Asynchronous GABA release occurs at output synapses of fast-spiking interneurons in human and rat neocortex and is elevated in epileptic tissues from both species. 2+ at presynaptic terminals but was independent of postsynaptic firing. Furthermore, AR at FS autapses was markedly elevated in human epileptic tissue as compared to non-epileptic tissue. In a rat model of epilepsy, we found similar elevation of AR at both FS autapses and synapses onto excitatory neurons. Further experiments and analysis showed that AR elevation in epileptic tissue may result from an increase in action potential amplitude in the FS neurons and elevation of residual Ca2+ concentration. Together, these results revealed that GABAergic AR occurred at both human and rat neocortex, and its elevation in epileptic tissue may contribute to the regulation of epileptiform activities.Down-regulation of GABAergic inhibition may result in the generation of epileptiform activities. Besides spike-triggered synchronous GABA release, changes in asynchronous release (AR) following high-frequency discharges may further regulate epileptiform activities. In brain slices obtained from surgically removed human neocortical tissues of patients with intractable epilepsy and brain tumor, we found that AR occurred at GABAergic output synapses of fast-spiking (FS) neurons and its strength depended on the type of connections, with FS autapses showing the strongest AR. In addition, we found that AR depended on residual Ca The balance between excitation and inhibition in the cerebral cortex is important for multiple brain functions. Down-regulation of GABA-induced inhibition disrupts this balance and may lead to epileptic seizures. Asynchronous release of GABA is known to occur at certain GABAergic synapses and represents release of inhibitory neurotransmitter that is not precisely timed to presynaptic action potentials. Whether asynchronous release is subject to change after the induction of epilepsy remains unclear. In this study, using simultaneous recordings from inhibitory fast-spiking neurons and excitatory pyramidal cells, we found that asynchronous release occurred at the output synapses of fast-spiking neurons in both human and rat neocortex. The occurrence of asynchronous release depended on the level of residual calcium at the presynaptic terminals but not on postsynaptic spiking. Further experiments using cortical tissue derived from human patients with intractable epilepsy and from a rat model of the disorder revealed an elevation of asynchronous release in epileptic cortex, possibly resulting from an increase in action potential amplitude of fast-spiking neurons and changes in calcium dynamics in their axon terminals. Taken together, these results demonstrate that asynchronous release is a fundamental property shared by neocortical fast-spiking neurons regardless of species, and the enhancement of asynchronous release in epileptic tissue suggests a role for it in regulating epileptic activities. A and GABAB receptors, leading to cortical inhibition During active states in the cerebral cortex, cortical neurons receive both excitatory and inhibitory synaptic inputs. Proper balance of these inputs Molecular and functional changes in GABA receptors Under most circumstances, action potential (AP) is initiated at the axon initial segment and propagates to the presynaptic terminals, triggering neurotransmitter release within milliseconds We obtained human cortical tissues from small brain blocks that were surgically removed to cure intractable epileptic patients and brain tumor patients. Since the surgery is considered a therapy of the last resort for patients that had frequently suffered severe epileptic seizures, the cortical tissue should have experienced chronic epileptiform activities. We found that although AP burst-evoked AR occurred in all GABAergic synapses of FS interneurons  in these human epileptic tissues, FS autapses exhibited the strongest AR among these synapses. Further experiments in rats revealed similar differences in AR at different synapses. Importantly, as compared with control tissues, AR is significantly stronger in epileptic tissues, indicating that AR at GABAergic synapses might be subjected to modulation by epileptic seizures and involved in regulating epileptiform activities.Human neocortical tissues from 52 patients (aged 5–42 y) with frontal or temporal lobe epilepsy were sliced and examined by electrophysiology within 2–10 h after surgical removal. Whole-cell recording was performed on single FS neurons or synaptically connected FS-FS and FS-PC pairs in layer 5.n = 85/392), we consistently observed elevated spontaneous synaptic events immediately after high-frequency firing evoked by DC current injection through the recording pipette in current-clamp mode (− pipette solution (75 mM Cl−), inhibitory postsynaptic potentials (IPSPs) were depolarizing events at the resting membrane potential (∼−70 mV). Consistent with previous findings in rodents hold = −70 mV) single AP could trigger an inward current in the same recorded cell that peaked within 2 ms and could be completely blocked by the bath application of picrotoxin , a GABAA receptor antagonist (n = 12 FS neurons). The amplitudes of these IPSCs were relatively large (255.8±53.6 pA) because we selectively examined the effects of PTX on FS neurons with obvious autaptic unitary IPSCs, ensuring accurate measurements of the IPSC kinetics after subtraction  of GABA at autapses made by single FS neurons on themselves. In about 22% of FS neurons tested (amp mode . By usintagonist . This inA receptor-mediated spontaneous IPSCs (sIPSCs)  in FS neurons were also completely abolished by PTX, indicating that these events were also GABA(sIPSCs) . In phys n = 74, . We thenn = 11). We found that high-frequency firing in presynaptic FS neurons also evoked long-lasting AR, detected as sIPSCs in the postsynaptic FS neuron  and 7.0±1.4 (p<0.01) when the number of APs were increased to 40 . Together, these results indicate that although weaker than in FS autapses, asynchronous GABA release also occurs in synaptically connected FS neurons, providing long-lasting inhibition within this population of cortical inhibitory neurons.In addition to their autaptic connections, FS neurons also form inhibitory synaptic connections onto other FS neurons . We founn = 61/66 pairs). In current-clamp mode, a train of APs evoked by step current injection in FS neuron evoked both autaptic (through FS autapses) and synaptic (through synapses from FS neuron to PC) AR in a stimulus intensity-dependent manner . As shown in p<0.001). In sharp contrast to autapses, PT-AR at FS-PC synapses had shorter duration (98.8±9.6 versus 187±11 ms) and less IPSC events  in response to presynaptic firing of 20 APs at 200 Hz. Cumulative frequency distribution of all these recordings revealed significant differences in the duration and total number of events of PT-AR between FS autapses and FS-PC synapses  evoked monosynaptic IPSCs in both FS neuron  and PC . Among 3To examine whether the strength of PT-AR depends on the size of AP-triggered synaptic responses, we normalized the PT-AR duration by the average peak amplitude of unitary IPSCs (synaptic strength). We found that although PT-AR duration in autaptic connections was longer than that in FS-PC connections, the normalized PT-AR duration showed no significant difference between the two types of connections , indicatWe next analyzed the dependence of AR differences between FS autapses and FS-PC synapses on the clinical parameters of patients . We found no obvious dependence of PT-AR duration differences in the two types of connections on these parameters ; that isTogether, these results demonstrated that, in human epileptic tissue, AR also occurred at FS-PC synaptic