Oxygen-glucose deprivation induces ATP release via maxi-anion channels in astrocytes ATP represents a major gliotransmitter that serves as a signaling molecule for the cross talk between glial and neuronal cells . ATP has been shown to be released by astrocytes in response to a number of stimuli under nonischemic conditions . In this study , using a luciferin-luciferase assay , we found that mouse astrocytes in primary culture also exhibit massive release of ATP in response to ischemic stress mimicked by oxygen-glucose deprivation -LRB- OGD -RRB- . Using a biosensor technique , the local ATP concentration at the surface of single astrocytes was found to increase to around 4 μM . The OGD-induced ATP release was inhibited by Gd3 + and arachidonic acid but not by blockers of volume-sensitive outwardly rectifying Cl − channels , cystic fibrosis transmembrane conductance regulator -LRB- CFTR -RRB- , multidrug resistance-related protein -LRB- MRP -RRB- , connexin or pannexin hemichannels , P2X7 receptors , and exocytotic vesicular transport . In cell-attached patches on single astrocytes , OGD caused activation of maxi-anion channels that were sensitive to Gd3 + and arachidonic acid . The channel was found to be permeable to ATP4 − with a permeability ratio of PATP/PCl = 0.11 . Thus , it is concluded that ischemic stress induces ATP release from astrocytes and that the maxi-anion channel may serve as a major ATP-releasing pathway under ischemic conditions . Introduction Astrocytes do not merely support , but actively regulate , neuronal functions by forming highly elaborate networks with other astrocytes and neurons -LSB- 1 -- 4 -RSB- . Astrocytes communicate with neurons by releasing extracellular signaling molecules such as ATP and glutamate , while they communicate with each other via gap junctions , in addition to these gliotransmitters . Astrocytic ATP release has been shown to be induced in response to receptor stimulation by UTP -LSB- 5 -RSB- , glutamate -LSB- 6 -RSB- , and noradrenaline -LSB- 7 -RSB- , to mechanical stimulation -LSB- 8 -- 10 -RSB- , to osmotic swelling -LSB- 11 -RSB- , and to exposure to low Ca2 + solution -LSB- 8 , 9 , 12 -RSB- . Although ischemic stress is known to induce ATP release from rat hippocampal slices -LSB- 13 -RSB- , it is not known whether astrocytes respond to ischemia with ATP release . The first purpose of the present study was to address this question . Astrocytes express maxi-anion channels with a large single-channel conductance of 200 -- 400 pS -LSB- 14 -- 16 -RSB- . Recently , we have demonstrated that astrocytic maxi-anion channels are activated by chemical ischemia and serve as one of the major pathways for glutamate release -LSB- 17 -RSB- . The maxi-anion channel was found to possess a pore with a size large enough to permeate ATP -LSB- 18 -RSB- and was found to actually conduct anionic forms of ATP in mammary C127 cells under hypotonic conditions -LSB- 19 -RSB- , in kidney macula densa cells in response to increased luminal NaCl concentrations -LSB- 20 -RSB- , and in cardiac myocytes under hypotonic or ischemic conditions -LSB- 21 -RSB- . Thus , there is a possibility that maxi-anion channels serve as one of the pathways for ATP release from astrocytes under ischemic conditions . The second purpose of this study was to test this possibility . Materials and methods Cells Astrocytes were obtained from neonatal mouse brain cortex and cultured , as previously described -LSB- 17 , 22 -RSB- , in Eagle 's MEM supplemented with L-glutamine , 100 U/ml penicillin , 0.1 mg/ml streptomycin , 10 % FBS , and 2.2 mg/ml NaHCO3 . HEK293 cells stably transfected with recombinant P2X2 purinergic receptors were prepared and cultured , as described elsewhere -LSB- 23 -RSB- . The HEK-P2X2 cells were used after dissociation into single cells and culturing in suspension for over 10 min . Solutions and chemicals The standard Ringer solution contained -LRB- mM -RRB- : 135 NaCl , 5 KCl , 2 CaCl2 , 1 MgCl2 , 5 Na-HEPES , 6 HEPES , and 5 glucose -LRB- pH 7.4 , 290 mosmol/kg H2O -RRB- . For inside-out experiments , we used standard Ringer solution both in the bath and in the pipette . The pipette solution for biosensor whole-cell experiments contained -LRB- mM -RRB- : 125 CsCl , 2 CaCl2 , 1 MgCl2 , 10 EGTA , and 5 HEPES -LRB- pCa 7.6 , 275 mosmol/kg H2O , pH 7.4 adjusted with CsOH -RRB- . For ATP conductance measurements in the inside-out configuration , the bath solution was substituted with a solution of 100 mM Na4ATP . Oxygen-glucose deprivation -LRB- OGD -RRB- stress was applied by switching the perfusate from the standard Ringer solution to the OGD solution . The OGD solution was prepared by replacing D-glucose in Ringer solution with 2-deoxyglucose and was continuously bubbled with 100 % argon gas for more than 1 h before and during experiments . The oxygen concentration -LRB- pO2 -RRB- measured using an oxygen sensor -LRB- LICOX A3R , GMS , Kiel-Mielkendorf , Germany -RRB- was 118.4 ± 1.1 mmHg -LRB- n = 8 -RRB- for standard Ringer solution exposed to air in the experimental perfusion chamber . Under OGD conditions , the oxygen concentration in the perfusate solution decreased to a stable value of 7.5 ± 0.6 mmHg -LRB- n = 9 -RRB- at the inlet and 40.1 ± 1.8 mmHg -LRB- n = 8 -RRB- at the outlet of the experimental patch-clamp chamber . GdCl3 was stored as a 50-mM stock solution in water and added directly to solutions immediately before each experiment . Arachidonic acid , phloretin , brefeldin A -LRB- BFA -RRB- , 1-octanol , probenecid , glibenclamide , and apyrase were purchased from Sigma-Aldrich -LRB- St. Louis , MO , USA -RRB- and BAPTA-AM from DOJINDO -LRB- Kumamoto , Japan -RRB- ; they were added to solutions immediately before use from stock solutions in DMSO . DMSO did not have any effect when added alone at concentrations employed in the present study -LRB- less than 0.1 % -RRB- . Osmolality of all solutions was measured using a freezing-point depression osmometer -LRB- OM802 , Vogel , Kevelaer , Germany -RRB- . Luciferin-luciferase ATP assay The bulk extracellular ATP concentration was measured by a luciferin-luciferase assay -LRB- ATP Luminescence Kit ; AF-2L1 , DKK-TOA , Tokyo , Japan -RRB- with an ATP analyzer -LRB- AF-100 , DKK-TOA , Tokyo , Japan -RRB- , as previously described -LSB- 19 , 21 , 24 -RSB- , using astrocytes cultured in 12 - or 24-well plates . After the culture medium was totally replaced with isotonic Ringer solution -LRB- 1,000 and 425 μl for 12 - and 24-well plates , respectively -RRB- , cells were incubated for 60 min at 37 °C . An aliquot -LRB- 100 μl -RRB- of the extracellular solution was collected as a control sample for background ATP release . An OGD challenge was then applied by gently removing most of the remaining extracellular solution -LRB- 875 and 300 μl for 12 - and 24-well plates , respectively -RRB- and adding the OGD solution -LRB- 1,000 and 400 μl for 12 - and 24-well plates , respectively -RRB- , and then placing the plates in an airtight incubator where normal air was replaced with 100 % argon . At the specified time points , the plate was carefully rocked again to ensure homogeneity of the extracellular solution , and samples -LRB- 20 and 50 μl for 12 - and 24-well plates , respectively -RRB- were collected from each well for the luminometric ATP assay . The ATP concentration was measured by mixing 20 or 50 μl of sample solution with 530 or 500 μl normal Ringer solution and 50 μl of luciferin-luciferase reagent . At this ratio , the ionic salt sensitivity of the luciferase reaction was negligible . When required , drugs were added to the OGD solution to give the final concentrations as indicated . Since Gd3 + is known to interfere with the luciferin-luciferase reaction -LSB- 25 -RSB- , we supplemented the luciferin-luciferase assay mixture with 600 μM EDTA when the samples contained Gd3 + . Other drugs employed in the present study had no significant effect on the luciferin-luciferase reaction . Biosensor ATP measurements To measure the local concentration of ATP released from single astrocytes , the biosensor technique was employed , as described previously -LSB- 23 -RSB- . ATP-dependent changes in whole-cell P2X2 receptor currents were recorded from a biosensor HEK-P2X2 cell at a holding potential of − 50 mV . Before the actual measurements of ATP release , a calibration curve was constructed by recording the biosensor responses to known concentrations of ATP . The biosensor cell in the whole-cell configuration was lifted by means of a micromanipulator