituted normality was not provided.

To measure L3 responses to changes in light intensity under dynam

To measure L3 responses to changes in light intensity under dynamic, continuous illumination we used the Gaussian flicker stimulus and described L3 responses using a linear-nonlinear (LN) model. Onalespib concentration This model consists of a linear filter and a static nonlinearity. The linear filter represents the temporal sensitivity of the neuron, while the nonlinearity captures other aspects of the cell’s response

such as gain, threshold, and saturation (Figure 4D; Chichilnisky, 2001, Clark et al., 2011 and Sakai et al., 1988). These studies revealed that the linear filter of L3 displayed a single lobe of negative polarity (Figure 4D). The neurotransmitter receptor that detects photoreceptor responses in arthropods is a histamine gated chloride channel. Thus, this inversion reflects the sign

inverting synapse between photoreceptors and L3. Consistently, L3 displayed an increase in intracellular calcium to contrast decrements and a decrease in calcium to contrast increments. Interestingly, the temporal characteristics of the L3 linear filter were qualitatively different from those measured in L1, L2, and L4 (Figure 4D; Clark et al., 2011). In particular, while the initial response lobes of the linear filters for L1, L2, and L4 all decayed rapidly, reaching baseline in less than 400 ms, the L3 filter took almost three times as long to decay to baseline. These results demonstrate that stimulus features that happened hundreds of milliseconds in the past www.selleckchem.com/products/VX-809.html contributed to the calcium signal in L3 (Figure 4D). Interestingly, the static nonlinearity revealed that the mean calcium signal of L3 had different gains for increases and decreases in luminance GPX6 (Figures 4D and S4). This form was well fit by two linear functions, one for response increments (R2 = 93.4) and one for response decrements (R2 = 94.8), with a higher slope for the latter (Figure S4). The full LN model matched the response of the cells more closely than the linear prediction (R2 = 0.67 and R2 = 0.63; Figure 4E), mainly improving predictions for strong calcium responses (Figure 4E, arrowheads).

The rectified properties of L3 were also apparent when the 200 ms delayed response to a given contrast was plotted (Figure S4). Thus, unlike L1, L2 and L4, which respond with similar gains to contrast increments and decrements, L3 is rectified and has a higher gain for contrast decrements. These physiological data suggest that L3 could be preferentially involved in dark edge motion detection. Given the intriguing physiological responses of L3 and the previously proposed role for L4 in motion detection, we tested the effect of silencing these neurons on behavioral responses in a single-fly assay. We measured behavioral responses of tethered flies walking on an air-cushioned ball, surrounded by three visual stimulus displays, which allowed tight control of the visual stimulus presentation (Figure S5, Buchner, 1976 and Clark et al., 2011).

, 1999 and Lou et al , 2008), but the isoform responsible for thi

, 1999 and Lou et al., 2008), but the isoform responsible for this enhancement was not known. A broad-spectrum PKC inhibitor, though with questionable selectivity (Lee et al., 2008), reduced enhancement by PDBu to ∼40% of control, and inclusion of a more selective PKC blocking peptide in the

presynaptic terminal reduced the enhancement to less than 20% of that observed in control conditions (Hori et al., 1999). Previous studies suggested that the calcium-insensitive isoform PKCɛ mediates this enhancement because it is present at the calyx of Held and is activated by phorbol esters (Saitoh et al., 2001). However, our observation that phorbol ester-induced potentiation of evoked EPSCs is reduced by ∼70% in PKCα/β double knockouts compared to controls indicate that these two isoforms account for the bulk of the contribution of PKCs to EPSC enhancement by phorbol esters. check details Moreover, our results are consistent with the observation that ∼50% of phorbol ester-induced potentiation in the hippocampus is impaired in PKCβ knockout mice (Weeber et al., 2000). The component of phorbol ester-induced

enhancement that is not mediated by PKCs is likely mediated by the synaptic protein Munc13, either as a result of phorbol esters directly activating Munc13, or as a result of phorbol ester binding to the N-terminal domain of Doc2α, thereby allowing it to interact with Munc13 (Hori et al., 1999 and Lou et al., 2008). Phorbol esters enhance mEPSC frequency ∼6-fold in wild-type animals Decitabine molecular weight (Figure 8). In the absence of PKCα

