Likewise, nose-touch-evoked calcium transients in FLP were significantly reduced, resembling in magnitude the responses in the RIH-ablated animals ( Figure S7); FLP harsh head touch responses, in contrast, were unaffected ( Figure S7). unc-7 loss-of-function

mutants showed partial defects in nose touch escape behavior ( Figures S7 and S8). These nose touch defects were rescued when a functional unc-7(+) transgene was expressed in the nose touch circuit using the cat-1 (expressed in the CEPs, RIH, and few other neurons) and egl-46 www.selleckchem.com/products/epacadostat-incb024360.html (expressed in FLP and PVD) promoters ( Figure 6B; Figures S7 and S8). unc-7(+) expression using either promoter alone did not result in phenotypic rescue (data not shown), suggesting that gap junction formation requires production of the innexin protein in both connected neurons. In contrast, mutations in unc-13, which impair synaptic transmission, did not detectably impair RIH nose touch responses ( Figure 6B). Together, these results support the hypothesis that signaling in the RIH-centered nose touch circuit is predominantly, if not exclusively, mediated by gap junctions. If signaling in the nose touch circuit is mediated primarily by gap junctions, information flow through RIH might be bidirectional: just as activation of neurons such as OLQ can indirectly excite FLP,

FLP activation could be able to excite OLQ. We examined this possibility by imaging OLQ calcium dynamics in response to mechanical stimuli sensed by FLP. We observed that harsh touch applied to the side of the head led to robust calcium transients in OLQ as well as RIH http://www.selleckchem.com/products/blz945.html (Figures 8B and 8C; Figure S7E). Mutations in the mechanosensory channel mec-10 eliminated OLQ and RIH responses to harsh head touch, and these responses could be rescued by FLP-specific expression of mec-10 ( Figures 8B and 8C; Figure S7E). Moreover,

ablation of RIH eliminated the harsh head-touch-evoked calcium transients in OLQ ( Figures 8B and 8C), indicating that the FLPs indirectly activate the OLQs through the RIH-centered network. We also tested the effect of the network on nose touch responses in OLQ. Interestingly, a mec-10 mutation significantly impaired OLQ and RIH calcium responses to nose touch; oxyclozanide these defects were rescued by mec-10(+) expression in FLP ( Figures 8B and 8D). Furthermore, ablation of RIH significantly reduced the responses of the OLQ neurons to nose touch ( Figures 8B and 8D). These results indicate that just as the nose touch responses of the FLPs depend on a combination of RIH-mediated network activity and cell-autonomous MEC-10 function, OLQ nose touch responses depend on both RIH-mediated network activity and cell-autonomous OSM-9 function. We have shown here how a network of interacting mechanosensory neurons detects nose touch stimuli and in response evokes escape behavior.

Such changes were absent in dorsal striatum (Figures S2A and S2B). Next, to examine potential similarities between

cocaine- and stress-induced regulation of histone methylation in this brain region, the standard 10 day chronic social defeat stress protocol was used (Berton et al., 2006). As previously reported (Krishnan et al., 2007 and Vialou et al., 2010), two distinguishable groups of defeated mice, susceptible and unsusceptible, were observed based on a measure of social avoidance, in which susceptible mice displayed decreased social interaction compared to both control and unsusceptible mice (Figure 2C). Ten days after the final defeat, susceptible and unsusceptible mice, as well as nondefeated controls, were analyzed for G9a protein levels in NAc. G9a expression was significantly decreased in NAc of susceptible

mice compared to unsusceptible animals (Figure 2D), which were no different from nonstressed controls. These effects were VX-770 in vivo not observed in dorsal striatum (Figure S2C). Consistent with a reduction in G9a expression in susceptible animals, global levels of H3K9me2 were decreased in NAc of these mice compared to both control and unsusceptible mice (Figure 2E). Additionally, Glp mRNA, similar to G9a mRNA, was significantly reduced in NAc of susceptible mice, a molecular response that was absent in unsusceptible mice ( Figure 2H). Numerous other repressive chromatin modifiers, such as Suv39h1, many of which form multimeric-binding complexes with G9a and GLP ( Fritsch et al., 2010), were also significantly induced in NAc of unsusceptible mice ( Figure 2H), indicating that increased repressive chromatin regulation ABT-263 concentration may contribute to proadaptive responses to stressful stimuli. In dorsal striatum, in contrast, H3K9me2 of was significantly increased after social stress in both susceptible and unsusceptible mice ( Figure S2D). Regulation of H3K9me2 in NAc after chronic social stress is specific for this mark, which is euchromatic, as global levels of the associated heterochromatic mark H3K9me3 were unaltered in NAc of both susceptible and unsusceptible mice ( Figure S3A). To extend these findings in mice to clinical