and positioned near a handmade local microperfusion device consisting of several inlet tubes . Each of the inlet tubes was filled with a different concentration of ATP solution ranging from 1 to 20 μM . To minimize the effect of desensitization , the sensor cell was exposed to ATP puffed every 3 min for 1 s to a maximum of five applications . During the measurements of ATP from an individual astrocyte under OGD stress , the biosensor cell was gently touched to the surface of the target cell . From the recorded currents , the concentration of ATP was calculated according to the calibration curve . Patch-clamp experiments and data analysis Patch-clamp experiments were performed , as described previously -LSB- 17 , 19 -RSB- , using an EPC-9 patch-clamp system -LRB- HEKA Electronics , Lambrecht/Pfalz , Germany -RRB- . The membrane potential was controlled by shifting the pipette potential -LRB- Vp -RRB- . Currents were sampled at 3 kHz and filtered at 1 kHz . Data acquisition and analysis were done using Pulse + PulseFit -LRB- HEKA Electronics , Lambrecht/Pfalz , Germany -RRB- . Whenever the bath Cl − concentration was altered , a salt bridge containing 3 M KCl in 2 % agarose was used to minimize variations of the bath electrode potential . Liquid junction potentials were corrected online . All experiments were performed at room temperature -LRB- 23 -- 25 °C -RRB- . Single-channel amplitudes were measured by manually placing a cursor at the open and closed channel levels . Background currents were subtracted and the mean patch currents were measured at the beginning -LRB- first 25 -- 30 ms -RRB- of current traces in order to minimize the contribution of voltage-dependent current inactivation . The reversal potentials were calculated by fitting I -- V curves to a second-order polynomial -LSB- 18 -RSB- or measured directly from ramp currents . Permeability ratios were calculated , as described previously -LSB- 19 , 21 -RSB- . Data were analyzed in Origin 6 or 7 -LRB- OriginLab Corporation , Northampton , MA , USA -RRB- . Pooled data are given as means ± SEM of n observations . Statistical differences of the data were evaluated by analysis of variance -LRB- ANOVA -RRB- and the paired or unpaired Student 's t-test where appropriate and were considered significant at P < 0.05 . Results OGD induces massive release of ATP from astrocytes Under control conditions , when astrocytes were incubated in standard Ringer solution , the basal release of ATP was very low and did not exceed 0.25 nM over 40 min incubation time -LRB- Fig. 1a : open circles -RRB- . In contrast , when cells were transferred to OGD conditions , the bulk ATP in the incubation medium rapidly increased from the basal level to a peak level of approximately 2 nM within 15 -- 20 min . After this period , the extracellular ATP concentration remained high up to 30 min of incubation and then slightly decreased at 40 min of incubation under OGD stress -LRB- Fig. 1a : filled circles -RRB- . Fig. 1OGD-induced ATP release from astrocytes detected by the luciferin-luciferase assay . a Time course of ATP release during incubation with control standard Ringer solution -LRB- open circles -RRB- and with the OGD solution -LRB- filled circles -RRB- . b Pharmacological profiles of ATP release measured 20 min after application of OGD solution . Each data point or column represents the mean ± SEM -LRB- vertical bar -RRB- . * Significantly different from the control OGD value in the absence of drugs at P < 0.05 In the following pharmacological experiments , we used an incubation time point of 20 min , when the net ATP release induced by OGD reached a steady-state level . Application of extracellular 50 μM Gd3 + nearly completely inhibited OGD-induced ATP release from cultured mouse astrocytes . Arachidonic acid -LRB- 20 μM -RRB- also significantly inhibited OGD-induced ATP release . In contrast , OGD-induced ATP release was not significantly affected by blockers of the volume-sensitive outwardly rectifying -LRB- VSOR -RRB- Cl − channel , phloretin -LRB- 100 μM -RRB- and glibenclamide -LRB- 200 μM -RRB- , of the CFTR Cl − channel , glibenclamide -LRB- 200 μM -RRB- , and of the transporter MRP , probenecid -LRB- 1 mM -RRB- . Also , the ATP release was not sensitive to inhibitors of connexin or pannexin hemichannels , 1-octanol -LRB- 2 mM -RRB- and carbenoxolone -LRB- 100 μM -RRB- , and a blocker of the P2X7 receptor , brilliant blue G -LRB- 1 μM -RRB- . Preincubation of the cells with an inhibitor of vesicular transport , brefeldin A -LRB- BFA , 5 μM -RRB- , for 120 min and together with an intracellular Ca2 + chelator , BAPTA-AM -LRB- 5 μM -RRB- , for 30 min had no significant effect on the OGD-induced release of ATP -LRB- Fig. 1b -RRB- . In order to detect ATP release at the surface of single astrocytes , we employed a biosensor ATP assay technique -LSB- 23 -RSB- . We first established the calibration curve by recording the responses of biosensor HEK-P2X2 cells to different known concentrations of ATP -LRB- Fig. 2a -RRB- . We then gently put the biosensor cell close to the surface of a target astrocyte and recorded the whole-cell current responses from the biosensor cell . As shown in Fig. 2b -LRB- top panel -RRB- , virtually no ATP-evoked currents were observed during perfusion of standard Ringer solution . However , inward current spikes were consistently recorded upon perfusion with OGD solution after a lag period of 7.2 ± 1.0 min -LRB- n = 5 -RRB- -LRB- Fig. 2b : middle panel -RRB- . The OGD-induced inward current responses were eliminated by application of an ATP-hydrolyzing enzyme , apyrase -LRB- Fig. 2b : bottom panel -RRB- . OGD-induced responses were never observed in biosensor cells alone which were positioned remote from target astrocytes . Using the calibration curve , the local ATP concentration was estimated to be 3.7 ± 0.1 μM at the surface of single astrocytes subjected to OGD stress . Fig. 2Cell surface ATP release induced by OGD from single astrocytes . a Calibration of ATP measurements by the biosensor technique . Top panel : representative current responses evoked by puff applications of 1 -- 20 μM ATP in a biosensor HEK-P2X2 cell under whole-cell clamp . Bottom panel : concentration-response curve of the ATP-induced currents recorded from biosensor cells . Each data point represents the mean ± SEM -LRB- vertical bar -RRB- . b Representative current traces recorded from biosensor cells in close proximity to target astrocytes after perfusion with standard Ringer solution -LRB- top panel -RRB- or with the OGD solution in the absence -LRB- middle panel -RRB- or presence -LRB- bottompanel -RRB- of 0.1 mg/ml apyrase , which was applied after current responses appeared . When apyrase was applied from the beginning of OGD application , no current response was observed in biosensor cells positioned next to astrocytes -LRB- data not shown -RRB- From these data , it is concluded that in response to OGD stress , Gd3 + - and arachidonic acid-sensitive ATP release from astrocytes is induced , increasing the extracellular ATP concentration to a level high enough to stimulate most types of purinergic receptors . OGD activates ATP-conductive maxi-anion channels in astrocytes The fact that both Gd3 + and arachidonic acid are potent blockers of maxi-anion channels in mouse astrocytes -LSB- 17 -RSB- suggests that OGD-induced ATP release involves the activity of maxi-anion channels . In cell-attached experiments , OGD stress actually induced activation of anion channels with a large conductance -LRB- Fig. 3a -RRB- , as seen to occur in a similar manner upon stimulation by hypotonicity or chemical ischemia in mouse astrocytes -LSB- 17 -RSB- . The unitary I -- V relationship for these events exhibited slight outward rectification , and the mean slope conductance was 427 ± 23 pS at +60 mV and 262 ± 21 pS at − 50 mV -LRB- Fig. 3b -RRB- . In cell-attached patches , OGD-induced activation of the maxi-anion channel started with a lag time of several minutes and reached its peak within 10 min -LRB- 8.8 ± 1.2 min , n = 6 -RRB- , as shown in Fig. 3c . Fig. 3OGD-induced single-channel activity of the maxi-anion channel in cell-attached patches on astrocytes . a Representative current trace recorded during application of alternating pulses -LRB- the protocol shown in inset above -RRB- . Note that voltage-dependent inactivation of channel activity took place shortly after application of step pulses to ± 50 mV . b Current-voltage -LRB- I -- V -RRB- relationship for unitary events of the maxi-anion