connections but was substantially weaker than that in FS autaptic connections.2+ in presynaptic terminals during trains of stimulation could be responsible for the occurrence of AR in hippocampus 2+ in the occurrence of AR in human neocortex. In the presence of EGTA-AM (100 µM), a membrane permeable Ca2+ chelator, FS autaptic, and FS-FS/FS-PC synaptic AR in human cortical tissues were completely blocked after 15-min drug application  in the train , suggesting that AR requires Ca2+ entry through Ca2+ channels. Together, these results indicate that AR was dependent on the buildup of intracellular Ca2+ during the train of stimulation.Further recordings showed that the replacement of normal ACSF with CaPrevious studies demonstrated that alteration of GABAergic inhibition could contribute to epileptogenesis. We speculated that the asynchronous GABA release from FS output synapses may undergo changes in the epileptic brain tissue. Furthermore, the findings in epileptic cortical tissues described above may be due to lengthy treatments of patients with anti-epileptic drugs. We thus performed recordings from FS neurons of surgically removed non-epileptic peri-tumor tissues and compared the AR properties with those of epileptic tissues.n = 20) were significantly lower than the values found in the epileptic tissue  and 37.3% (n = 38/102) for control and pilocarpine-treated rats, respectively, much higher than those in the opposite directions .Next, we sought to examine whether or not there are differences in AR between FS autapses and FS-PC synapses in cortical slices obtained from adult rats and whether AR is subjected to change after the induction of epileptic seizures . In thesn = 47). These values increased to 110±9 ms and 9.7±1.1 events, respectively, when the number of APs was increased to 40 , and the total number of PT-AR events was 5.6±1.4 and 5.0±1.2 for control (firing in FS neuron only) and paired firing , respectively. This result indicates that postsynaptic spiking is not required for the occurrence of AR from presynaptic FS neurons.Because postsynaptic spiking may cause an elevation of intracellular Can = 25 and 32, respectively; t test, p = 0.07) in FS-PC pairs, indicating that the basic neurotransmission was unchanged in this model of epilepsy.Next, we analyzed the strength of AR and synchronous release in pilocarpine-treated rats. Similar to control rats, we also observed that the strength of PT-AR was different between FS autapses and FS-PC synapses, with stronger PT-AR detected at FS autapses . No signp = 0.002 and p = 0.005, n = 35) and to 126±16 ms and 9.6±1.7 events in FS-PC synapses . Cumulative frequency distributions of recordings obtained from epileptic animals by PT-AR duration and events showed a rightward shift in comparison with those from control rats  to 7.6±0.9%  for FS neurons firing 20 APs at 200 Hz. Similar results were obtained when FS neurons fired 40 APs  and FS-PC synapses (n = 10 pairs). Consistent with the results described above  and the half width showed no significant difference, all APs during the train in pilocarpine rats exhibited larger peak amplitude and integrated area than those in control rats . Further analysis revealed that AP waveform changes correlated well with the AR strength; PT-AR duration and events increases with increasing AP amplitude and area . Although we observed no significant TTX effect on the synaptic strength (average amplitude of the first IPSCs), the success rate, and the total integrated charge of IPSCs during the train stimulation (40 or 60 APs at 200 Hz) in FS-PC pairs  2+ entry during APs 2+ after a train of stimulation. Indeed, when we perfused the slice with TTX at a low concentration that showed no effect on the synchronous synaptic transmission but slightly decreased the amplitude of APs, PT-AR duration and number of events were significantly reduced (Previous findings  reduced . These f2+ source (voltage-gated Ca2+ channels) and the sensor of exocytosis may differ at different types of synaptic connections and thus determine the occurrence and the strength of AR. At the hippocampal cholecystokinin interneuron terminals, this distance is large, leading to long-lasting intracellular Ca2+ transient and thus asynchronous vesicle release; whereas at parvalbumin interneuron terminals, the Ca2+ channels locate closely with sensors, allowing fast and precise synchronous release of vesicles 2+ source and the sensor of exocytosis in FS neuron terminals 2+ sensors The distance between the Ca+ channel density in these neurons, consistent with homeostatic regulation in response to epileptic activities.Changes in AR could also be explained by the homeostatic regulation of synaptic strength and intrinsic neuronal property in response to the epileptic activity. Previous studies have shown that changes in network activities can lead to alterations of synaptic strength in both excitatory and inhibitory synapses, leading to adjustment of the firing rate of individual neurons within a physiological range Because it is not possible to obtain normal brain tissue from healthy humans, we used the discarded peri-tumor tissues from patients with brain tumors who exhibited no clinical symptoms of epileptic seizures. Recordings from these non-epileptic tissues were considered as control for the effects of epileptic activities or lengthy treatment of anti-epileptic drugs. Our observation of AR occurrence in both control and epileptic human tissues indicated that AR is a fundamental property of human neocortical FS neurons and not due to drug treatment. Furthermore, our results clearly show that AR at FS autaptic connections in the epileptic tissue was substantially stronger than that in the non-epileptic tissue . SimilarFurther analysis on clinical parameters  revealedOur results demonstrate that AR in FS neuronal terminals occurs not only in rat but also in human neocortex, indicating that AR is a fundamental property of the cerebral cortex and participates in cortical functions. In a normal brain, asynchronous GABA release after high-frequency firing provides long-lasting inhibition and enables gain control of the postsynaptic neurons Previous studies mainly focused on the alteration of basic neurotransmitter release at GABAergic synapses in epileptic brain tissue. Given that cortical neurons encounter large depolarizations and high-frequency discharges during epileptiform activities, it is important to investigate the changes in AR evoked by high-frequency discharges. Interestingly, we found no significant difference in the amplitude of unitary IPSCs between control and epileptic animals. With high-frequency stimulation of FS neurons, we did observe an increase in asynchronous GABA release in the rat model as well as in human epileptic tissues. Therefore, our results suggest that asynchronous GABA release in FS interneurons could be a target for the development of novel anti-epilepsy drugs.The protocols for handling and using the human brain tissue had been approved by the Biomedical Research Ethics Committee of Shanghai Institutes for Biological Sciences (License No. ER-SIBS-221004). The use and care of animals complied with the guidelines of the Animal Advisory Committee at the Shanghai Institutes for Biological Sciences.2 and 5% CO2) sucrose solution  in which the NaCl was substituted with equiosmolar sucrose and dextrose was reduced to 10 mM. We then sliced the brain tissue in this sucrose solution. In the case of brain tumor, we only used the peri-tumor tissue that had clear cortical layers (from layer 1 to 6) and white matter.Human neocortical slices were prepared from brain tissues that had to be removed surgically to cure intractable epileptic seizure and brain tumor. Prior to the surgery, all patients and relatives or their legal representatives