and PKCβ, this enhancement is reduced by ∼50% (compare black and purple traces in Figure 8I). This result agrees with previous observations using pharmacology (Lou et al., 2008 and Oleskevich and Walmsley, 2000) and suggests that PKC plays a less important role in potentiating spontaneous release compared to evoked release. In double knockout animals, the impairment of the phorbol ester-induced increase in mEPSC frequency (Figure 8I), although moderate, contrasts with the lack of effect on tuclazepam tetanus-induced increase in mEPSC frequency (Figure 9C). Further studies are needed to understand this potential difference in the regulation of spontaneous activity. PKCα (in 129S2 genetic background) and PKCβ (in C57BL/6J genetic background) single knockout animals, generated by M. Leitges (Leitges et al., 1996 and Leitges et al., 2002), were bred together to obtain offspring heterozygous for both genes (het-het animals). Crosses of het-het animals generated α+/+ β+/+ (WT), α−/− β+/+ (αKO), α+/+ β−/− (βKO), and α−/− β−/− (double knockout) animals with a frequency of 1:16 each. All animal handling and procedures abided by the guidelines of the Harvard Medical Area Standing Committee on Animals.

Finally, rapid automatized naming was assessed using the object a

Finally, rapid automatized naming was assessed using the object and digit sheets from the PhAB (Frederickson et al., 1997) and two custom-made color

sheets. The dependent FK228 supplier variable is the total time taken to name all items on each sheet, irrespective of errors. Auditory steady state responses (ASSR) were induced using an amplitude-modulated white noise presented binaurally at 65 dB SPL. The sound modulation rate linearly increased from 10 to 80 Hz in 5.4 s (Poulsen et al., 2007) (Figures 1A and 1B). The sound was surrounded by 0.3 s of unmodulated white noise to prevent onset and offset effects. This 6 s (5.4 + 2 × 0.3 s) stimulus was presented 80 times (silent interstimuli interval (ISI): 2–4 s) during two sessions of 40 trials each. Participants watched a mute wildlife documentary during the experiment to ensure sustained vigilance (Wilson et al., 2007) and were asked to blink during ISI to prevent artifacts during the stimuli. Data were preprocessed and analyzed using in-house software (http://cogimage.dsi.cnrs.fr/logiciels/). ASSR were recorded using a CTF Systems MEG device, with 151 radial gradiometers over the scalp and 29 reference gradiometers and magnetometers

for ambient field correction (1,250 Hz sampling rate; 300 Hz online low pass filter). Trials contaminated with eye movements, blinks, and cardiac or muscular activity were rejected offline Selleck Tariquidar (Gratton et al., 1983). Remaining MEG trials were averaged from 0.3 s pre- to 6.3 s poststimulus onset and high-pass filtered at 0.26 Hz. PD184352 (CI-1040) After a baseline adjustment on the 300 ms prestimulus period, the averaged signal was used to compute cortical current maps with BrainStorm (http://neuroimage.usc.edu/brainstorm) using the weighted minimum-norm estimation approach. We estimated 15,000 cortically distributed current dipoles, whose locations (but not orientations) were constrained onto the gray

matter surface of either the individual brain images when available or the BrainStorm generic brain model built from the standard brain of the Montreal Neurological Institute (MNI colin27). Individual structural MRI could be acquired (Tim-Trio, Siemens; 7 min anatomical T1-weighted magnetization-prepared rapid acquisition gradient echo sequence, 176 slices, field of view = 256, voxel size = 1 × 1 × 1 mm) in 29 participants (14 controls/15 dyslexics: subjects 1–9, 11, 12, 14–18, 20–22, 25–35, and 38; see green frame in Figure S1). Head and cortex meshes were extracted with BrainVISA version 4.0 (http://brainvisa.info/). When individual MRI images could be used, source estimates were in a second step projected onto the standard MNI colin27: after realignment and deformation of the subject cortex surface to the Colin cortex surface, the sources amplitudes are interpolated from the subject surface to the Colin surface using Shepard’s method (weighted combination of a few nearest neighbors).