channel . The reversal potential was − 4.6 ± 1.9 mV . Each data point represents the mean ± SEM -LRB- vertical bar -RRB- . c Time course of OGD-induced activation of the maxi-anion channel . The mean patch currents recorded at ± 50 mV are plotted as a function of time The single-channel activity induced by OGD persisted after excision of the patch -LRB- Fig. 4 : control -RRB- . In the inside-out mode , however , the channel activity was blocked by extracellular Gd3 + -LRB- 50 μM -RRB- in backfilled pipettes and by intracellular -LRB- bath -RRB- application of 20 μM arachidonic acid -LRB- Fig. 4 : Gd3 + and arachidonate -RRB- . In contrast , phloretin -LRB- 100 μM -RRB- failed to affect the maxi-anion channel activity -LRB- Fig. 4 : phloretin -RRB- . Fig. 4Pharmacological profile of OGD-activated maxi-anion channel events in inside-out patches excised from astrocytes . a Representative current traces recorded during application of alternating pulses -LRB- the protocol shown in inset above -RRB- in the absence -LRB- control -RRB- and presence of Gd3 + -LRB- 50 μM in the pipette -RRB- , arachidonic acid -LRB- 20 μM in the bath -RRB- , and phloretin -LRB- 100 μM in the bath -RRB- . b Effects of Gd3 + , arachidonic acid , and phloretin on the OGD-induced single-channel activity recorded at ± 25 mV . Data are normalized to the mean currents before application of drugs . Each column represents the mean ± SEM -LRB- vertical bar -RRB- . * Significantly different from the control value at P < 0.05 In inside-out patches , when the bath solution was changed from standard Ringer solution to a 100 mM Na4ATP solution , the reversal potential shifted from 0 mV to − 20.6 ± 0.9 mV -LRB- n = 6 -RRB- ; from this result , a PATP/PCl value was estimated to be 0.11 ± 0.01 -LRB- Fig. 5 -RRB- . Fig. 5Representative ramp I -- V records of single-channel events of the maxi-anion channel preactivated by OGD in an inside-out patch exposed to standard Ringer solution -LRB- chloride -RRB- and one exposed to 100 mM Na4ATP solution -LRB- ATP -RRB- . The inward currents recorded in the ATP-exposed patch represent ATP4 − currents . The amplitude of outward currents became smaller when ATP4 − was present in the bath , suggesting a blocking of currents by ATP4 − , as observed in mammary C127 cells -LSB- 19 -RSB- These results indicate that in astrocytes OGD stress activates a maxi-anion channel which can conduct anionic forms of ATP and is sensitive to Gd3 + and arachidonic acid . Discussion In the brain , bidirectional communication takes place between glial cells and neurons . Astrocytes clear synaptically released glutamate from the synaptic cleft , receive signals from neurons at certain receptors , and release molecules , called gliotransmitters , that modulate synaptic transmission , protect neurons from impairment , or repair neuronal tissues after damage . ATP and glutamate represent major gliotransmitters . It has been reported that astrocytes release ATP in response to a number of stimuli , including receptor activation -LSB- 5 -- 7 -RSB- , mechanical or osmotic perturbation -LSB- 8 -- 11 -RSB- , and deprivation of extracellular Ca2 + -LSB- 8 , 9 , 12 -RSB- . The present study demonstrated that astrocytes also respond to ischemic stress mimicked by OGD with ATP release -LRB- Figs. 1a and 2b -RRB- . ATP released from astrocytes has been shown to regulate glutamatergic synaptic transmission -LSB- 6 , 7 , 10 -RSB- , inhibit excitability of retinal neurons -LSB- 26 -RSB- , activate microglia -LSB- 27 , 28 -RSB- , modulate some functions of astrocytes -LSB- 11 -RSB- , and protect astrocytes themselves from H2O2-induced cell death -LSB- 29 -RSB- . Pathophysiological functions of ischemia-induced ATP release from astrocytes remain to be elucidated . There has been much controversy about the pathway of nonlytic release of ATP from a variety of cell types -LSB- 30 , 31 -RSB- . The candidate pathways proposed for astrocytic ATP release include connexin hemichannels -LSB- 8 -RSB- , MRP -LSB- 11 -RSB- , P2X7 receptors -LSB- 12 -RSB- , and exocytotic vesicular transport -LSB- 9 -RSB- . In the present study , however , OGD-induced ATP release was shown to be insensitive to blocking agents for connexin hemichannels -LRB- 1-octanol , carbenoxolone -RRB- , MRP -LRB- probenecid -RRB- , P2X7 receptors -LRB- brilliant blue G -RRB- , and exocytosis -LRB- BFA plus BAPTA-AM -RRB- -LRB- Fig. 1b -RRB- . Recently , it was reported that pannexins form ATP-conductive channels when expressed in Xenopus oocytes -LSB- 32 -RSB- . However , a possible involvement of the pannexin hemichannel in OGD-induced ATP release from astrocytes can be ruled out by the present observation -LRB- Fig. 1b -RRB- of insensitivity to carbenoxolone , which is a potent blocker of not only connexin but also pannexin hemichannels as well -LSB- 33 -RSB- . Although VSOR anion channels were shown to be permeable to ATP at large positive potentials in endothelial cells -LSB- 34 -RSB- , potent VSOR blockers -LRB- phloretin , glibenclamide -RRB- failed to affect OGD-induced ATP release from astrocytes in the present study -LRB- Fig. 1b -RRB- . In contrast , Gd3 + and arachidonic acid were found to prominently suppress ATP release from mouse astrocytes subjected to OGD -LRB- Fig. 1b -RRB- . Our previous study -LSB- 17 -RSB- showed that in mouse astrocytes , maxi-anion channels are activated by chemical ischemia and are sensitive to both Gd3 + and arachidonic acid . The present study demonstrated that maxi-anion channels are activated by OGD -LRB- Fig. 3 -RRB- and that OGD-activated maxi-anion channels are sensitive to Gd3 + and arachidonic acid -LRB- Fig. 4 -RRB- and can conduct anionic forms of ATP -LRB- Fig. 5 -RRB- . On balance , it appears that the maxi-anion channel serves as a major pathway for OGD-induced ATP release from mouse astrocytes in primary culture . Our preliminary study has shown that in mouse hippocampal slices , astrocytes also respond to OGD with the activation of maxi-anion channels -LRB- H.-T . Liu , R.Z. Sabirov , Y. Okada , unpublished observations -RRB- . Since ischemia-like stress was reported to induce ATP release from hippocampal slices -LSB- 13 -RSB- , it is highly possible that the maxi-anion channel also provides a pathway for ischemia-induced ATP release from astrocytes in slice preparations .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O B O O O O

maxi-anion channel
oxygen-glucose deprivation


Impaired performance of alpha7 nicotinic receptor knockout mice in the five-choice serial reaction time task Rationale Nicotinic receptors have been implicated in attentional performance . Nicotine can improve attention in animals and humans , but knowledge about relevant receptor subtypes is very limited . Introduction Nicotinic acetylcholine receptors are pentameric cation channels , so-called because of the actions of nicotine in binding to these receptors and bringing about channel opening . Twelve subunits of neuronal nicotinic receptors have been identified , α2-10 and β2-4 . From these subunits the two most common nicotinic receptors formed in the brain are the heteromeric α4β2 and the homomeric α7 nicotinic receptors , at approximately 85 and 10 % of total nicotinic receptors , respectively . The α7 nicotinic receptors are especially highly expressed in the hippocampus where , as well as mediating cholinergic neurotransmission , their high Ca + + permeability -LRB- Seguela et al. 1993 -RRB- engenders the capacity to modulate glutamatergic and GABAergic neurotransmission both pre - and post-synaptically -LRB- Berg and Conroy 2002 -RRB- . This study aims to shed further light on the role of α7 receptors in behaviour and in the behavioural effects of nicotine . Nicotine is the second most commonly abused drug in the world after alcohol ; cigarette smoking , with its well-established risks of chronic disease , constitutes a great burden on societies worldwide . Considerable efforts have been expended in investigating the function of nicotinic receptors in nicotine addiction and in other neuropsychiatric conditions , and in mediating the cognitive effects of nicotine . For example , cigarette smoking is approximately twice as common in patients with schizophrenia , as it is within the general population . The α7 nicotinic receptor has been implicated in the pathogenesis of schizophrenia and more specifically in the P50 sensory gating deficit measurable in approximately 90 % of schizophrenics , about 50 % of their relatives and about 10 % of control subjects -LRB- Waldo