had provided written informed consent. Brain tissues from 52 human patients with frontal or temporal lobe epilepsy (aged from 5 to 42 y) and two patients with brain tumors (57 and 69 y), who showed no clinical symptoms of epileptic seizures, were used in this study. A small block of the discarded tissue was immediately immersed into an ice-cold oxygenated  rats and pilocarpine-treated epileptic rats. A classic protocol was utilized to produce a pilocarpine model of epilepsy 4, 2 CaCl2, 26 NaHCO3, 1.25 NaH2PO4, and 25 dextrose . Whole-cell recordings were achieved using a Multiclamp 700B amplifier (Molecular Devices). Signals were filtered at 10 kHz and then sampled by Micro 1401 mkII  at 20 kHz using Spike 2 acquisition software. The impedance of patch pipettes was 5–7 MΩ with an internal solution containing in mM: 71 KCl, 72 Kgluconate, 2 MgCl2, 10 HEPES, 0.025 BAPTA, and 2 Na2ATP . The reversal potential of Cl− was −15 mV. Alexa Fluor 488 (50 µM) and biocytin (0.2%) were added to the internal solution for visualizing the morphology of recorded neurons. AP trains with varying AP number and frequency were evoked by step current injection (in current-clamp mode) or trains of voltage commands (voltage-clamp mode). Liquid junction potential (9 mV) has not been corrected for the membrane potential shown in the text and figures.Neocortical tissues from human patients, control rats, and pilocarpine-treated rats were utilized for patch clamp recordings. Slices with a thickness of 350 µm were cut in the ice-cold sucrose ACSF from a block of brain tissue with a vibratome . After slicing, the slices were immediately transferred to an incubation beaker and incubated at 35.5°C for about 1 h and then room temperature until use. Recordings were performed in a submerged-style chamber at 36°C mounted under an infrared-differential interference contrast (IR-DIC) microscope . The ACSF contained in mM: 126 NaCl, 2.5 KCl, 2 MgSOPCs and FS neurons were identified by their morphology and firing properties. PCs had pyramid-shaped soma and a single thick apical dendrite, exhibiting a regular firing pattern with adapting APs in response to a steady depolarization, whereas FS interneurons had a non-pyramidal soma and multiple primary dendrites and fired APs at very high frequencies (up to 500 Hz) without adaptation. After recording, the neurons were further identified using DAB-staining.A receptor antagonist, Tocris), DL-2-Amino-5-phosphonopentanoic acid  and 6-Cyano-7-nitroquinoxaline-2,3-dione , and EGTA-AM  were applied through bath perfusion.Picrotoxin . The current trace of the postsynaptic neuron was transformed to slope for event detection. Individual AR events were detected by setting a slope threshold. The frequencies of spontaneous IPSCs during the 4-s period before the train stimuli were defined as the baseline frequency. PT-AR frequency was calculated with a bin size of 50 ms. The end of AR was defined as the time point of the last IPSC before AR frequencies reached the baseline frequency. The PT-AR duration was the time between the cessation of the train stimulation and the end of AR. To investigate the strength of asynchronous GABA release from FS neurons, we only measured the duration and number of spontaneous IPSC events after the train stimulation because it was difficult to detect the asynchronous events that occurred during the train, due to the mixture of synchronous and asynchronous release in response to presynaptic high-frequency discharges. For single AP-evoked unitary IPSCs, we obtained the peak amplitude by taking the differences between the peak and the baseline current. The rise time of unitary IPSCs was measured as the time from 10% to 90% of the peak amplitude; the decay time constant was obtained by fitting the decay phase with a single exponential function. The threshold of an action potential (AP) was defined as the membrane potential when dV/dt reached 20 V/s. The peak amplitude of an AP was measured as the voltage difference between the peak and the threshold. AP area was the integrated area above the level of AP threshold .quantal), which was used for deconvolution of the postsynaptic currents. We then performed the deconvolution with the following equation: Release rate = F−1[F (IPSC)/F], where F is the discrete Fourier transform In order to calculate the quanta number during and after the train stimulation, we performed deconvolution analysis as described in previous studies t test. Two-sample Kolmogorov-Smirnov (K-S) test was performed if the data were not normally distributed. In order to study the intensity-dependent properties of synchronous release and PT-AR, two-way ANOVA analysis was utilized to test whether the number or frequency of APs in FS neurons had an effect on PT-AR.Values were given as mean ± s.e.m., and error bars in figures also indicate the s.e.m. Significance of differences was assessed by two sample Student's Figure S1p<0.001). No significant differences were detected between male and female patients .  No obvious correlation was observed between PT-AR duration and patients' age and time since seizure onset. (D–F) The difference in PT-AR duration occurred regardless of the causes of seizure, occurrence frequency, and seizure duration. Only those patients with seizure occurrence frequency higher than once a week but lower than once a day (1/w≤f<1/d) showed a small AR difference between the two types of connections. Note that patients with longer seizure duration (≥3 min) had larger PT-AR duration than those with shorter seizures (<3 min). * p<0.05; ** p<0.01; *** p<0.001.Correlation between PT-AR duration and clinical parameters. (A) Significant difference in AR duration between autaptic and FS-PC connections was observed in both male and female patients Click here for additional data file.Figure S2Postsynaptic spiking had no effect on AR strength. (A) Example recording from an FS-PC pair. Control: only presynaptic FS neuron was stimulated. Paired firing: Both FS neuron and PC were stimulated simultaneously. (B) Expanded traces (shadowed parts shown in A) for clarity. (C) Group data from 6 FS-PC pairs showing no significant differences in PT-AR duration (left) and total number of events (right) between control and paired firing. In this experiment, APV (50 µM) and CNQX (20 µM) were applied in the bath to block the fast glutamatergic transmission.(TIF)Click here for additional data file.Figure S3Comparing the ratio of PT-AR to total release in FS-PC synapses of control and pilocarpine-treated rats. (A) Calculation of the quanta released during (Train) and after (PT-AR) the train stimulation (40 APs at 200 Hz in FS neurons). The total release is the sum of Train and PT-AR. (B–C) Bar plots of the number of FS-PC pairs versus PT-AR ratio. Note the differences between control and pilocarpine rats. The mean PT-AR ratios for different groups were indicated.(TIF)Click here for additional data file.Figure S4p<0.05; ** p<0.01; *** p<0.001.AR evoked by prolonged high-frequency firing. (A) Left, example trace showing prolonged stimulation (300 APs at 150 Hz) in a human FS neuron caused AR at both autaptic and FS-PC synaptic connections. Right, group data indicate that PT-AR frequency at autapses was significantly higher than FS-PC synapses.  Example FS-PC pair recording in control and pilocarpine-treated rats.  Group data showing the significant enhancement of PT-AR frequency in model animals (bin size: 200 ms). * (TIF)Click here for additional data file.Figure S5n = 9 FS neurons). * p<0.05; ** p<0.01.Changes in AP waveform after the bath application of a low concentration of TTX. (A) Phase plot of the first APs evoked by 500-ms current injections pre and post the application of TTX (100 nM). (B) Changes in AP parameters pre and post TTX ((TIF)Click here for additional data file.