depression, we evaluated G9a and Glp levels, as well as other repressive chromatin modifiers, in NAc of postmortem human depressed patients (all symptomatic at their time of death; see Supplemental Experimental Procedures for detailed methods on tissue collection). Similar to results observed in mice susceptible to social stress, G9a ( Figure 2F) and Glp ( Figure 2H) mRNA levels were significantly reduced in these patients. Numerous other enzymes involved in transcriptional repression—shown to be significantly induced in NAc of unsusceptible mice—were also downregulated in NAc of human depressed subjects ( Figure 2H). Consistent with decreased G9a and Glp expression in NAc of depressed humans, global levels of H3K9me2 were also significantly reduced ( Figure 2G).

6–9.8) for 45 min at 4 °C

and washed four times before samples were applied. Sera were applied in serial two-fold or triple-fold dilutions and a mouse control serum sample positive for A/Sidney/5/97 or A/Beijing/262/95 H1N1 was included on each plate. For detection of SIgA, 100 µl of the lavage was used undiluted in the first well and subsequently serial two-fold diluted. The plates were incubated for 1.5 h at 4 °C, washed 3 times and incubated for 1 h at 4 °C with anti-mouse Ig-HRP conjugates (Southern Biotech). After incubation, the plates were washed 3–4 times and incubated for 30 min with Bortezomib clinical trial 100 µl staining solution (1 tablet of OPD (o-Phenylenediamine dihydrochloride) dissolved in 100 ml 0.05 M phosphate-citrate buffer pH 5.0

and 40 µl H2O2). After incubation the reaction was stopped by adding 50 µl 2 M H2SO4 per sample and the absorbance was determined at 492 nm. The IAV-specific IFN-? T-cell and IAV-specific B-cell response in the spleen and local draining cervical lymph nodes (CLN) or inguinal lymph nodes (ILN) after i.n. BLP-SV or i.m. SV vaccination, respectively, was assessed by ELISPOT. For detection of IAV-specific CB-839 B-cells, cells were directly cultured in high protein binding filter plates (MultiScreen-IP, Millipore) that were pre-coated with Vaxigrip® suspension for injection: strains 2009/2010, Sanofi Pasteur MSD, lot: E7068 at 1 µg per well dissolved in 50 µl of PBS. For detection of IAV-specific IFN-? T-cells, cells were cultured in the presence of HA antigen or IMDM (Gibco, Invitrogen) medium as a control that was supplemented with heat-inactivated 5% FCS (Bodinco,

The Netherlands), 5 × 10-5 M 2-mercaptoethanol, penicillin (100 units/ml) and streptomycin (100 µg/ml) (Gibco, Germany) for 72 h at 37 °C in high protein binding filter plates (MultiScreen-IP, Millipore) that were pre-coated with a rat anti-mouse IFN-? monoclonal antibody (clone AN-18, purchased at BD Biosciences, Pharmingen) at Mephenoxalone 0.1 µg per well dissolved in 50 µl of PBS for 48 h at 37 °C. After incubation, spot forming units of IAV-specific B- and T-cells were detected with goat-anti-mouse IgG-biotin (Sigma) and Avidin-AP (Sigma). Plates were developed with NBT-BCIP (Roche) and analyzed by using the Aelvis spotreader and software. Data are shown as IAV-specific IFN-? T-cell or the IAV-specific B-cell count per 106 cells above background. Single cell suspensions were prepared from spleen and draining lymph nodes and cells were cultured for 72 h in the presence of ConA at 2.5 µg/ml or IMDM (Gibco, Invitrogen) at 37 °C. Analyzing the culture supernatants assessed the amount of cytokine secreted during a 72 h T-cell re-stimulation. Briefly, fluoresceinated microbeads coated with capture antibodies for simultaneous detection of IL-17A (TC11-18H10) and IL-5 (TRFK5) were added to 50 µl of culture supernatant. Cytokines were detected by biotinylated antibodies IL17 (DuoSet ELISA kit, R&D systems Europe Ltd, the U.K.