et al. 1991 -RRB- . The sensory gating deficit in schizophrenia is thought to reflect an input dysfunction that allows greater passage of information through the hippocampus for processing by other brain areas -LRB- Freedman et al. 2002 -RRB- . The density of α7 nicotinic receptors in hippocampus and cingulate cortex from schizophrenics is considerably reduced -LRB- Freedman et al. 1995 ; Marutle et al. 2001 -RRB- . It might , therefore , be expected that performance of tasks requiring sustained attention would require α7 nicotinic receptors . Attempts to investigate the functional role of α7 nicotinic receptors at the behavioural level have most often involved the use of selective agonists and antagonists to these receptors . Another avenue of research that has recently become possible uses genetically modified mice with specific deletions of genes for neuronal nicotinic receptors . Mice with targeted deletion of the gene for the α7 nicotinic receptor lack α-bungarotoxin binding sites in the hippocampus and fast , rapidly desensitizing nicotinic currents in hippocampal neurons -LRB- Orr-Urtreger et al. 1997 -RRB- . Viability and brain morphology appear normal in these mice ; of special interest is the lack of any impairment of sensorimotor gating as measured with both auditory and tactile stimuli in the prepulse inhibition paradigm -LRB- Paylor et al. 1998 -RRB- . This result is consistent with the lack of improvement in prepulse inhibition with nicotinic α7 agonists -LRB- Olivier et al. 2001 ; Schreiber et al. 2002 -RRB- , although such agonists are able to improve auditory gating in animal models of sensory gating deficit -LRB- O'Neill et al. 2003 -RRB- . In other behavioural studies , α7 − / − mice have not proved to be considerably different in behaviour from wild-type littermates -LRB- Paylor et al. 1998 -RRB- . In locomotion , α7 − / − mice had a tendency towards less horizontal and vertical activity and spent significantly more time in the centre of the open field than the wild-type mice , but in the light -- dark test of anxiety there were no significant differences between the two groups -LRB- Paylor et al. 1998 -RRB- . In the delayed matching-to-place task in the Morris water maze , α7 − / − mice were slightly impaired in comparison with wild-types , as shown by a longer time to find the hidden platform -LRB- Fernandes et al. 2005 -RRB- . Nicotine-induced locomotor depression , hypothermia , seizures , drug discrimination and tolerance were unchanged in α7 − / − mice -LRB- Franceschini et al. 2002 ; Tritto et al. 2004 ; Stolerman et al. 2004 ; Naylor et al. 2005 -RRB- . However , the α7 − / − mice were less sensitive to impairments in contextual learning brought about by ethanol -LRB- Wehner et al. 2004 -RRB- . The series of experiments presented in this paper use the five-choice serial reaction time task -LRB- 5-CSRTT -RRB- , a commonly used model of attentional performance that is considered analogous to the continuous performance test used in human research . The test requires responses to visual stimuli presented in one of five locations in one wall of an operant conditioning chamber . In a previous study Young et al. -LRB- 2004 -RRB- found that α7 − / − mice took longer to acquire the task than wild-type C57 Bl/6J mice and made more omission errors , but they were not different with respect to accuracy or the latencies of correct responses . Young et al. -LRB- 2004 -RRB- also reported significant improvements in accuracy and reductions in omission errors at several doses of nicotine in C57 Bl/6J mice , including a reduction in the latency of correct responses with a 0.3-mg / kg dose of nicotine . In this paper , the first published report of nicotine improving the performance of mice in the 5-CSRTT and the effects seen were not dose-related , but the findings were important and need replication and investigation under a wider range of conditions . Several studies have reported that nicotine enhances the attentional performance of rats in the 5-CSRTT -LRB- Hahn and Stolerman 2002 ; Hahn et al. 2002 , 2003a -RRB- , although the use of selective nicotinic agonists and antagonists has provided no evidence for involvement of the α7 receptor . The relatively specific α7 agonist AR-R17779 had no effect upon task performance in an experimental procedure in which administration of nicotine , epibatidine and isoarecolone all improved performance -LRB- Hahn et al. 2003a -RRB- . These agonists all have much higher relative affinities than AR-R17779 for heteromeric nicotinic receptors . The α4β2 nicotinic agonist SIB1765F has also been shown to improve 5-CSRTT performance in rats -LRB- Grottick and Higgins 2000 -RRB- . The α7 antagonist methyllycaconitine did not block the effects of nicotine on response latency and anticipatory responding , whereas dihydro-β-erythroidine , an antagonist at heteromeric receptors , did block the effect of nicotine on these measures -LRB- Blondel et al. 2000 ; Grottick and Higgins 2000 -RRB- . However , the α7 agonist GTS-21 has been reported to improve performance in various tests of attention and memory in healthy human volunteers when given in repeated doses -LRB- Kitagawa et al. 2003 -RRB- and in a test of memory in monkeys 24 h after administration -LRB- Briggs et al. 1997 -RRB- . The small number of drugs investigated and their limited selectivity indicates a need for investigations on the role of α7 receptors in attention by another approach , such as the α7 − / − mouse . The aims of the present study were , therefore , to investigate the attentional performance of α7 − / − mice using the 5-CSRTT and to determine whether the gene deletion influenced any effects of nicotine on performance . If the α7 nicotinic receptor is important in attentional mechanisms then it should be possible to confirm the attentional deficits reported by Young et al. -LRB- 2004 -RRB- in α7 − / − mice . If nicotine improves attention , at least in part , through these receptors then it should also be possible to show that nicotine-induced attentional enhancement is diminished in such animals . The procedures used in the initial stages of training were based on those of Patel et al. -LRB- 2006 -RRB- because they were close to those used in studies of rats where nicotine improved performance -LRB- Hahn et al. 2002 ; Hahn and Stolerman 2002 -RRB- . Mice were also trained with both fixed and variable inter-trial intervals -LRB- ITI -RRB- so that α7 − / − and wild-type animals could be compared for their abilities in coping with the temporally unpredictable stimuli in the variable ITI condition . In the later stages of the study , some parameters were modified to make the training procedure more similar to that of Young et al. -LRB- 2004 -RRB- so as to facilitate comparisons and to clarify reasons for certain differences in the results obtained . Materials and methods Animals The animals used were descendants of α7 − / − mice produced by Orr-Urtreger et al. -LRB- 1997 -RRB- , bred from heterozygotes purchased from the Jackson Laboratory , USA -LRB- B6.129S7-Chrna7tm1 Bay , stock no. 003232 -RRB- . The mice were at backcross generation N8 , the background strain was C57BL/6 , donor strain 129S7 via ABI 2.1 ES cell line ; subsequent breeding was always carried out using heterozygous pairs . Twenty-four male α7 − / − mice and 24 male wild-type littermates were used in initial training . Genotypes were determined with the methods used by Stolerman et al. -LRB- 2004 -RRB- , and all mice were injected with TROVAN sterile transponder chips -LRB- Mid Fingerprint , Dorset UK -RRB- at age 10 weeks . These groups were divided into sub-groups of 12 mice each trained with fixed and variable ITI . One wild-type mouse was eliminated from the study at an early stage due to an unresolvable eye infection . Table 1 lists the subsequent parameter changes that took place as training proceeded . Table 1Parameters for all experimentsStudyStimulus durationLimited holdFixed ITIVariable ITITime-outAnticipatory time-outSessions to criteria1a60022 -LRB- 0.25 -- 3.75 -RRB- 00760044 -LRB- 0.5 -- 7.5 -RRB- 20210044 -LRB- 0.5 -- 7.5 -RRB- 2025144 -LRB- 0.5 -- 7.5 -RRB- 2042444 -LRB- 0.5 -- 7.5 -RRB- 2071544 -LRB- 0.5 -- 7.5 -RRB- 20281b154NA20NA2a12NA4 -LRB- 0.5 -- 7.5 -RRB- 22152b12NA4 -LRB- 0.5 -- 7.5 -RRB- 22NAStudies 1a and 2a refer to experiments looking at baseline -LRB- undrugged -RRB- performance at initial and final parameters , whereas studies 1b and 2b refer to the first and second of the experiments with nicotine . For experiment 