Importantly, no effects on motor function and balance were observed in mice after treatment with ar-turmerone even after administering a dose 500-fold higher than the effective dose in the 6-Hz model. In addition, quantification of its concentration in mouse brains revealed rapid absorption after i.p. administration, capacity to cross the BBB and long-term brain residence. Hence, our results provide additional information on the anticonvulsant properties of ar-turmerone and support further evaluation towards elucidating its mechanism of action, bioavailability, toxicity and potential clinical application. In a previous study, we uncovered the anticonvulsant properties of turmeric oil and its sesquiterpenoids  in both zebrafish and mouse models of chemically-induced seizures using pentylenetetrazole (PTZ). In this follow-up study, we aimed at evaluating the anticonvulsant activity of ar-turmerone further. A more in-depth anticonvulsant evaluation of ar-turmerone was therefore carried out in the i.v. PTZ and 6-Hz mouse models. The potential toxic effects of ar-turmerone were evaluated using the beam walking test to assess mouse motor function and balance. In addition, determination of the concentration-time profile of ar-turmerone was carried out for a more extended evaluation of its bioavailability in the mouse brain. Ar-turmerone displayed anticonvulsant properties in both acute seizure models in mice and modulated the expression patterns of two seizure-related genes ( Epilepsy is a common neurological disorder characterized by spontaneous recurrent focal or generalized seizures that affect the central nervous system in diverse ways. To date, epilepsy affects about 50 million people worldwide  2]. Alt. Alt2]. Curcuma longa  [11 [1110]  13] [14 [1413] . Howeverma longa . The rhi50 B.C.) . Present50 B.C.)  24] [25 [25Curcu50 B.C.) . Convers50 B.C.)  27]  [31 [3130]  33]. Fu. Fu33]. c-fos and bdnf in zebrafish larval brains  [39 [39c-fosl brains  41] . NM. NM45]. Straub tail)   starting from 32 - 64 mA  45]. If. If45]. C57Bl/6 mice were randomly divided into control and treatment groups of five mice each. The methodology for this assay was based on the one described in our previous study . BrieflyC57BI/6 mice (10 weeks old) were divided in groups of five. Blinded evaluation was performed to lessen observer bias. All mice were trained until proficiency to walk along a wooden beam without pausing was achieved. The ‘beam walking test’ apparatus consists of a flat bench 70 x 35 cm) with a wooden beam  located 31.5 cm above the bench and held to it on two posts. Two points on the beam were labeled as ‘start’ and ‘end’ areas to allow timing of the animal when walking across the beam. ‘Start –point’ was the site where the mouse was located to begin the test; the ‘end-point’, the place in front of a dark goal box (20 x 10 x 10.5 cm) with food pellets inside. Following training, every mouse was i.v. administered vehicle (PEG200:DMSO 1:1), sample (ar-turmerone 50 mg/kg) or positive control (diazepam 1 mg/kg) according to the treatment group. Ten minutes after administration, the mouse was placed on the tip of the beam at the ‘start-point’ facing towards the beam. The number of footslips, falls and total time on beam (from ‘start’ to ‘end’ points) were noted  cm with  48]. If. If48]. Three NMRI mice per dosage group were analyzed at each time-point. A dose of 50 mg/kg of ar-turmerone in PEG200:DMSO 1:1 was administered intraperitoneally (i.p.). Dose and route of administration were selected on the basis of the effectiveness of ar-turmerone to control seizure generation in the 6-Hz psychomotor seizure test.Transcardial perfusion was applied to mice after defined time-points of drug administration  in order to obtain bloodless brain samples. Brains were separated by dissection, washed with saline solution to remove excess surface blood and dried on filter paper. Brains were individually weighed and mixed with hexane (1.5-fold of weight) using a homogenizer . Hexane brain extract (supernatant) was collected after centrifugation at 2000 rpm (10 min). Hexane was selected due to its efficiency in the extraction of lipophilic analytes from brain tissue  50]. . 50]. An aliquot of 100 µL of brain extract was dried by passing a slow stream of nitrogen over the sample at room temperature to eliminate the solvent. Residue was dissolved in 100 µL methanol. Solution was vortexed (1 min) and centrifuged at 10.000 rpm (10 min). For HPLC analysis, an aliquot of 20 µL of the clear supernatant was injected. Internal standard (IS) propylparaben was added to obtain a final concentration of 0.25 µg/mL.Elite HPLC System [VWR Hitachi] equipped with diode array detection (DAD). Chromatographic separation was performed on a Phenomenex reversed phase column type Luna 3u C18  attached to an Phenomenex guard column C18 (4 × 3 mm). The column was operated at a flow rate of 1 ml/min at room temperature (detection wavelength: 240 nm). Samples were analyzed using gradient elution consisting of HPLC grade acetonitrile and Milli-Q purified water according to Mehrotra et al.  [57 [5746]  60] [61 [6160]  63] [64 [6463]  66] sup sup66] sA receptor occupancy (e.g 30% for diazepam) in contrast with rotarod performance which is affected only at much higher levels (72%) of receptor occupancy . It. ItA recccupancy . The safccupancy . With reccupancy . Of courccupancy  72] , brain-derived neurotrophic factor [bdnf] and interleukin-10 [il10]), was analyzed in zebrafish brains after treatment with ar-turmerone in the presence and absence of PTZ. Genes for this evaluation were selected on the basis of their key role in seizure-related pathogenesis. The proto-oncogene c-fos is upregulated significantly in the brain in response to triggering stimuli (e.g. seizures) in both zebrafish and rodent models  after PTZ exposure. This observation is in line with the reported upregulation of BDNF [protein and mRNA] at the hippocampal level in seizure animal models [bdnf in the PTZ-only treated group appears to be due to the proconvulsant properties of PTZ. However, the upregulation of bdnf expression in the case of ar-turmerone-treated larvae does not appear to be due to an intrinsic proconvulsant activity of the compound as c-fos expression is significantly downregulated after combined treatment with ar-turmerone and PTZ. Thus, it could be inferred that the increase in bdnf expression in ar-turmerone + PTZ treated larvae was not only ’triggered’ by PTZ but also related to an intrinsic effect of ar-turmerone. Although additional confirmatory studies are clearly warranted, these findings suggest that ar-turmerone may modulate anti-depressant activity mediated by enhancement of BNDF mRNA expression in the brain. In addition to the rodent studies, modulation of the seizure-related markers . Future experiments using other models  are still necessary to characterize the potential clinical application of ar-turmerone further. Hence, the present study supports continued research for investigating the anticonvulsant properties of ar-turmerone, likely as an alternative for pharmaco-resistant cases and/or clinical cases of adverse effects with currently available AEDs.