Although Mab and Mad occupy the same cortical territory as mouse CFA and RFA (Tennant et al., 2011), important differences exist between them. First, Mab and Mad are contiguous and equal in area, whereas CFA is larger than RFA and they are separated buy ABT-737 by a representation of the neck (Tennant et al., 2011). Second, RFA is not apparent in all experiments or animals (Tennant et al., 2011), whereas Mab and Mad almost always co-occur. It is interesting

to note that in rats, mapping with short stimulus durations produces maps that include RFA and CFA, whereas long (500 ms) durations reveal maps containing movement representations similar to Mab and Mad (Ramanathan et al., 2006). Primate motor cortex is commonly described as a hierarchical arrangement of primary motor cortex, premotor areas, and supplementary motor cortex where premotor areas can facilitate motor output from primary motor

cortex (Cerri et al., 2003). It has been suggested based on their connectivity that rodent RFA and CFA are homologous to premotor and primary motor cortex, respectively (Rouiller et al., 1993). Our observation that Mad expands after http://www.selleckchem.com/products/pci-32765.html application of GABA receptor antagonists but Mab does not suggests that these regions may be differentially regulated Adenosine by feed-forward or lateral inhibition. Coupled with the relatively longer latencies for movements evoked from the more caudal Mad region, this could be viewed as evidence for a hierarchical arrangement of mouse motor cortex. Although intracortical connections are obviously critical for motor function, it is also known that multiple motor cortical regions project in parallel to the spinal cord (Rouiller et al., 1993 and Dum

and Strick, 2002). This implies that multiple motor regions can contribute directly to movement, and may not be arranged hierarchically (Graziano and Aflalo, 2007). This view is corroborated by the results of our experiments with glutamate and GABA receptor antagonists, which demonstrated that the Mab and Mad representations could function independently after a diminution of intracortical synaptic transmission. If multiple motor regions do not form a hierarchical chain, they may instead encode various behaviors or postures (Graziano et al., 2002 and Graziano et al., 2005). This is consistent with our observation that stimulation of Mab and Mad drives limb movements to different end positions in space. This result could be produced with optogenetic or electrical stimulation, suggesting that it is not an artifact of passive electrical current spread from the stimulation site (Strick, 2002).

05, one-way ANOVA, Kruskal-Wallis with Dunn’s multiple comparison test). However, the response during all waking periods remained significantly different from wake 1 (p < 0.01, one-way ANOVA, Dunnett’s multiple comparison test). The overall mean amplitude for a first wake episode (n = 13, from 4 different GSK-3 inhibitor cats) was 0.297mV ± 0.176mV and it was 0.328mV ± 0.177mV during wake 2; this difference

was highly significant (Figure 1G, p < 0.001, Wilcoxon matched-pairs signed-rank test). Even relatively short periods of SWS were sufficient to enhance responses in the second wake episode (Figure 1H). We obtained six intracellular recordings from somatosensory cortical neurons in which we recorded evoked responses to

medial lemniscus stimuli during two consecutives wake episodes separated by a period of SWS, although it was not always the first and second wake episodes (Figure 3). As expected, membrane potential recordings showed the presence of prolonged silent states during SWS, which were absent during wake (Figure 3A). Responses were highly variable during SWS (data not shown), while they were stable during wake (Figures 3B and 3D). In five out of the six intracellular recordings in which we were able to obtain stable recording throughout wake-SWS-wake transitions (85 recording sessions, near 200 wake-SWS-wake transitions, 2 animals), the response amplitude was increased in the second wake episode (post-SWS) as compared to the first episode (pre-SWS); however, due to the small number of recordings, the difference was not significant (Figure 3C, p = 0.2, Wilcoxon matched-pairs see more signed-rank test). Therefore, intracellular recordings support field potential observations. To characterize the mechanisms implicated in the potentiation of evoked responses, we performed whole-cell recordings from layer II/III pyramidal regular-spiking neurons in vitro. From in vivo LFP recordings, we extracted the timing of a single unit firing recorded Bay 11-7085 during SWS (Figure 4A) and during wake (Figure 5A) and used that timing to build sleep-like ( Figure 4B) and wake-like ( Figure 5B) patterns

of synaptic stimulation; the timing of slow waves was also detected to build the intracellular hyperpolarizing current pulse stimulation pattern replicating hyperpolarizing (silent) states of SWS ( Figure 4B, see Experimental Procedures). The stimulation protocols are detailed in Figure S2. The mean membrane potential of neurons recorded in vitro was maintained to about −65mV to mimic the membrane potential of cortical neurons during wake or active phases of SWS. Minimal intensity stimuli were applied in the vicinity of recorded neurons. The sleep-like pattern of synaptic stimulation induced a transient facilitation only ( Figure 4C; 0.640mV ± 0.245mV in control versus 0.817mV ± 144mV in the first minute after conditioning, p < 0.05, Mann-Whitney test).