But the upper right quadrant—where low-contrast SD is greater tha

But the upper right quadrant—where low-contrast SD is greater than high-contrast SD—is the most populated, and many of the cells in this quadrant lie along the diagonal; their SD ratios changed little during inactivation. To evaluate the overall trend in the plot of Figure 2C, we can compare these data to what the two models for the origin of contrast dependent variability might predict. A thalamic origin predicts that cortical inactivation would have no effect on the SD ratio: the SD ratio would be identical

for intact and inactivated cortex, and all of the points would lie along the diagonal (red). A cortical origin predicts that cortical inactivation would abolish much of the difference in variability between low and high contrast. The SD

ratio would therefore be reduced toward 1, and the points would lie along a horizontal line at 1 (blue). We can test these 5-FU datasheet two predictions statistically. Since the fit to the points in Figure 2C is not significantly different from the diagonal (p = 0.71, paired t test) but was significantly different from a horizontal line at a value of 1 (p < 0.001), the data favor a thalamic origin for contrast-dependent changes in response variability. In addition to the control experiments of Chung and Ferster (1998), five observations confirmed that the electrical stimulus was effective in inactivating the cortex. First, find more mean spiking activity (pooled across all trials) was reduced more than 40-fold to 0.007 spike/trial, and peak spike rates were reduced 30-fold after cortical inactivation. Second, mean Vm responses to high-contrast preferred gratings were smaller after inactivation (52% reduction on average, n = 35), likely from the suppression of intracortical activity. Third, a marked hyperpolarization of Vm was evident immediately following the shock artifact, suggesting that the shock evoked a large inhibitory potential, which is likely one of the mechanisms by which spiking activity is silenced. Fourth, as noted above, Vm variability

immediately following the shock, but before the visual response, was markedly lower than the resting variability. In the time window between 5–10 ms following the shock, the trial-to-trial SD of the membrane 3-mercaptopyruvate sulfurtransferase potential was reduced relative to the resting cortex by 39% (p < 0.01, paired t test), pooled across all stimulus types (Figure 2B). Fifth, as observed previously (Finn et al., 2007), the fraction of thalamic inputs to these simple cells was highly correlated to their DC-Null/DC-Pref ratios (Figure S3B). Here, DC-Null and DC-Pref are the mean depolarizations evoked by high-contrast gratings at the null and preferred orientations. Because LGN responses are themselves not tuned to orientation, this ratio for the LGN input to a simple cell should be 1. Conversely, because the spike responses of cortical cells are orientation specific, this ratio for cortical inputs to a simple cell should be 0.

elegans may suggest the existence of similar mechanisms in the no

elegans may suggest the existence of similar mechanisms in the nociceptive and somatosensory pathways of larger nervous systems. A complete strain list and descriptions of plasmid and strain constructions are in Supplemental Experimental Procedures. Laser ablations were carried out using a standard protocol (Bargmann and Avery, 1995). The RIHs, OLQs, and FLPs were ablated in the early L1 stage, usually check details within 3–4 hr after

hatching; the PVD cells were ablated at a slightly later stage, near the end of L1. Loss of the ablated cell was confirmed by observing loss of cameleon fluorescence in the adult animal. Optical recordings were performed essentially as described (Kerr et al., 2000 and Kerr, 2006) on a Zeiss Axioskop 2 upright compound microscope equipped with a Dual View beam splitter and a UNIBLITZ Shutter. Fluorescence images were acquired using MetaVue 6.2. Filter-dichroic pairs were excitation, 400–440; excitation dichroic 455; CFP emission, 465–495; emission dichroic 505; YFP emission, 520–550. Individual adult worms (∼24 hr past L4) were glued with Nexaband S/C cyanoacrylate glue to pads composed of 2% agarose in extracellular saline (145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 5 mM MgCl2, 20 mM D-glucose, 10 mM HEPES buffer [pH 7.2]). Serotonin was also included at a concentration of 5 mM for nose touch-imaging

