Changes in response latencies and magnitudes of BF-matched (A1 and A2) and BF-unmatched (B1 and B2) CN neurons to the time interval from onset of the ES_AI.

<p>Data presented were sampled with protocol 1. The grey bars represent ES_AI.</p

The changes in BFs (A) and response magnitudes (B) of CN neurons induced by ES_AI as the function of BF differences between the stimulated AI and measured CN neurons.

<p>These changes of BF-matched and BF-unmatched CN neurons were clearly different. The line in A is a regression line. Open circles represent data sampled with protocol 1 and filled circles represent data sampled with protocol 2. The grey boxes and bars in B represent the mean ± SD of percentage changes in spike number following ES_AI.</p

Effects of sham stimulation of the AI on the auditory responses of CN neuron.

<p><b>A</b>. Raster plot of typical changes in auditory responses of a CN neuron when a stimulating electrode was placed in the AI but no electrical stimulation was given. <b>B</b>. Averaged spike numbers calculated within a 10-minute window during the observation period. <b>C</b>. Averaged response latencies calculated within a 10-minute window during the observation period. The asterisk represents the BF of the AI neuron where the electrode was placed. Arrowheads indicate supplementary injection of ketamine and xylazine. N: no electrical stimulation.</p

Effect of ES_AI on the auditory responses of a BF-matched CN neuron sampled with protocol 1.

<p><b>A</b>. Raster plot of typical changes in auditory responses of a CN neuron with the same BF as that of AI neuron before, during, and after ES_AI. The responses to 19–20 kHz tone markedly increased following ES_AI. <b>B–D</b>. Changes in the latencies of this neuron in response to 19 kHz, 20 kHz and 21 kHz tones following ES_AI. The response latencies were reduced by ES_AI. The asterisk represents the BF of the stimulated AI neurons. The grey lines in B–D represent ES_AI.</p

Effect of ES_AI on the auditory responses of a BF-unmatched CN neuron sampled with protocol 1.

<p><b>A</b>. Raster plotting of a typical change in auditory responses of a CN neuron that had a BF different from that of AI neurons before, during, and after ES_AI. The responses to 12–13 kHz decreased and then ceased whereas the responses to 14 kHz appeared and gradually increased following electrical stimulation of AI neuron tuned 16 kHz. <b>B–D</b>. Changes in the latencies of this neuron in response to 12 kHz, 13 kHz and 14 kHz tones following ES_AI. The response latencies to 12–13 kHz tones gradually increased before the cessation of auditory responses and recovered with extremely long latencies. The changes in response latencies were similar to that of BF-matched neurons depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014038#pone-0014038-g001" target="_blank">Fig. 1</a>. The asterisk represents the BF of the stimulated AI neurons. The grey lines in B–D represent ES_AI.</p

Effects of electrical stimulation of non-AI cortical area on the auditory responses of CN neuron.

<p><b>A</b>. Raster plot of typical changes in auditory responses of a CN neuron before, during and after electrical stimulation. <b>B</b>. Averaged spike numbers calculated within a 10-minute window during the observation period. <b>C</b>. Averaged response latencies calculated within a 10-minute window during the observation period. Arrowheads indicate supplementary injection of ketamine and xylazine. ES: electrical stimulation. N: no electrical stimulation.</p

Time required for 10% change, maximum changes, and 50% recovery in response latencies and magnitudes of the BF-matched and BF-unmatched neurons after ES_AI.

<p>Data presented were sampled with protocol 1. *: <i>p</i><0.001, compared between matched and unmatched neurons.</p

Increases in antigen-specific IgG by PorB is dependent on TLR2 and MyD88.

<p>(a) Concentration of IgG antibody to Ova in C57Bl/6 mice as measured by ELISA in serum on from day 42. WT C57Bl/6, TLR2 ^-/- and MyD88 ^-/- mice were vaccinated on days 0, 14 and 28 with Ova, Ova + PorB or sham (PBS). Vaccines that included PorB showed significantly less elevation in IgG over vaccines that did not include PorB in TLR2 ^-/- mice, and no detectable effect in MyD88 ^-/- mice. None of the mice had detectable antibodies to Ova prior to vaccination (data not shown). (b) OD of specific subtypes of anti-Ova IgG at a 1:50 dilution of serum as measured by ELISA. In WT mice, IgG1 and IgG2b were the dominant subtypes indicative of a Th2-type response. These in turn decreased in the TLR2 ^-/- mice, consistent with the total IgG data. Data represents one of two experiments. * p<0.05.</p

IL-1β induction by PorB requires ATP to activate the inflammasome.

<p>WT BMDMs were stimulated with PorB, LPS, TNFα, or Pam₃CSK₄ at the indicated concentrations for 5 hours. After 5 hours, ATP was added to half the cells for 30 minutes. Supernatants were collected and analyzed by IL-1β ELISA. IL-1β release was not observed for PorB, LPS or Pam₃CSK₄ in the absence of ATP. In the presence of ATP, significantly increased IL-1β release was observed for all three TLR ligands (p<0.05). </p

Upregulation of co-stimulatory cell-surface proteins by PorB is partially TLR2 dependent.

<p>WT and TLR2 KO BMDMs were stimulated in culture with PorB, Pam3CSK4 or LOS, then stained for markers of activation and examined by flow cytometry. In WT cells, PorB increased surface expression of CD14, CD40, CD54, CD69 and CD86 as compared to unstimulated cells. Knockout cells had decreased expression of CD14, CD40 and CD86, but still had higher expression of CD54 and CD69 when compared to basal levels. In the latter two cases, the TLR2 ligand Pam3CSK4 had no effect on TLR2 -/- cells. Histograms are from one representative sample of at least 3. Data represents one of two experiments.</p