2% ± 21%

n = 6) despite baseline levels (101.2% ± 6.3%) similar to the untreated animals. These results demonstrate that 20 min of visual conditioning is sufficient to increase transcription under control of the BDNF exon IV promoter in an NMDAr-dependent manner in tectal neurons in the intact animal ( Figures 1B and 1C). Next, we tested whether this enhanced BDNF exon IV promoter activity led to a change in BDNF protein levels in the tectum. At 5 hr after visual conditioning, midbrains including the optic tectum, were surgically isolated and homogenized for western blotting. Blots were probed with an antibody that recognizes both the immature and mature forms of BDNF. Visual conditioning led to an increase in the ratio of proBDNF to mBDNF (control: 0.04 ± 0.01, conditioned: 0.26 ± 0.04; Figures selleck 1D and 1E, n = 3 repeats, 5 animals per condition for each experiment). Because the antibody gave several bands, we confirmed the identity

of the BDNF bands by introducing a BDNF antisense Morpholino (BDNF MO) oligonucleotide, 3 MA fluorescently tagged with lissamine rhodamine. At 5 hr postconditioning, brains that had been previously electroporated with the BDNF MO showed reduced expression of proBDNF compared with brains electroporated with a scrambled MO or conditioned animals without MO treatment (Figure 1F, n = 2 experiments, 4-5 animals per experiment). As a retrograde spread of plasticity from the tectum to the eye has been reported (Du et al., 2009), we also assayed proBDNF levels in the eyes of conditioned animals. However, conditioning did not induce a detectable change

in proBDNF levels in the eye (Figure S1 available online). Thus, the activation of the BDNF exon IV promoter by visual conditioning resulted in increased proBDNF protein levels in the tectum. The activity-dependent regulation of BDNF levels is significant, as BDNF has been reported to modulate the susceptibility of synapses to undergo plasticity. In the hippocampus, proBDNF has been shown to facilitate LTD and in Xenopus mBDNF is thought to be required for retinotectal LTP ( Du et al., 2009, Mu and Poo, 2006 and Woo et al., 2005). To determine if the proBDNF synthesized in response to visual conditioning affected old retinotectal plasticity, we first examined the effects of visual conditioning in a plasticity protocol designed to enhance stimulus direction sensitivity of tectal neurons, believed to engage both LTP and LTD at tectal cell synapses ( Engert et al., 2002, Mu and Poo, 2006 and Zhou et al., 2003). To increase proBDNF levels, animals were visually conditioned and then returned to their normal visual environments. At 4–6 hr postconditioning, animals received three bouts of training with a moving bar projected onto the retina. The training bouts were delivered at 4 min intervals. This spaced training protocol is designed to induce direction selectivity in tectal neurons as previously described (Engert et al., 2002 and Zhou et al., 2003). (n.b.

In the flexible value procedure (Figure 1A), the saccade to one object was followed by a reward and the other object was associated with no reward, and this contingency

was reversed frequently. To examine the short-term behavioral learning, we measured the target acquisition time after a go cue (the disappearance of the fixation point). As the value of each object changed blockwise, the target acquisition time changed accordingly: the monkeys made saccades more quickly to the high-valued object Dabrafenib clinical trial than the low-valued object (Figure 1B) (difference of target acquisition time: 57.7 ms, p < 0.001, two-tailed t test). On choice trials (see Experimental Procedures), the monkeys mostly chose the high-valued object (average: 83.9% ± 0.8%). These saccades can be called “controlled saccades,” because they were controlled by reinforcing feedbacks delivered just after the saccades. During learning of stable values (Figure 1C), the saccades to a set of objects were always followed by a reward (high valued) and the saccades to a different set of objects were always followed by no reward (low valued), and this was repeated across days (see Figure S2 for detail). To examine the long-term behavioral memory, we used a free-looking task (Figure 1D) and a free-viewing procedure (Figure S2D). These tests were done at least 1 day after the learning session, and the saccades were followed by no reward. Yet, see more the monkey made saccades to the objects

automatically and did so more likely to high-valued objects than low-valued objects. The preference to the high-valued objects emerged slowly across several daily learning sessions and then remained stable after four daily sessions of learning (Figure S2D), as reported previously (Yasuda et al., 2012). Therefore, to analyze the neuronal and behavioral coding of stable object values, we used fractal objects that the monkey had learned for more than four daily sessions. Cytidine deaminase When such well-learned objects were used in the free-looking task, the likelihood of saccades to high-valued