1a , where ITI values are shown for both fixed and variable ITI mode , these refer to settings for different groups of mice . All times stated are in seconds . For variable ITI the mean , smallest and largest values are shown.NA Not applicable Food restriction was started at age 11 -- 12 weeks and behavioural training began a week later . The mice were kept at 80 % of their free-feeding weights as calculated from a previously obtained growth curve for animals from the same colony . The animals were weighed daily and fed accordingly . All animals had access to water while in their home cages and were individually housed in a room at 21 °C and on a 12-h light/dark cycle , with lights coming on at 0700 hours . The studies complied with local ethical requirements and were carried out in accordance with the Animals -LRB- Experimental Procedures -RRB- Act , 1986 . Apparatus Sound-insulated and ventilated enclosures containing aluminium operant chambers were used -LRB- CENES , Cambridge , UK -RRB- . The chambers measured 14 cm across × 12 cm high × 11 -- 13 cm deep . The back wall of the chamber was curved so that each nose-poke hole was equidistant from the feeder tray at the centre of the front of the chamber . There were five nose-poke holes 1 cm in diameter , 2 cm above the floor , 1.5 cm apart and 1.5 cm deep . Each hole had a green-light-emitting diode at the end and a photoelectric cell at the opening . The two house lights were situated 10 cm up the sides of the chamber . An Acorn computer running Arachnid software -LRB- Paul Fray , Cambridge , UK -RRB- under RISC OS collected the data . Training procedure The mice were trained for 5 days each week , Monday to Friday . The training procedure was based on that of Patel et al. -LRB- 2006 -RRB- and was modified from that of Humby et al. -LRB- 1999 -RRB- so as to resemble more closely the procedures in previous work carried out with the 5-CSRTT in rats -LRB- Bizarro et al. 2004 -RRB- . After the mice were habituated to handling , training began with four sessions of 5 , 10 , 15 and 15 min for the mice to habituate to the test chambers . The house lights were not illuminated at this stage . Before the next two sessions that were 15 min each in duration , 15 -- 20 food pellets were placed in feeder trays before the start of each session . This was followed by two sessions of 15 - and 20-min duration , respectively , in which food pellets were presented according to fixed-time schedules of 15 and 30 s. Nose-poke training then started with four 30-min sessions of non-spatial training with the stimulus duration set at 60 s and with an ITI of 2 s . As noted above , subgroups of mice were trained with ITI of fixed duration , whereas for other groups , individual ITI varied around the mean value -LRB- for details , see Table 1 -RRB- . In non-spatial training sessions , all five holes were illuminated simultaneously and a nose-poke in any hole was reinforced by delivery of a food pellet . For the first of these sessions , 25 mg food pellets -LRB- PJPPP-0020 , Sandown Chemical , UK -RRB- were placed at the front of each of the nose-poke holes to encourage the mice to nose-poke . For spatial training , the house lights were on at all times except during time-outs . Each session lasted for 30 min and there was no limit to the number of trials in a session . A stimulus was randomly presented in one of the five holes and a response in the correct hole during the stimulus duration or the limited hold -LRB- LH -RRB- period after the stimulus was reinforced and followed by a 10-s allowance for the time taken to eat the food pellet . This was sufficient for mice to consume the food and return to attending to the task ; the next ITI began at the end of the 10-s allowance for eating . In sessions in which there was a time-out , this was initiated by a nose-poke response into an incorrect hole during the stimulus duration or LH . Premature responses during the ITI in experiment 1 were recorded but had no programmed consequences . Time-outs involved turning off the lights for 2 s and were followed immediately by the start of the next ITI . Experiment 1a : initial acquisition of attentional task Table 1 describes the stages of training , with criteria for progression to the next stage being that the mean percentage of correct responses for each group was greater than 70 % and the mean response latency for each group was less than the stimulus duration for two consecutive days . The training parameters were not advanced if the mice had not been trained the day before . Acquisition was considered to be complete when the mean percentages of correct responses was stable for two consecutive weeks . Experiment 1b : first set of nicotine tests For testing the effects of nicotine , a reduced number of 15 wild-type and 15 α7 − / − mice were used because it was known from previous studies that this was a sufficiently large group size for assessing drug effects -LRB- Humby et al. 1999 ; Hahn and Stolerman 2002 -RRB- . The selected animals were those that had all been trained at a similar time of day previously and no other selection criterion was used . Up to this stage , some mice had been trained with fixed and some with variable ITI . All selected mice were switched to fixed ITI mode before drug testing started to reduce the number of independent variables ; all other parameters remained unchanged . These mice were given saline injections subcutaneously in the flank after training on each of the 3 days before the first nicotine test day to allow habituation to the procedure to take place . There were four test days on consecutive Tuesdays and Fridays . Mice were allocated to a dosing schedule for injection of saline or 0.001 , 0.01 or 0.1 mg/kg nicotine 10 min before the start of each test session ; each mouse was tested once at each dose by the end of the study and dosing sequences were random . Young et al. -LRB- 2004 -RRB- found that these doses of nicotine significantly improved the task performance of nicotine-naïve mice . Experiment 2a : retraining after changing parameters Seven weeks after the end of experiment 1b , retraining of the same 30 mice commenced , followed by alterations of experimental parameters to make them resemble more closely those used by Young et al. -LRB- 2004 -RRB- . The changes entailed reducing the LH from 5 to 2 s , using variable ITI mode only , and including a 2-s time-out for anticipatory responses . House lights were extinguished during the time-outs during which no stimuli were presented , and the presentation of the next stimulus was delayed by the duration of the time-out . Although mice originally trained with fixed ITI mode were at this time trained exclusively in variable ITI mode , there were no differences in performance as a function of this variation in training history . Table 1 lists the details of all the changes in task parameters . The animals were trained under the altered parameters for 7 weeks , by which time performance was relatively stable . One α7 − / − mouse developed an unusual behavioural phenotype . This mouse was markedly hyperactive in its home cage , required approximately twice as much food as the other animals to maintain its weight and made anticipatory responses at a rate 5 -- 10 times greater than the next most impulsive mouse . No data from this mouse are presented . Experiment 2b : second set of nicotine tests A second experiment with nicotine began immediately after the end of experiment 2a . The mice were injected with saline for 3 days before the start of the test sessions so that they might habituate to the injection procedure . There were eight test sessions on consecutive Tuesdays and Fridays . Mice were allocated to a dosing schedule for injection of saline or 0.001 , 0.003 , 0.01 , 0.03 , 0.1 , 0.3 or 1 mg/kg nicotine 10 min before the start of each test session . Each mouse was tested once at each dose by the end of the study and dosing sequences were random . Data analyses The six behavioural measures selected for analysis were as follows : Accuracy -LRB- percentage of correct responses -RRB- : 100 × correct responses / -LRB- correct + incorrect responses -RRB- Omissions -LRB- percentage of omission errors -RRB- : 100 × omission errors/stimuli presentedLatency of correct responses : the mean time between stimulus onset and a nose-poke in the correct holeLatency of incorrect responses : the mean time between stimulus onset and a nose-poke in any incorrect holeReinforcers earned : equal to the number of correct responsesAnticipatory