Introduction: Clarification of the relationship between external stimuli and brain response has been an important topic in neuroscience and brain rehabilitation. In the current study, using functional near infrared spectroscopy (fNIRS), we attempted to investigate cortical activation patterns generated during execution of a rehabilitation robotic hand.Methods: Ten normal subjects were recruited for this study. Passive movements of the right fingers were performed using a rehabilitation robotic hand at a frequency of 0.5 Hz. We measured values of oxy-hemoglobin (HbO), deoxy-hemoglobin (HbR) and total-hemoglobin (HbT) in five regions of interest: the primary sensory-motor cortex (SM1), hand somatotopy of the contralateral SM1, supplementary motor area (SMA), premotor cortex (PMC), and prefrontal cortex (PFC).Results: HbO and HbT values indicated significant activation in the left SM1, left SMA, left PMC, and left PFC during execution of the rehabilitation robotic hand . By contrast, HbR value indicated significant activation only in the hand somatotopic area of the left SM1 .Conclusions: Our results appear to indicate that execution of the rehabilitation robotic hand could induce cortical activation. In recent decades, many rehabilitation robots have been developed for patients with brain injury , Positron Emission Tomography, and functional near infrared spectroscopy (fNIRS) are available for use in brain activation studies by external stimuli  with no history of neurological, physical, or psychiatric illness were recruited for this study. All subjects were asked to complete a questionnaire in order to confirm that they had no history of neurological, physical, or psychiatric illness. They understood the purpose of the study and provided written, informed consent prior to participation. The study protocol was approved by our Institutional Review Board.The rehabilitation robotic hand consists of two moving parts for four fingers (from second to fifth finger) and thumb Figures . The movAll subjects were asked to sit comfortably on a chair in an upright position during conduct of the experiment. They were instructed to relax their hands maximally and not to move the hand voluntarily or imagine the movements during performance of passive movements, which were executed by the robotic hand. The subject’s right hand was fixed at the rehabilitation robotic hand. Using a block paradigm design -movement by the robot (20 s)-resting (20 s)-movement by the robot (20 s)-resting (20 s)-movement by the robot (20 s)) at a frequency of 0.5 Hz, flexion-extension movements of the right fingers (from second to fifth) and abduction-adduction of the right thumb were performed by the rehabilitation robotic hand. During performance of passive hand movements, one experimenter confirmed that there had been no movement of the shoulder, elbow and wrist, and another experimenter observed changes in cortical activities using the monitor screen of the fNIRS system on a real-time basis , with continuous wave laser diodes with wavelengths of 780, 805, and 830 nm, was used for recording of cortical activity at a sampling rate of 10 Hz; we employed a 49-channel system with 30 optodes (15 light sources and 15 detectors). Based on the modified Beer–Lambert law, we acquired values for oxy-hemoglobin (HbO), deoxy-hemoglobin (HbR), and total-hemoglobin (HbT: mmol) following changes in levels of cortical concentration  based on the Brodmann area (BA) and anatomical locations of brain areas: SM1 , the hand somatotopic area of the SM1 , PMC , SMA , medial boundary: midline between the right and left hemispheres, lateral boundary: the line 15 mm lateral from the midline between the right and left hemispheres), and the prefrontal cortex (PFC)  Figure .p < 0.01). In addition, a significant increase of HbT value was also observed for the left PMC and PFC during performance of passive movements of the right fingers. By contrast, in terms of HbR value, only the hand somatotopic area of the left SM1 showed a significant decrease, compared with other ROIs   Figure . In the ) Figure .HbO, HbR and HbT, which measure neural activity indirectly by detecting hemodynamic changes of the underlying cerebral cortex, are the most commonly used parameters of fNIRS  and primary somatosensory cortex (S1) as a result of passive movements (Mima et al., Therefore, we believe that our results indicating activation of the SM1 by the rehabilitation robotic hand are consistent with those reported in previous studies. The pathway of M1 activation by somatosensory stimulation has not been clearly elucidated. Previously, it was thought that an afferent input arrives at the M1 through the S1 (Forss et al., Although many rehabilitation robots have been developed for patients with brain injury, studies on cortical activation during execution of the robots are limited (Aisen et al., In conclusion, we investigated cortical activation patterns during execution of our rehabilitation robotic hand; according to our results, the contralateral SM1, along with the contralateral PMC, contralateral SMA, and contralateral PFC were activated. Our results appear to suggest that execution of the rehabilitation robotic hand could induce cortical activation; therefore, we believe that our results would be helpful in research on development of rehabilitation robots. In addition, fNIRS could be a useful tool in research on the cortical effect of rehabilitation robots. For the clinical application, conduct of further studies on the optimal conditions for cortical activation and robot-assisted rehabilitation therapy by decoding of movement intention will be necessary (Gomez-Rodriguez et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PTEN, (10q23.3), is a negative regulator of the phosphatidylinositol 3-kinase (PIK3)/AKT survival pathway and a tumor suppressor frequently deleted in PCa. The androgen receptor (AR) signalling pathway is known to play an important role in PCa and its blockade constitutes a commonly used treatment modality. In this study, we assessed the deletion status of PTEN along with AR expression levels in 43 primary PCa specimens with clinical follow-up.Prostate cancer (PCa), a leading cause of cancer death in North American men, displays a broad range of clinical outcome from relatively indolent to lethal metastatic disease. Several genomic alterations have been identified in PCa which may serve as predictors of progression. In Situ Hybridization (FISH) was done on formalin fixed paraffin embedded (FFPE) PCa samples to examine the deletion status of PTEN. AR expression levels were determined using immunohistochemistry (IHC).Fluorescence PTEN deletion. Kaplan-Meier analysis showed an association with disease recurrence (P=0.03). Concurrently, IHC staining for AR found significantly lower levels of AR expression within those tumors deleted for PTEN (P<0.05). To validate these observations we interrogated a copy number alteration and gene expression profiling dataset of 64 PCa samples, 17 of which were PTEN deleted. We confirmed the predictive value of PTEN deletion in disease recurrence (P=0.03). PTEN deletion was also linked to diminished expression of PTEN (P<0.01) and AR (P=0.02). Furthermore, gene set enrichment analysis revealed a diminished expression of genes downstream of AR signalling in PTEN deleted tumors.Using FISH, we found 18 cases of PTEN deleted tumors expressing low levels of AR may represent a worse prognostic subset of PCa establishing a challenge for therapeutic management.Altogether, our data suggest that  Prostate cancer (PCa) strongly affects the male population, and is classified as the most commonly diagnosed cancer and a leading cause of cancer death in North American men. The curAndrogens are required for maintaining the homeostasis of the normal prostate epithelium. Their effect is mediated via the androgen receptors (AR), a member of the nuclear superfamily of steroid receptor, acting as a transcription factor in prostate cell nuclei. PCa cells have retained the ability to proliferate upon stimulation with androgens, resulting in tumor growth. Thus, PPTEN) is a tumor suppressor gene on 10q23.3 locus that acts by negatively regulating the PI3K/AKT pathway[PTEN was shown to be haploinsufficient in tumor suppression[PTEN genomic deletion has been detected in human tissues representing all stages of PCa development and progression including High Grade Prostatic Intraepithelial Neoplasia (HGPIN), primary PCa and at higher frequency in metastatic PCa and CRPC[in situ hybridization (FISH), PTEN deletion status of primary PCa has been associated with poor outcome[PTEN and PI3K/AKT activation can modulate AR activity and contribute to CRPC[The phosphatidylinositol 3-kinase (PI3K)/AKT signal transduction pathway contributes to cancer growth and survival, and is activated in a broad range of human malignancies including PCa. The pho pathway. In animpression. PTEN geand CRPC-15. Usin outcome. Previou to