experiments. Worms used for calcium imaging had similar levels of cameleon expression in sensory neurons as inferred from initial fluorescence intensity. Acquisitions were taken at 28 Hz (35 ms exposure time)

with VEGFR inhibitor 4 × 4 or 2 × 2 binning, using a 63× Zeiss Achroplan water-immersion objective. Thermal stimulation was applied as described (Chatzigeorgiou et al., 2010b). The nose touch stimulator was a needle with a 50 μm diameter made of a drawn glass Oxalosuccinic acid capillary with the tip rounded to ∼10 μm on a flame. We positioned the stimulator using a motorized stage (Polytec/PI M-111.1DG microtranslation stage with C-862 Mercury II controller). The needle was placed perpendicular to the worm’s body at a distance of 150 μm from the side of the nose. In the “on” phase, the glass tip was moved toward the worm so that it could probe ∼8 μm into the side of the worm’s nose on the cilia and held on the cilia for 1 s, and in the “off “ phase the needle was returned to its original position. To visualize the harsh head touch response in FLP, the same nose touch setup was used, but the probe was aligned in a more posterior position between the two bulbs of the pharynx. The probe was displaced ∼24 μm at a raised speed of 2.8 mm/s. The stimulus was a buzz (i.e., the probe was displaced 2.5 μm in and out for the duration of the stimulus) lasting ∼1 s. To obtain single images we used a Zeis LSM 510 Meta confocal microscope with a 40× objective. Images were exported as single TIFF files. To measure the intensity of the fluorescence, we imported the TIFF image in ImageJ.

In no cell type is their function more vital than in neurons, in

In no cell type is their function more vital than in neurons, in which limited glycolysis causes the cell to rely on

oxidative phosphorylation for ATP production. In this study, we used dynamic imaging techniques to explore the mitochondrial pathophysiology in a VCP knockdown (VCP KD) human dopaminergic neuroblastoma cell line (SH-SY5Y) and in fibroblasts from patients carrying three independent pathogenic mutations in the VCP gene. We demonstrate that VCP deficiency induces Sotrastaurin the uncoupling of respiration from oxidative phosphorylation. This results in decreased mitochondrial membrane potential, leading to higher respiration and lower ATP levels due to reduced ATP production. These findings define a mechanism whereby VCP dysfunction may cause cell death and highlight pathophysiological events that may occur in IBMPFD. Mitochondrial membrane potential (ΔΨm) is an indicator of mitochondrial health and function. To study VCP implication in mitochondrial function, we transiently silenced the VCP gene using siRNA in CH5424802 research buy SH-SY5Y human neuroblastoma cells (see Figure S1A available online) and shRNA in mouse primary cortical cultures ( Figure 1D). Additionally, stable populations of VCP KD SH-SY5Y cells were generated using shRNA ( Figure S1B). ΔΨm was measured in VCP-deficient SH-SY5Y cells ( Figure 1A), in human fibroblasts from three patients with independent VCP mutations (R155C, R155H, and R191Q;

for donors’ details see Figure S1E and Table S1) and age-matched controls ( Figure 1B), and in primary neurons and astrocytes ( Figures 1C and 1D). A significant decrease in ΔΨm was observed in all VCP-deficient cell models studied (SH-SY5Y cells = 72% ± 8%, n > 20 cells in 3 independent experiments compared to either untransfected cells or cells transfected with scramble (SCR) control siRNA; primary neurons = 62% ± 9% and primary astrocytes = 74% ± 4%, n ≥ 5 cells in 3 independent experiments compared to cells transfected