objects was significantly higher than to low-valued ones (Figure 1D, right) (difference of automatic looking: 18.9%, p < 0.01, two-tailed t test). These saccades can be called “automatic saccades,” because they were not followed by any reinforcing feedbacks delivered just after the saccades. To test whether the caudate nucleus controls the saccade behavior to choose high-valued objects, we recorded spike activity of single neurons in the caudate nucleus using the flexible and stable value procedures. We first found that many neurons in the caudate nucleus responded to visual objects, confirming previous studies (Brown et al., 1995, Caan et al., 1984, Rolls et al., 1983 and Yamamoto et al., 2012). The ratios of neurons that responded to fractal objects relative to the encountered neurons in the three caudate regions (Figure 3A) were: head 163/845 (19.3%), body 109/381 (28.6%), and tail 107/205 (52.2%).

For testing C9ORF72 iPSN sensitivity to glutamate-induced excitotoxicity, healthy control and C9ORF72 iPSNs were treated with various concentrations of L-glutamate (1, 3, 10, 30, 100 μM) for 2–8 hr. At the appropriate time point, cells were incubated with 1 μM

propidium iodide and 1 μM calcein AM (Invitrogen) for 30 min to visualize dead and live cells, respectively. For ASO rescue experiments, iPSNs were treated with ASOs for 72 hr and then treated with L-glutamate prior to dead/live cell quantification. ADARB2 fusion protein was expressed and purified from Rosetta II cells following gateway expression system (Invitrogen) and GSTrap HP column purification. Increasing concentrations of purified protein was incubated selleck chemicals Selleckchem Dinaciclib with 10 nM Cy5-labeled RNA. The fraction of RNA shifted, due to ADARB2 binding, was densitometrically quantified in ImageJ (NIH). Z-stack images were

taken on a Zeiss Axioimager with the Apotome tool or a Zeiss LSM510-meta single-point laser scanning confocal microscope matched exposure times or laser settings and normalized within their respective experiment. Statistical analysis was performed using the Student’s t test or one-way analysis of variance with the Turkey’s or Dunnet’s post-hoc test and the Prism 6 software (GraphPad Software, Inc.). Additional details are provided in the Supplemental Experimental Procedures. C.J.D designed, performed, and analyzed the experiments and the OME ASO; maintained, treated, and characterized the C9ORF72 fibroblasts and iPSNs; and wrote the manuscript.

P.Z. optimized and performed Southern blot methodologies and characterized repeat lengths of the cell and tissue used in these data sets. J.T.P. aided in iPSN differentiation, iPSN characterization, and siRNA and RNA-FISH experiments. A.R.H. and J.W. generated and purified ADARB2 protein and performed the EMSA experiments. N.A.M. analyzed and categorized microarray data. S.V. maintained C9ORF72 cell lines, aided in RNA isolation, tested antibody specificity, and optimized and performed the RNA-coIP experiments. E.L.D. differentiated and maintained all iPSCs cells to the neuronal stage and quantified iPSC differentiation efficacy. E.M.P. performed the proteome array assay. B.H. performed imaging analysis for proteome array hits. D.M.F. aided in blinded imaging and gene expression old analysis. N.M. and P.T. provided fibroblast or iPSC lines. B.T. screened human tissue for C9ORF72 expanded repeat and provided critical input for experimental design and strategies. F.R. and C.F.B. designed and provided MOE ASOs and performed initial ASO screening as well as providing input on experimental ASO strategies. S.B. provided the proteome array and aided in analysis and interpretation. R.S. and J.D.R. oversaw project development, experimental design, data interpretation, and manuscript writing. This work was funded by grants from NIH (J.D.R.), P2ALS (J.D.R.), Muscular Dystrophy Association (J.D.R.