CRPC-18. ThesPTEN gene using Fluorescent in situ hybridization (FISH) and AR expression by immunohistochemistry (IHC). An existing PCa microarray dataset of DNA CNAs by array comparative genomic hybridization (CGH) and corresponding gene expression profiling were used to validate these findings.In this study, we surveyed PCa samples for genomic DNA copy number alterations (CNAs) of the This study was conducted with the written consent of the participants and approved by the Research Ethics Board of the McGill University Health Centre (study BMD-10-115).Formalin fixed paraffin embedded (FFPE) blocks (n = 43) of primary tumors and adjacent benign tissues from radical prostatectomy were retrieved from the Department of Pathology. Duplicate tissue cores (1mm diameter) were assembled into tissue microarrays (TMAs). Haematoxylin and eosin (H&E)-stained TMA sections were reviewed to determine the presence of representative areas of the original samples. The clinicopathologic features of the cohort are summarized in Table PTEN gene on chromosome 10q23.3 region and the commercially available CEP10 Spectrum Green probe , which spans the 10p11.1-q11.1 centromeric region. RP11-383D9 DNA was labeled with Spectrum Orange-dUTP  using the Nick Translation Reagent Kit (Abbott Molecular). The 5 μm TMAs sections were de-paraffinized in 6 changes of xylene before immersion in 95% ethanol. The slides were then placed in 0.2 N HCl solution at room temperature (RT°) for 20 min followed by a 2-hour incubation at 80°C in 10 mM citric acid buffer (pH 6) for pre-treatment. Specimens were digested in 0.1 mg/ml protease I (Abbott Molecular), and then fixed for 10 min in formalin before dehydration in an ethanol series. The two probes and target DNA were co-denatured at 73°C for 6 min and left to hybridize at 37°C O/N using the ThermoBrite system (Abbott Molecular). Post-hybridization washes were performed in 2xSSC and 0.3% NP40/0.4xSSC at 73°C for 2 min and 1 min respectively, followed by a 30 sec incubation at RT° in 2xSSC.Dual-color FISH was carried out on TMA sections using the BAC clone RP11-383D9  mapping to the DAPI III, Abbott Molecular) staining of nuclei with reference to the corresponding H&E-stained tissue identified the areas of adenocarcinoma. Using hybridization in 30 benign control cores, 10q23.3 deletion was defined as ≥15%  of tImmunostaining of AR on TMAs sections was performed using a mouse anti-AR antibody  and the Envision detection kit . The 5 μm TMAs sections were de-paraffinized in a series of xylene and hydrated in a graded series ethanol solutions. Heat-induced antigen retrieval was performed by immersing the slides in 10 mM citric acid buffer solution (pH 6) and boiling for 30 min using microwave energy. The slides were left in solution to cool down for 30 min at room temperature. Endogenous peroxydase activity was blocked for 5 minutes (Dako). After a 60 min block with 10% normal goat serum in PBS (Dako), the primary antibody (1:50 dilution in Dako antibody diluent) was used for two hours at room temperature. Chromogenic detection was carried out using a peroxidase-conjugated secondary antibody (30 min) and DAB reagents (10 min) provided with the Envision detection kit. Tissue sections were counterstained with Meyer’s Haematoxylin .PTEN deleted and non deleted specimens categories with the Mann–Whitney U-Test (http://elegans.som.vcu.edu/~leon/stats/utest.html).Nuclear staining was assessed by two independent observers using the H-score method described in,22. BriePTEN genomic status as reported in the corresponding array CGH study[Analysis was perfGH study. Two andGH study) from thGH study. LapointPTEN at chromosome 10q23.3 on TMAs representing 43 cases of human PCa with clinical follow-up. The clinicopathologic characteristics of the study subjects are summarized in Table PTEN . To further evaluate the clinical significance of the PTEN deletion, we stratified the 43 cases based on their PTEN deletion status and performed a Kaplan-Meier survival analysis. Figure PTEN deletion was associated with a significant shorter time to recurrence (P=0.03).We used FISH to assess the genomic status of PTEN deletion compared to those with no deletion of PTEN . AR immunostaining of specimens with different H-scores are shown as examples in Figure PTEN deletion status was assessed by array CGH[PTEN which was significantly associated with a reduced levels of PTEN mRNA  identified as NELSON_RESPONSE_TO_ANDROGEN_UP,[PTEN compared to those with a deletion (FDR of 0.01). To further confirm this result, we tested a second set of androgen regulated genes reported by DePrimo et al.[PTEN deletion  is within the range of previous reports[PTEN genomic deletion and poor outcome of PCa reported earlier[PTEN deletion at high frequency in CRPC samples[The frequency of  reports,12,14,15 samples, in circ samples and its  samples,28. FurtPTEN deletion. We found a similar association between PTEN deletion and AR transcript levels in a PCa microarray dataset. The differential expression of AR according to the PTEN tumor status has not been well documented so far. A pilot IHC study has found a positive correlation between AR and PTEN expression[PTEN deletion status and AR expression[The intriguing finding of our study was the reduced levels of AR expression quantified by H-score in tumors harboring a pression. In contpression in CRPC pression, therebyin vitro studies in cell lines derived from advanced PCa suggested that PTEN could act as suppressor of AR activity[PTEN null murine cells did not affect AR expression, but upregulated the AR transcriptional activity[PTEN null murine prostate cells had a reduced AR protein levels compared to wild-type PTEN cells and the AR protein levels were partly restored by the PI3K/mTOR inhibitor BEZ235[PTEN deleted tumors. A shown by Lin et al., it is also possible that PTEN interacts directly with AR and promotes its degradation[PTEN deletion in human tumors is associated with lower AR expression and transcriptional activity need to be further explored.Previous activity,32. It wactivity. In modeactivity. Anotherradation. UnderlyPTEN deleted tumor cells are expected to be less responsive to androgen ablation treatment. In support of this hypothesis, it was reported that CRPC and early biochemical recurrence were associated with reduced immunoreactivity of PTEN and AR in the PCa samples harvested before treatment initiation[PTEN deleted tumor.Given their reduced levels of AR expression, the itiation. The addSome previous studies have found that low levels of AR were associated with PCa recurrence,37 whilePTEN deleted PCa by microarray analysis[PTEN include genes expressed in normal prostate luminal epithelium such as KLK3 (PSA), TMPRSS2, and NKX3-1. Of interest, the list includes AZGP1 previously reported as a surrogate marker for subtype-1 tumors, a favourable prognostic subclass of PCa defined by gene expression pattern analysis[PTEN status in subtype-1 tumors from the array CGH data may, at least in part, explain their androgen-regulated gene expression feature and good clinical outcome.During the course of our study, two groups also showed a reduced expression of androgen regulated genes in human analysis,35. In oanalysis. AZGP1 panalysis,41. Prevanalysis. ConsistPTEN deletion is associated with PCa recurrence and may thus serve as prognostic marker. As proposed, the low expression of AR and its target genes associated with PTEN deletion may have consequences on response to androgen ablation therapy and may be an indication for the introduction of additional therapeutic modalities.Although limited by the small sample size of this study, our preliminary data support that The authors declare that they have no competing interests.Conceived and designed the experiments: KC, JE, JL. Performed the experiments: KC, JE. Analyzed the data: KC, JE, FB, JL. Contributed materials/clinical data: AA. Wrote the paper: KC, JE, SC, JL. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/543/prepub