response rate : the mean number of responses per second during the ITI calculated by using the following formula : total number of responses in all ITI of a session/number of trials/ITI length in seconds . The accuracy measure was not calculated when the sum of correct and incorrect responses was 15 or less . For analysis of variance -LRB- ANOVA -RRB- , percentage data for accuracy and omissions were arc-sine transformed , latencies were log-transformed and anticipatory response data were subject to square root transformation , as in previous studies -LRB- Hahn et al. 2002 , 2003a ; Patel et al. 2006 -RRB- . For post hoc tests , the Bonferroni correction for multiple comparisons was applied to t tests . Graphs show untransformed data . Results for each measure were analysed by ANOVA for repeated measures using Unistat 5.0 for Excel , three-factor ANOVA was used to examine data from experiment 1a and two-factor ANOVA was used for experiments 1b , 2a and 2b . Further details of the factors are given below , at relevant places in the Results section . Drugs Nicotine hydrogen tartrate -LRB- Sigma , UK -RRB- was dissolved in isotonic saline with pH adjusted to 7 with NaOH solution . All injections were given s.c. in the flank in a volume of 1 ml/100 g and all doses were those of the nicotine base . Results Experiment 1a : initial acquisition of attentional task There were no significant differences between any of the groups with respect to any parameter of performance at the first stage of training with the stimulus duration set at 60 s and the ITI at 2 s. Figure 1a -RRB- shows the similarity in the changes in accuracy for α7 − / − and wild-type mice over these first seven sessions . It can be seen that the data show signs that the mice began to learn the task from the very earliest stage of training . Results for other variables are not shown because they were similarly negative with respect to genotype . Fig. 1Experiment 1a : accuracy of 5-CSRTT performance for wild-type -LRB- ● -RRB- and α7 − / − mice -LRB- ○ -RRB- during the first seven sessions of training in the 5-CSRTT . Results show main effects of genotype and training day as means ± SEM for groups of 23 mice . Subgroups of mice with each genotype were trained with fixed and variable inter-trial intervals without effect on performance -LRB- data not shown -RRB- During subsequent stages of training there were progressively greater demands made upon the animals ' ability by altering task parameters , such as reducing the stimulus duration -LRB- Table 1 -RRB- . Differences between the genotypes then emerged , including changes in accuracy , response latencies and anticipatory responding . The data for intermediate stages of training are not shown for brevity ; they largely replicated the observations made when the final set of task parameters was in operation . The performance of the animals was relatively stable during the last 10 days of training under the final parameters and these data were selected for analysis and are presented next . Data for the final 10 days of training were examined by three-factor analysis of variance , the factors being genotype , ITI mode , and days . This analysis yielded main effects of genotype on accuracy -LSB- F -LRB- 1 , 42 -RRB- = 8.67 , p < 0.01 -RSB- , reinforcers earned -LSB- F -LRB- 1 , 42 -RRB- = 12.3 , p < 0.01 -RSB- , anticipatory response rate -LSB- F -LRB- 1 , 42 -RRB- = 4.50 , p < 0.05 -RSB- and arguably , on correct response latency -LSB- F -LRB- 1 , 42 -RRB- = 3.96 , p = 0.053 -RSB- . Figure 2a shows that α7 − / − mice were much less accurate than wild-types , were slightly slower to make a correct response , earned fewer reinforcers and responded more frequently during the ITI . Neither the three-way -LRB- group × ITI mode × day -RRB- nor any two-way interaction was significant for any of the parameters measured . For changes across the ten successive days of training , omission errors -LSB- F -LRB- 9 , 378 -RRB- = 4.99 , p < 0.0001 -RSB- , reinforcers earned -LSB- F -LRB- 9 , 378 -RRB- = 3.28 , p < 0.001 -RSB- and anticipatory response rate -LSB- F -LRB- 9 , 378 -RRB- = 2.11 , p < 0.05 -RSB- showed significant effects . Most of these effects did not show consistent patterns over days -LRB- Fig. 2 -RRB- and are not considered further . Fig. 2Experiment 1a : main effects of genotype and day of training on performance of wild-type -LRB- ● -RRB- and α7 − / − mice -LRB- ○ -RRB- during the last 10 days before drug testing began -LRB- n = 23 -RRB- . Subgroups of mice of each genotype were trained in fixed -LRB- F -RRB- and variable -LRB- V -RRB- inter-trial interval modes ; inset bar graphs illustrate the significant main effects of this variable . All data shown are means ± SEM -LRB- n = 23 -RRB- For ITI mode , there were significant main effects for two variables , correct response latency -LSB- F -LRB- 1 , 42 -RRB- = 9.27 , p < 0.01 -RSB- and reinforcers earned -LSB- F -LRB- 1 , 42 -RRB- = 6.25 , p < 0.02 -RSB- , with those mice trained on variable ITI mode being slower to make correct responses and obtaining fewer reinforcers . The inset bar graphs in Fig. 2c and e illustrate the significant main effects of ITI mode . Experiment 1b : first set of nicotine tests Data were examined with two-factor analysis of variance , the factors being genotype and drug -LRB- nicotine -RRB- dose . There were no significant effects of nicotine -LRB- 0.001 -- 0.1 mg/kg -RRB- upon any measure of performance -LSB- largest F -LRB- 3 , 78 -RRB- = 2.32 , p = 0.082 , for correct response latency -RSB- . The main effect of genotype was significant for accuracy -LSB- F -LRB- 1 , 26 -RRB- = 4.42 , p < 0.05 -RSB- and reinforcers earned -LSB- F -LRB- 1 , 26 -RRB- = 6.55 , p < 0.02 -RSB- . Table 2 shows means ± SEM . for both wild-type and α7 − / − mice , for each attentional parameter , over the 30-min sessions . As in experiment 1a , α7 − / − mice were less accurate than wild-types and earned fewer reinforcers . Genotypes did not differ significantly for other variables , although inspection of Table 2 reveals trends for omission errors , correct response latencies and anticipatory response rate that were in the same direction as the effects seen in experiment 1a . There were no significant interactions between genotype and nicotine . Table 2Mean ± SEM effects of nicotine -LRB- 0.001 -- 0.1 mg/kg -RRB- on attentional parameters in the 5-CSRTT in α7 knockout -LRB- KO -RRB- mice and their wild-type -LRB- WT -RRB- littermatesAttentional parametersNicotine -LRB- mg/kg -RRB- Accuracy -LRB- percent correct -RRB- Omission errors -LRB- percent omissions -RRB- Correct response latency -LRB- s -RRB- WTKOWTKOWTKO0 .088.9 ± 1.784.7 ± 2.327.4 ± 2.830.5 ± 1.80.79 ± 0.030.85 ± 0.030.00189.7 ± 1.384.2 ± 2.725.7 ± 2.230.3 ± 1.70.78 ± 0.020.84 ± 0.040.0188.0 ± 2.181.7 ± 2.527.4 ± 2.331.5 ± 2.40.80 ± 0.020.82 ± 0.020.1088.8 ± 2.083.2 ± 1.826.1 ± 2.529.6 ± 2.40.81 ± 0.020.89 ± 0.03 Incorrect response latency -LRB- s -RRB- Reinforcers earned -LRB- 30 min -RRB- Anticipatory responses/sWTKOWTKOWTKO0 .02.27 ± 0.192.25 ± 0.2289.0 ± 2.982.7 ± 2.50.042 ± 0.0100.052 ± 0.0100.0012.05 ± 0.242.45 ± 0.1190.8 ± 2.582.9 ± 3.00.038 ± 0.0080.059 ± 0.0100.012.32 ± 0.162.16 ± 0.1488.1 ± 2.080.3 ± 2.70.045 ± 0.0130.061 ± 0.0130.102.32 ± 0.202.41 ± 0.1590.1 ± 2.982.6 ± 2.30.041 ± 0.0110.063 ± 0.013 Experiment 2a : retraining after changing parameters After the parameter changes listed in Table 1 , animals were retrained on the task for several weeks and the relatively stable performance during the last ten sessions of training before tests with nicotine was selected for analysis . Data were examined by means of two-factor analysis of variance , the factors being genotype and day of training . The analysis yielded significant effects of genotype on omission errors -LSB- F -LRB- 1 , 29 -RRB- = 20.4 , p < 0.001 -RSB- and reinforcers earned -LSB- F -LRB- 1 , 29 -RRB- = 19.7 , p < 0.001 -RSB- , but not on accuracy , correct or incorrect response latency or anticipatory response rate -LSB- maximum F -LRB- 1 , 29 -RRB- = 1.73 -RSB- . The α7 − / − mice omitted responses to a greater percentage of stimuli and earned fewer reinforcers than wild-types ; these effects were clearly manifested during each of the 10 days of training -LRB- Fig. 3b , e -RRB- . Fig. 3Experiment 2a : main effects of genotype and training day on performance of wild-type -LRB- ● -RRB- and α7 − / − mice -LRB- ○ -RRB- after reduction of limited hold from 5 to 2 s and introduction of 2-s time-out for anticipatory responding -LRB- all mice trained in variable ITI mode -RRB- . Results shown are for the last 10 days of training with the preceding altered parameters before the second set of tests with nicotine . Data shown are means ± SEM -LRB- n = 14 -- 15 -RRB- There were also small but significant differences between correct response latencies and anticipatory responding over days . Anticipatory responding occurred at a lower rate in the later stages of training -LSB- F -LRB- 9 , 241 -RRB- = 7.07 , p < 0.0001 -RSB- ; the latency of correct responses also varied over days , but no clear pattern was evident -LSB- F -LRB- 9 , 241 -RRB- = 2.12 , p < 0.05 -RSB- . The day of training had no effect on any other parameter . There were no significant genotype × day interactions . Experiment 2b : second set of nicotine tests These data were examined by two-factor analysis of variance , the factors being genotype and drug -LRB- dose of nicotine -RRB- . There were no effects of genotype -LSB- F -LRB- 1 , 27 -RRB- = 2.79 -RSB- or drug -LSB- F -LRB- 7 , 179 -RRB- = 1.44 -RSB- on the percentage of correct responses ; nor was there a significant group × genotype interaction -LSB- F -LRB- 7 , 179 -RRB- = 0.97 -RSB- . For omission errors there was a significant effect of genotype -LSB- F -LRB- 1 , 27 -RRB- = 16.7 , p < 0.0001 -RSB- and of drug -LSB- F -LRB- 7 , 189 -RRB- = 82.4 , p < 0.0001 -RSB- and additionally , a drug × group interaction was observed -LSB- F -LRB- 7 , 189 -RRB- = 6.40 , p < 0.0001 -RSB- . From Fig. 4b it can be seen that the effect of genotype arises from the slightly larger overall number of omission errors made by the α7 − / − mice . In view of the significant interaction , this difference was examined separately at each dose of nicotine by means of t tests with Bonferroni corrections for multiple comparisons . These analyses yielded significant differences between the genotypes at nicotine doses of 0.003 , 0.01 , 0.1 and 1.0 mg/kg ; in all cases the percentage of omission errors was greater in the α7 − / − than in the wild-type animals . Nicotine increased omission errors markedly at the largest dose tested , and this effect was greater in the α7 − / − than in the wild-type mice . Fig. 4Experiment 2b : main effects of genotype and nicotine dose on performance of wild-type -LRB- ● -RRB- and α7 − / − mice -LRB- ○ -RRB- after reduction of limited hold from 5 to 2 s and introduction of 2-s time-out for anticipatory responding -LRB- all mice trained in variable ITI mode -RRB- . Data shown are means ± SEM -LRB- n = 14 -- 15 -RRB- . Asterisks represent differences between genotypes at each dose of nicotine determined by t tests with Bonferroni correction ; * p < 0.05 , ** p < 0.01 -RRB- There was no main effect of genotype on correct latency -LSB- F -LRB- 1 , 27 -RRB- = 1.47 -RSB- . The main effect of drug on this measure was significant -LSB- F -LRB- 7 , 183 -RRB- = 22.4 , p < 0.0001 -RSB- and this appeared to be entirely due to an increase in latency at the largest dose of nicotine -LRB- Fig. 4c -RRB- . The interaction was not significant -LSB- F -LRB- 7 , 183 -RRB- = 1.63 -RSB- . There was also a significant effect of drug on incorrect latencies -LSB- F -LRB- 7 , 166 -RRB- = 2.23 , p < 0.05 -RRB- , although its origin is not obvious from Fig. 4d and there was no dose -- response relationship . With respect to reinforcers earned , there were significant effects of genotype -LSB- F -LRB- 1 , 27 -RRB- = 20.7 , p < 0.0001 -RSB- , drug -LSB- F -LRB- 7 , 189 -RRB- = 82.0 , p < 0.0001 -RSB- and a group × drug interaction -LSB- F -LRB- 7 , 189 -RRB- = 8.71 , p < 0.0001 -RSB- . The overall number of reinforcers earned was slightly greater in wild-type than in α7 − / − mice , and Fig. 4e shows that this difference was significant at four of the eight dose levels of nicotine that were tested . Nicotine itself decreased the number of reinforcers at the largest dose tested and this effect was greater in α7 − / − than in wild-type mice . Finally , there was a significant effect of nicotine -LSB- F -LRB- 7 , 189 -RRB- = 15.8 , p < 0.0001 -RSB- but not genotype -LSB- F -LRB- 1 , 27 -RRB- = 1.10 -RSB- on anticipatory responding . The effect of drug may be attributed to a decrease in the rate of responding at the 1.0 mg/kg dose of nicotine -LRB- Fig. 4f -RRB- . A significant drug × genotype interaction was seen -LSB- F -LRB- 7 , 189 -RRB- = 2.39 , p < 0.05 -RSB- , but post hoc tests did not identify a significant difference between the genotypes at any dose level of nicotine . Discussion The five-choice serial reaction time task revealed impairments in the performance of the α7 nicotinic receptor knockout mice on a variety of measures , thus supporting the view that these receptors play a role in attentional performance . Analysis through variation in task parameters suggested that the primary deficit was an increase in omission errors ; a general slowing in performance would have been reflected in corresponding increases in response latencies and decreases in anticipatory responding . Thus , the α7 − / − mice failed to detect significantly more stimuli than the wild-types , but when they did detect a stimulus they responded to it quickly and correctly . These findings confirm and extend the key observations of Young et al. -LRB- 2004 -RRB- and support the emerging consensus that although α7 − / − mice show deficits in the performance of several cognitive tasks , the differences from controls are modest and are manifest only with specific tasks used under precisely defined conditions -LRB- Paylor et al. 1998 ; Wehner et al. 2004 ; Fernandes et al. 2005 -RRB- . We now discuss in detail some differences between our results and those of Young et al. -LRB- 2004 -RRB- and indicate how they can be reconciled by considering parametric variations in the procedures used . In the present study , α7 − / − mice did not differ from wild types until the later stages of training , where the more stringent task parameters placed greater demands upon their cognitive ability -LRB- Fig. 2 -RRB- . These differences in the baseline -LRB- undrugged -RRB- performance of the α7 − / − mice and wild-type mice were largely replicated in our first study on the response to nicotine -LRB- Fig. 3 -RRB- . The impaired accuracy and increased response latency suggested an attentional impairment , whereas the increase in anticipatory responding suggested greater impulsivity . Keller et al. -LRB- 2005 -RRB- also found that the amount of anticipatory responding was increased in α7 − / − mice , although they used a different behavioural procedure . There is often an inverse relationship between accuracy and impulsivity in the 5-CSRTT , and from a comparison of inbred mouse strains it was argued that a decrease in accuracy could be secondary to increased impulsivity -LRB- Patel et al. 2006 -RRB- . The observed change in impulsivity may have contributed to the decrease in accuracy in experiment 1a , which should , therefore , be interpreted cautiously . We did not observe slow acquisition of task performance like that seen by Young et al. -LRB- 2004 -RRB- ; a primary effect on anticipatory responding may have contributed to their finding , too , rather than a deficit in learning ability . This interpretation is supported by the absence of impairments in α7 − / − mice in a variety of other learning tasks -LRB- Paylor et al. 1998 -RRB- and is especially likely with the definition of accuracy used by Young et al. -LRB- 2004 -RRB- , which included the number of anticipatory responses in the denominator for calculation of the proportion of correct responses . During experiment 1a we did not observe any difference between α7 − / − and wild-type mice with respect to omission errors . Interestingly , Young et al. -LRB- 2004 -RRB- reported an increase in omission errors in the α7 − / − mice . The difference in durations of the limited holds -LRB- LH -RRB- used in the two studies may explain this apparent disagreement . The LH , the period in which responses are recorded as being correct or incorrect following stimulus termination , was 2 s in Young et al. -LRB- 2004 -RRB- and 5 s in the final stage of experiment 1a . When , with a long LH , a stimulus is not detected , ` impulsive ' responses in the period of the LH are more likely to be recorded as incorrect than as correct responses , as there are four times more incorrect response options than there are correct options . The increased impulsivity observed in α7 − / − mice may , therefore , have contributed to the observed reduction in accuracy . At the