with SCR control shRNA; fibroblasts from patient 1 = 86% ± 2%, n = 7; fibroblasts from patient 2 = 85% ± 2%, n = 8; fibroblasts from patient 3 = 91% ± 2%, n = 5, compared to age-matched control fibroblasts) ( Figures 1A–1C). Overexpression of R155H, Bumetanide R191Q, and R155C VCP mutants in SH-SY5Y cells is associated with a significant reduction in the TMRM signal (TMRM in cells overexpressing R155H VCP = 73% ± 3%; R191Q VCP = 65% ± 3%; R155C VCP = 62% ± 13% compared to overexpressed WT VCP; n ≥ 3), confirming that the three pathogenic VCP mutations have a dominant-negative effect ( Figure 1E). Re-expression of WT, but not mutant VCP, rescued the TMRM signal in a clonal population of stable VCP KD SH-SY5Y cells (untransfected cells = 61% ± 9%; WT VCP = 97% ± 7%; R155H VCP = 62% ± 4%; R191Q VCP = 60% ± 1%; R155C VCP = 74% ± 5% compared to SCR SH-SY5Y control cells; n ≥ 3) ( Figures 1F and S1D). In healthy cells, ΔΨm is maintained by mitochondrial respiration.

For this purpose, we patched pairs of sGFP cells and PCs, selecte

For this purpose, we patched pairs of sGFP cells and PCs, selected randomly from a local population,

to identify connected ones. From 40 pairs, 70% (n = 28) were monosynaptically connected and 30% (n = 12) were unconnected EPZ-6438 in vivo (Figures 5A–5C). The morphology of one reconstructed connected sGFP cell-PC pair showed a typical Martinotti morphology, with an axonal projection of a Martinotti cell toward the pia (Figure 5A). This large axon and the PC dendrites overlapped, with many potential connection sites. There was no significant difference between the distribution of intersomatic distances for connected (61.9 ± 5.3 μm, n = 23) or unconnected (73.6 ± 9.4 μm, n = 9) sGFP-PC pairs (p = 0.26, t test; Figure 5D). Intersomatic distances

between sGFP cells and PCs were within 150 μm (average of 65.0 ± 4.5, n = 32) and the probability of being connected was homogeneously high from 0 to 150 μm (Figure 5E). In fact, the average connection probability observed for these recorded pairs (0.7) was similar to the probability observed in RuBi-Glutamate mapping experiments within a 200 μm radius from the PC (0.71 ± 0.03, n = 61). In addition, analyzing the connection probability within 150 μm intersomatic distances from the optical mapping experiments, to match with the distances considered with the paired those recordings, also demonstrated a similar connection probability (0.73 ± 0.04, n = SCH772984 61). Altogether, these results from the optical mapping and paired recording experiments indicate a dense inhibitory connectivity from the somatostatin-expressing interneurons within local circuits (<200 μm distance from the PC). Intracortical connectivity

has long been assumed to be vertically organized (“chains” or “columns” [Lorente de Nó, 1949 and Mountcastle, 1982]), and this columnar organization has been observed in the frontal cortex as well (Isseroff et al., 1984). Therefore, we reanalyzed the optical mapping data, measuring the distances between the soma of the sGFP interneurons and a line through the recorded PCs, perpendicular to the pial surface (Figure 6A); this line would correspond to the axis of a hypothetical column. In this analysis, we observed a narrower distribution of the distances of connected interneurons, on average located at ∼100 μm from a putative columnar axis (Figures 6B and 6C). Unconnected cells were located on average at >200 μm from this axis, displaying a broad distribution of distances. False positives showed a peak centered at <100 μm of from the axis (Figure 6C). This analysis showed therefore that sGFP cells located within the same “column” of the considered PCs are more likely to contact and modulate them.

What exactly has gone

What exactly has gone selleck products wrong with the state generation process due to the cholinergic manipulations? Here, the comprehensive set of metaphoric hoops through which the rats were made to jump becomes key to

narrowing down the options, highlighting the utility of using the incisive behavioral manipulations that animal learning theorists have spent decades developing. To understand what went wrong, it is useful to first review what aspects of learning were not disrupted by cholinergic manipulations: in addition to intact goal-directed learning, the comprehensive battery of tests shows that new state formation was not completely abolished. This is evident in the test following the third challenge, extinction training, in which exposure to one of the outcomes led to reinstatement of responding. Reinstatement indicates that extinction training did not simply overwrite and erase previous associations between actions and outcomes (Gershman et al., 2010), but rather reward omission caused rats in both groups to create a new state (Figure 1, state 4). However, reinstatement in cholinergically impaired rats was far from normal: these rats reinstated both actions ( Figure 1, right). One possible