This paper undertakes a direct, comprehensive assessment of the long-term sustainability of desertification rehabilitation in China under a plausible but worst case scenario where governmental interventions, in the form of payments for environmental services (PES), will cease. The analysis is based on household behavior as well as experimental data. Our econometric results highlight the main obstacles to the sustainability of rehabilitation programs subsequent to cessation of government intervention, including specific shortfalls in households’ preference for a free ride, budget constraints, attitudes, tolerance of and responsibility for desertification, and dissatisfaction with governmental actions. We conclude that desertification rehabilitation is not sustainable in China without continued governmental intervention. The results of this study are intended to support policy makers as they consider future directions for rehabilitation sustainability. Desertification is land degradation in arid, semi-arid and dry subhumid areas  2 area of desertification in 2000, and 1.6 million km2 of susceptible arid and semiarid areas China is one of the most severely affected countries with a 385,700 kmThe commonly accepted cause of desertification is the interaction of a set of natural  and anthropogenic factors with different temporal and spatial variability The rehabilitation of desertified lands is converting the desertification areas into productive ones, especially in the agro-pastoral zone The Sloping Land Conversion Program (SLCP) was enacted in 2000 by the Chinese government to convert 32 million hectares of sloped land into forest land over 10 years, with a budgetary outlay of over US$30 billion and affecting 60 million households The grazing prohibition program, begun in 2003, is another major initiative implemented in the desertified areas of north China. It possesses the same dual goals as SLCP. This 10-year program has a budget of US$3 billion and benefits 5.5 million rural households Early indications show that the environmental results of these programs in combating desertification are remarkable, although their social impacts can be controversial The origin, causes, impacts and processes of desertification, and the methods available to combat it in China have been discussed at great length In contrast to other governmental actions in environmental protection, these PES programs depend on private contracts with individual rural households, pursuing personal interests. The government is concerned about positive externalities – the improvement of the environment and social wellbeing through contracting with and compensating individual rural households. In order to achieve both the governmental goals and individual interests, contracts should impose compatible incentives. However, the interests of the two parties are not always compatible, and may even be opposite if compensation is not appropriate or after it has expired.Earlier studies have considered only rural residents of desertified areas and have ignored the behaviors and preferences of their urban residents. People who are urban residents according to China’s special resident registration system also suffer from desertification, but they have usually been overlooked and cannot benefit directly from the poverty amelioration functions of the PES programs.The timeliness of this sustainability study lies in the fact that the PES programs will soon be closed or modified. Thus, the questions must be asked: “How can residents of desertified areas maintain what has been achieved?” and “How can desertification be reduced in the future?” Ultimately, it is the households in the desertified areas who must directly face the problem in the long run. But what will be their decision when facing the end of governmental support? There is a timely opportunity to capture the knowledge of those households and their experience of the initial programs to date.This paper therefore undertakes a comprehensive assessment of sustainability, based on household behavior, intentions and preferences under a plausible but worst case post-PES scenario: where the PES program will be terminated. The purpose is to identify the likely impacts of PES program termination on household behaviors and thus on the longer-term sustainability of rehabilitation of desertification. The analysis is based on data obtained from a carefully designed survey that was conducted in a typical agro-pastoral zone in China. Both household and village level data were collected via in-person interviews with randomly selected households and with village leaders.Q) is a function of governmental intervention S, household i’s response to the intervention i(S)R, and natural factors N as follows Household behaviors may be explicated in the context of the following closed models: Desertification rehabilitation , where stage I indicates a worse condition of desertification, and stage II indicates a better rehabilitation condition. A clarification is that rehabilitation is not yet completely accomplished at stage II. The model supposes that a household will pay This CE model is used to investigate individual preference towards different product packages on the basis of the economic theories of Utility Maximization and Value. Originally used for individual behavior studies in selecting commercial products For the experiment at hand, we adopt WTP (willingness to pay) and WTA (willingness to accept) to be proxies of household behaviors towards environmental protection. WTP can be defined as the maximum price which the household will be willing to pay for further desertification rehabilitation and WTA as the minimum price at which the household wishes to give up further rehabilitation. Specifically, WTA represents a pecuniary preferred compensation for the difference of PES programs’ performance on rehabilitation and individual expectation. The pecuniary number that households give or accept is the result of behaviors with rational preference and decision. WTP and WTA are functions of a household’s behaviors as follows:The disparity between WTP and WTA is an additional measure that can provide insight to household behavior. It has been theoretically and empirically documented, and is generally larger for non-market goods and smaller for ordinary goods E106°30′–107°47′, N37°4′–38°10′), is a county of Ningxia province located in the arid and semiarid region of northwest China. Geographically, it is a typical transition zone between the Mu Us Desert and the Loess Plateau with a climate that varies from semiarid to arid, and vegetation that varies from steppes to desert steppes. Annual precipitation is 250–350 mm, much less than the annual evaporation of approximately 2,900 mm The study area, Yanchi  were compensated for their direct contributions to environmental protection, there is a need to consider the reactions of other residents (town and city). The results of this study are intended to support policy makers as they consider future directions for rehabilitation sustainability.The sustainability of desertification rehabilitation is analyzed under the plausible scenario that after the end of government programs, residents themselves will contribute to continued rehabilitation attempts without external support. As mentioned, both the rural and urban residents in the desertification area are troubled by this environment, but the rural residents suffer more because their living is directly restrained by desertification. While PES programs offer rural residents both an improved environment and monetary compensation, urban residents benefit from the improved environment, but receive no compensation. In our hypothesis, stopping of PES programs pushes both rural and urban residents to face a dilemma of whether or not to continue to fund desertification rehabilitation in the absence of government support.During data collection, respondents were first asked if they were satisfied with the results of current PES programs  and if they were personally willing to sponsor a new, similar program. Those who were less than satisfied were then asked to select a pecuniary preferred compensation for the difference between the perceived success of current PES programs and their individual expectation, which is defined as WTA. Then respondents who agreed to pay for a new program of desertification rehabilitation were requested to select from a range of options their preferred pecuniary contribution toward future rehabilitation. This is defined as WTP.a and Vd). This group was also offered an open choice of numeric pecuniary WTP (experiment Vb), to investigate the embedding effect, reliability and internal consistency of the different results from experiment Va. The other group was only asked to make a binary choice for the acceptance or rejection of randomly designated pecuniary contribution options (experiment Vc).Two groups of households were interviewed under a stratified random sampling strategy covering 8 townships from the north to the south . Each group was comprised of households both living in the rural region and living in town . One group was queried regarding a range of numeric pecuniary contribution options for WTP and WTA  0) is that there is no significant difference in the two elicitation treatments. A priori it is expected that the null hypothesis will not be rejected.The null hypothesis  a, Vb and Vd is represented by:i indicates the investigated household, h is a vector of demographic variables, x denotes a vector of impact factors such as profession, income, severity of desertification and the interaction of profession and severity of desertification, and m is a vector of perception of desertification rehabilitation including Q1, Q2 and Q3 . It is necessary to state that (1) the robustness of variables is highly dependent on data availability and the model’s significance; and (2) severity of desertification cannot be numerically