same time , an increase in omission errors such as that reported by Young et al. -LRB- 2004 -RRB- may have been masked , as training with a long limited hold gives the animals more time to make an impulsive response . The trend -LRB- p < 0.06 -RRB- towards longer correct response latencies in α7 − / − mice in experiment 1a might also be explained by late ` correct ' impulsive responses rather than slower reactions . Both groups had short response latencies and the differences between them were eliminated when impulsive responses were punished and the LH was reduced in experiment 2a . Additional and more detailed data on the temporal and spatial patterning of errors and systematic studies of the effects of punishment and of varying LH is needed to establish the validity of this interpretation . In an attempt to understand the origin of the differences between the present results and those of Young et al. -LRB- 2004 -RRB- , we retrained the mice with the LH reduced from 5 to 2 s , used only variable ITI and introduced a 2-s time-out for anticipatory responses ; these task parameters resembled closely those of Young et al. -LRB- 2004 -RRB- . In experiment 2a , carried out under these altered conditions , there was a clear increase in omission errors in the α7 − / − mice . In contrast , the percentage of correct responses was similar in wild-type and α7 − / − animals and the previously observed differences in the latencies of correct responses and in anticipatory response rates were no longer seen . This pattern of deficits in α7 − / − mice was indistinguishable from that observed by Young et al. -LRB- 2004 -RRB- . The absence of a difference in anticipatory responding between genotypes may have been associated with the reduction in its rate as a consequence of the time-out contingency in experiment 2a ; the use of variable ITI in the retraining phase may also have been important because the initial difference between the wild-type and α7 − / − mice in anticipatory responding was greater in animals trained with fixed interval than with variable interval -LRB- Fig. 2 -RRB- . This observation might reflect impaired utilisation of temporally predictable stimuli in the α7 − / − mice , rather than differences in impulsivity per se . Nevertheless , caution is needed because the altered performance after parametric changes may also have been associated with the longer training and greater age of the animals at this stage . In experiment 1b , the absence of any significant effect of nicotine even in wild-type mice could have been attributed to the parameters of the schedule maintaining the behaviour . Nevertheless , the doses of nicotine used were within the range reported to improve accuracy and reduce omission errors in mice -LRB- Young et al. 2004 -RRB- . It , therefore , seemed possible that a nicotine effect would emerge when the training parameters were adjusted to resemble those used by Young et al. -LRB- 2004 -RRB- . However , this was not the case and in experiment 2b ; no dose of nicotine improved performance on any variable . The largest dose of nicotine impaired performance ; omission errors and the latencies of correct responses increased , whereas reinforcers earned and anticipatory responses decreased -LRB- Fig. 4f -RRB- . This pattern of results can be attributed to a general deficit in operant response rates produced by the very large -LRB- 1 mg/kg -RRB- dose of nicotine . The lack of change in accuracy is compatible with explanations in terms of motor impairment rather than an attentional deficit . The data in Fig. 4a indicate that these effects of nicotine were also present in the α7 − / − mice and , in some cases , their magnitude was increased -LRB- e.g. omission errors -RRB- . The reason for this unexpected observation is unclear ; it may represent a little more than a minor enhancement at certain doses of nicotine of the significant main effect of genotype on omission errors . Why was there no improvement in attentional performance by any dose of nicotine ? A ceiling effect associated with the very high baseline accuracy offers only a partial explanation because the baseline rate of omission errors was high -LRB- 30 % -RRB- , but there was still no nicotine-induced improvement on either measure . Prior exposure to nicotine might be relevant , but Young et al. -LRB- 2004 -RRB- obtained positive effects of nicotine at 0.003 and 0.3 mg/kg in mice previously exposed to the drug and in rats , nicotine-induced attentional enhancement did not show tolerance during 6 weeks of daily exposure to the drug -LRB- Hahn and Stolerman 2002 -RRB- . The parameters for training and testing during experiment 2b were also very similar to those used by Young et al. -LRB- 2004 -RRB- . On the basis of the available information it appears that nicotine-induced improvement of performance in wild-type mice with a C57BL/6 background is not a robust phenomenon . This aspect of the study contrasts with the good agreement of the present results with those of Young et al. -LRB- 2004 -RRB- with respect to the performance of α7 − / − and wild-type mice in experiments 1a and 2a . Comparisons between C57BL/6 and DBA/2 mice in the 5-CSRTT have also been carried out in two different laboratories , and there was excellent agreement between the results ; in each case , the DBA/2 mice were the poorer performers , exhibiting increased impulsivity and lower accuracy -LRB- Greco et al. 2005 ; Patel et al. 2006 -RRB- . DBA/2 mice had reduced levels of α7 expression and impaired sensory gating that was corrected by α7 agonists -LRB- Stevens et al. 1998 -RRB- . The present work did not investigate the mechanisms underlying the impaired performance of α7 − / − mice , but some suggestions can be made . Investigations of brain areas important for attentional performance using localised lesions and drug administration have demonstrated the involvement of the medial prefrontal cortex and D1 dopamine receptor activation within this area -LRB- Muir et al. 1996 ; Granon et al. 2000 ; Hahn et al. 2003b -RRB- . Dopamine utilisation in the frontal cortex also appears to be reduced in individual rats that perform badly in the task -LRB- Puumala and Sirvio 1998 -RRB- , whereas the α7 agonist GTS-21 elevated dopamine and noradrenaline concentrations in the fronto-parietal cortex , which is implicated in attentional function -LRB- Summers et al. 1997 -RRB- ; it is possible that a reduction or lack of α7 receptors might lead to a deficit in dopamine levels in the fronto-parietal cortex , although changes in neurotransmitter systems other than dopamine may also contribute . It can be suggested , speculatively , that the α7 − / − mouse may have some value as a model for very specific aspects of the cognitive dysfunctions in schizophrenia . Diminished frontal dopamine has been hypothesised to be important in schizophrenia -LRB- Davids et al. 2003 ; Laruelle et al. 2003 -RRB- , and post-mortem studies have shown diminished numbers of α7 receptors in brains of schizophrenics . The α7 − / − mouse has not been tested for an auditory sensory gating deficit , the endophenotype of schizophrenia in which the α7 nicotinic receptor has been strongly implicated -LRB- Freedman et al. 2002 -RRB- and which may offer a cognitive explanation for the attentional deficits observed . In conclusion , this study confirms that α7 − / − mice exhibit a weakened ability to detect visual stimuli in a test of sustained attention , suggesting that the presence of α7 receptors is necessary for normal task performance . Manipulating task parameters can alter the apparent nature of the deficit , suggesting a need to compare different strains of mice under more than one set of conditions to characterise them adequately . While this observation encourages further investigation of the value of α7 receptor agonists in neuropsychiatric states such as dementia -LRB- Kitagawa et al. 2003 -RRB- and schizophrenia -LRB- Martin et al. 2004 -RRB- , the lack of effect with nicotine in the present experiments is a major concern . Identification of a protocol for assessing attention-enhancing effects of nicotine in mice may require further modifications of protocols or the use of different strains of animal .

O O O O B I B B O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O 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O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B I B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O

addiction
schizophrenia
nicotinic receptor
nicotine
mice
knockout
attention