Under these conditions, locomotion was correlated with an increase in both ge and gi for all cells tested (Figures 3F and 3G; Table Birinapant cost 1). Interestingly, the balance of excitation and inhibition (E/I balance) was also modulated by behavioral state, reflected by a depolarization in the reversal potential of the visually evoked conductances (Figure 3H; Table 1). Together, both an increase in total conductance and a shift in the E/I balance toward excitation would be expected to produce larger

subthreshold depolarizations, consistent with our current-clamp data and the increased spiking during locomotion reported here and in previous studies (Figure S2; Ayaz et al., 2013 and Niell and Stryker, 2010). To examine whether the stationary and moving states are relevant for visual behavior, we trained mice to perform a visual detection task and analyzed their performance during the two conditions. Mice learned to lick for a water reward during the presentation PD0332991 research buy of drifting gratings of varying contrasts (low: 9%/16%; medium:

27%/50%; high: 81%/100%) and to withhold licking for the presentation of a gray screen (Figures 4A and 4B; Figures S4A and S4B). Injection of the GABAA-receptor agonist muscimol into V1 (n = 4) significantly impaired behavioral performance compared to saline controls (n = 4; Figure 4C; Figure S4C), indicating that the visual cortex was necessary for this task. Interestingly, locomotion was correlated with a significant increase in the hit rates for both low- and medium-contrast gratings. False alarm rates also increased during locomotion, though this effect was driven primarily by one animal and did not reach significance (Figure 4D; Table 2). An increase in hit rates could reflect an overall increase in licking

and not improved perception. To distinguish between these possibilities, we computed the discriminability (d′) for each contrast (Figure 4E; Table 2), a metric that is invariant to the behavioral criterion (Wickens, 2002). For each mouse tested, d′ was enhanced during locomotion for the low-contrast Astemizole condition; however, no significant effect was observed for the medium- and high-contrast conditions, likely reflecting a saturation of performance (Figure 4F; Table 2). Notably, moving trials were evenly distributed across the entire behavioral session (Figure S4D), and performance during the first and second halves of the sessions did not differ (p > 0.1, Wilcoxon signed-rank test). Thus, the improvement in d′ during movement did not reflect a correlation between locomotion and motivation. Here we present three main findings. First, we show large-amplitude, low-frequency membrane potential fluctuations in V1 during quiet wakefulness that are abolished during locomotion. This decrease in membrane potential variability results in reduced spontaneous firing rates during locomotion.

Ester, aldehyde and alcohol analogues were all shown to be incapable of inducing the decarboxylase system. However, a low level of apparent decarboxylation of these compounds was detected using pre-induced enzyme suggesting that they may be poor, indirect substrates, probably following a low level of oxidation to the corresponding carboxylic acid. Sorbic acid and

cinnamic acid both contain an alkenyl bond between carbons 2 and 3, which has the trans (E)-configuration ( Table 1). This bond is essential. Removal of this alkenyl bond in either of the carboxylic ABT-888 nmr acids abolished all activities as decarboxylase inducer or substrate. The structures of these acids are shown in Table 2. The effect learn more of the C2–C3 alkenyl bond may simply be due to its unsaturation or its geometry, or a combination of both. Analogues of the alkenyl fragment between C2 and C3, such as a triple bond or a cyclopropane ring (e.g. phenylpropiolic acid and 2-phenylcyclopropanecarboxylic acid, SD entries 44,62), did not substitute for the alkene bond. The importance of the trans (E)-configuration of the C2–C3 alkene bond was examined by comparing cis (Z)-2,4-methoxycinnamic acid with its trans (E)-isomer. The trans (E)-isomer was active

both as an inducer and as a substrate while the cis (Z)-isomer was inactive (SD entries 79,82). Sorbic acid also contains alkene unsaturation between Adenosine C4 and C5 and cinnamic acid is substituted by a phenyl ring at C3. Removal of the C4–C5 unsaturation in sorbic acid again abolished all activity as inducer or substrate for Pad-decarboxylation (Table 2; 2-hexenoic acid). While the trans (E)-configuration at C4–C5 in sorbic acid is important for activity, cinnamic acid, together with the furan and thiophene analogues shown in Table 2, contain additional or extended unsaturation at C3. This extended unsaturation, however, allows the molecules to assume a similar shape to that found in sorbic acid, which presumably is one reason why the furan and thiophene analogues are successfully decarboxylated as Pad substrates and inducers in whole conidia.

All of the compounds that were found to decarboxylate with high activity carried substituents beyond C5 in their structures. In cinnamic acid, this extension formed part of the aromatic ring and sorbic acid accommodated a methyl group at C5. Removal of this additional substitution at C5 resulted in substantial loss of decarboxylation activity, as demonstrated by the low level of activity against trans (E)-2,4-pentadienoic acid (SD entry 10). This carboxylic acid contains all of the significant features mentioned previously, and would seem to fit into a site that would accommodate sorbic acid, yet it was decarboxylated poorly, and is particularly poor as an inducer. This feature strongly indicates that a carbon substituent at C5 in sorbic acid is a pre-requisite for induction.