characterized at this time. Because the sample is geographically stratified north to south, we can use different towns as a proxy for that variable. WTP and WTA in experiments Va, Vb and Vd. The parameters of interest in model (7) are 1α, 2α and 3α, which show the effects of household actions on desertification rehabilitation. To test the effects of household behaviors on desertification rehabilitation, an estimation strategy for experiments Vc is proposed as:i’s response  for a randomly assigned value of WTP. Definitions of other variables remain the same as model (7). The interaction of profession and severity of desertification is employed only in this model.To test if a given household acts similarly under a yes/no dichotomous choice condition, the estimate model for experiment Viratio (i = 1 and 2 for Va and Vb respectively), and iz is a vector of the other documented controls to explain the disparity including income effect, initial endowment effect, substitution, study design and uncertainty.The model that was used to estimate what will impact the disparity of WTA and WTP is:Income effect is presented as annual income. Initial endowment will be represented by a dummy variable in comparison of household income between 2011 and 2001 when the main interventions were launched. Because it is impossible to identify the exact household endowment in 2001, we use the dummy variable to roughly distinguish people whose income has been reduced by desertification or not. That means that the initial endowment is 1 if household income is more in 2010 than in 2001, and 0 if it is less.Any substitution effect will be excluded since desertification and its rehabilitation are pure public goods without substitutes. Because many researchers have argued that the effect of study design is not so clear e.g. , it willa) and the open choice test (Vb), rejecting the null hypothesis . Gender, profession and income are of statistical significance for experiment Va, whereas age, profession and income are of statistical significance for experiment Vb. The interaction of profession and severity of desertification is considered but finally rejected as less than significant. The perception represented by Q3 is also rejected in experiment Va and experiment Vb for a similar reason.The Ordinary Least Squares (OLS) regression results of experiment Va. Willingness to contribute is significantly less by 1.63% as age increases in experiment Vb.Specifically, as indicated by the value of log(WTP), men’s willingness to contribute to future remediation efforts is significantly higher (46.22%) than for women in experiment Va and Vb, respectively. It is also found that the log(income) will significantly augment the log(WTP), at 53.15% and 47.13% for both experiment Va and experiment Vb.Professions are roughly classified into a) those which are directly affected by desertification and with unstable income (e.g. farmers), and b) those which are not directly impacted by desertification, with or without out stable income . Log(WTP) of those in occupation a) is significantly higher by 73.12% and 46.46% than in occupation b) in experiment VIncome’s effect on WTP has been widely documented in other studies dV). We find that more suffering from desertification will increase log(WTA) up to 83.62%; and the less the praise of governmental rehabilitation programs, the more compensation will be required, with an increase of log(WTA) of 0.16%. Once again, due to the lack of more clear measures of the impact of desertification in the study area, the geographic location of different towns was used to represent the severity level.To get the model of significance we used a backward strategy in WTA regression (c. A Linear Probability Model (LPM) estimate and then a logit estimate are presented. Panel A, B and C represent the same variables as above. Both estimates show a significant positive relation of gender, income and Q1, and a significant negative relation of profession with the dependent variable. We also looked at the interaction between profession and severity of desertification (Desert×prof) here, which shows a significant positive relation with the dependent variable.In detail, the probability for men to accept the random assigned WTP is 22.54% less than women. One whose profession is type a) also exhibits a high likelihood to accept (27.39%), and higher income will increase the probability of acceptance up to 6.32%. But the interaction of profession and severity of desertification shows that as desertification severity increases, the probability for residents holding job type a) to accept the random assigned WTP bid increases by 17.48%. In addition, awareness of environmental improvement significantly also increases the probability of acceptance (5.81%). Here only the LPM results are presented. Results from the logit model seem not so robust.a and experiment Vb,though the dependent variable here is probability to accept a randomly assigned WTP. The introduction of the interaction (Desert×prof) indicates that the severity of desertification somehow reverses the impact of profession on the probability to accept a randomly assigned WTP. What should be emphasized is that the results in this analysis address the probability to accept a randomly assigned value, but are not a measure of the desire of the resident to pay. So it is possible that when the residents of the more seriously degraded regions express a lower probability to accept the value, it may be that the given value is too large for them to afford.These results are in accord with results of experiment V1 and ratio2 are given in 3 is not significant in experiments Va and Vb, but is significant in experiment Vd, so it is included here.Ordinary least squares (OLS) results of ratio1 and Ratio2 respectively. It has the same effect as income which also decreases the two ratios with values of 7.4634 and 8.0644. Using age as a proxy for learning experience of the value of desertification rehabilitation, it is found that the higher the age the higher the disparity of WTA and WTP. The disparity is 0.1738 and 0.2708 for ratio1 and ratio2. Q1 and Q2 showed significant effects for ratio1 respectively at 2.2103 and 4.0472, but not for ratio2.Profession type b) significantly narrows the disparity, with values of 5.529 and 8.0658 for RatioWe also investigated the reasons that some residents choose not to contribute see . Nearly China has suffered from desertification for many years ex ante assessment of the long run sustainability of desertification rehabilitation in China by examining household behavior under a plausible but worst case scenario where the PES programs will be terminated. As a result of the analysis, the following conclusions can be cautiously drawn: (1) Income is still the most constraining factor for households to contribute funds for continuing desertification rehabilitation in the absence of government support. (2) Residents whose profession can be directly impacted by desertification hold a stronger willingness to ameliorate the problem. (3) Variations due to the local severity of desertification are not conspicuous. (4) Households seem indifferent to the causes of desertification and ignorant of the governmental contribution to desertification rehabilitation, though sometimes they praise the improvement of environmental quality. (5) Young persons and males tend to value environmental remediation higher than older persons and females. (6) Respondents are not well satisfied with the accomplishments of governmental actions to date.Our analysis provides a comprehensive, We argue that rehabilitation cannot be sustained by relying on household contributions without governmental input, because we found that households prefer to free ride to some extent, and because they are usually constrained by household budget. The evidence also indicates that (1) Those whose profession is not directly impacted by desertification are richer but have less concern for environmental improvement; (2) The younger generation and males care most about environmental problems. But they tend to emigrate to the towns, and thereby reduce their passion to combat desertification in the rural regions; (3) Although the endemic desertification is derived in part from human contributions, individual households do not take into account the impact of their behavioral decisions on environmental protection; and remarkably, (4) Residents are not well satisfied with accomplished governmental actions to date.Our study has some limitations. Without panel data, we used cross-sectional data 10 years after the PESs’ implementation. Due to differences in sampling strategy and site selection, some of our findings may differ from others. But our study site is a traditional transitional zone in geography, climate and economic activity, which implies that our analysis may be more broadly applicable.File S1The Questionnaires used in this study.(DOC)Click here for additional data file.