explanation for this pattern of results (option A in Figure 1, bottom) is that upon reinstatement the rats erroneously retrieved Cisplatin Dipeptidyl peptidase two states—the most recent, postreversal state (state 3 in Figure 1), in which the right lever was mapped to sucrose and the left to pellets, and the state from initial training (state 1 in Figure 1), in which the lever to reward mapping was the reverse. This may, in fact, sound familiar to world travelers: a foolproof policy for safe street-crossing in some countries is to look left-right-left-right repeatedly, that is, to act upon both pre-travel and in-travel states. Such a retrieval deficit could also explain the lack of specificity of the postreversal devaluation test, in which cholinergically impaired

rats devalued both actions rather than only the one associated with the satiated outcome ( Figure 1, third column). Finally, it can also explain the intermediate level of responding in the contingency degradation test ( Figure 1, second column) by assuming that the new state (state 2, in which not pressing was associated with the outcome) was retrieved together with the old state (state 1). The deficit in reinstatement was observed even when cholinergic function was disrupted only during learning, yet this does not rule out a retrieval deficit, as retrieval of the appropriate states is also necessary during learning. That is, in order to learn, on every trial, the rat must retrieve and update associations within the current state.

075 s, spatial resolution: 0 33 mm, table speed: 458 mm/s; ferret

075 s, spatial resolution: 0.33 mm, table speed: 458 mm/s; ferret thorax acquisition times ≈0.22 s; enables accurate Modulators scanning of living ferrets without the necessity of breath-holding, respiratory gating, or electrocardiogram (ECG)-triggering) as previously described [28] and [29]. Briefly, all animals of group 1 (saline; infection control), group 2 (TIV; parenteral control) and of group 4 (nasal Endocine™ formulated split antigen, 15 μg HA) were scanned 6 days prior to virus inoculation (day 64) to define the uninfected baseline status of Galunisertib the respiratory system, and after challenge on 1, 2, 3 and 4 days

post inoculation (dpi). During in vivo scanning the anesthetized ferrets were positioned in dorsal recumbency selleck chemicals in a perspex biosafety container of approximately 8.3 l capacity that was custom designed and built (Tecnilab-BMI). The post-infectious reductions in aerated lung volumes were measured from 3-dimensional CT reconstructs using lower and upper thresholds in substance densities of −870 to −430 Hounsfield units (HU). Differences between the groups immunized with the Endocine™

adjuvanted H1N1/California/2009 vaccine preparations (groups 3–6) were analyzed statistically using the Kruskal–Wallis test. Differences between the sham (saline) immunized control group and the immunized groups were statistically analyzed using the two-tailed Mann–Whitney test. One intranasal immunization with Endocine™ adjuvanted split, or whole virus antigen induced high homologous HI antibody titers: in all ferrets of groups 3 and 5 (5 and 30 μg HA split antigen; titers 160–1120 and 400–3200, respectively) and in 5 out of 6 ferrets of groups 4 and 6 (15 μg HA split and whole virus antigen at; titers

≤5–5760 and 5–1280, respectively). A second immunization increased HI antibody titers in all ferrets, Cell press irrespective of antigen and antigen dose (groups 3–6, titers 1120–2560, 1120–5760, 640–3840 and 100–2880, respectively) (Fig. 1A). A third intranasal immunization did not substantially boost the HI immune response further (groups 3–6, titers 1280–3840, 1920–4480, 1280–3200 and 160–2560, respectively). The differences in HI antibody titers between the 3 split antigen HA doses (groups 3, 4 and 5) were not significant (p > 0.05). However, mean HI antibody titers in group 4 (15 μg HA split antigen) were significantly higher than those in group 6 (15 μg HA whole virus antigen); p = 0.01 and p = 0.02 after 2 and 3 immunizations, respectively. Cross-reactive HI antibodies were measured against the distant H1N1 viruses A/Swine/Ned/25/80, H1N1 A/Swine/Italy/14432/76 and H1N1 A/New Jersey/08/76 (Fig. 1B–D, respectively). The highest cross-reactive HI antibody titers were measured in group 4 (15 μg HA split antigen) after 2 immunizations.