a number of inborn [ 15 ] and acquired [ 613 ] neuropathologies are associated with impaired function of the mitochondrial 2-oxoglutarate dehydrogenase multienzyme complex ( ogdhc ) . ogdhc comprises multiple copies of the three catalytic components : the thiamine diphosphate - dependent 2-oxoglutarate dehydrogenase ( e1o ) , the lipoyl - bearing dihydrolipoamide succinyl transferase ( e2o ) , and the fad - binding dihydrolipoamide dehydrogenase ( e3 ) . coupled action of the components is required for an important regulatory step in the mitochondrial tricarboxylic acid cycle , the oxidative decarboxylation of 2-oxoglutarate ( reaction 1 , where r = ch2ch2cooh ) generating energy in the form of nadh , and macroergic compound succinyl - coa : 
 ( 1 ) 
   to reveal molecular mechanisms of the association between neuropathologies and ogdhc function , we introduced specific inhibitors of ogdhc , which have been successfully applied in recent years for cellular [ 1619 ] , tissue , and animal   studies . having the phosphonate residue instead of the leaving carboxyl group of 2-oxoglutarate , these synthetic inhibitors target the starting and rate - limiting e1o component of ogdhc in a highly specific manner , imitating transition state of the e1o - catalyzed step [ 15 , 22 ] . hence , application of such phosphonate analogs of 2-oxoglutarate allows modeling the states when a decrease in the ogdhc activity is observed . in particular , such a decrease occurs in brains of patients with neurodegenerative diseases , including alzheimer 's [ 9 , 10 , 13 ] and parkinson 's [ 6 , 11 ] diseases , wernicke - korsakoff syndrome , and progressive supranuclear palsy . it is important to note that 2-oxoglutarate is the glutamate precursor in the glutamate synthesis from glucose de novo . in view of this , the irreversible degradation of 2-oxoglutarate by ogdhc ( reaction 1 ) is intimately related to the synthesis / degradation of excitatory ( glutamate ) and inhibitory ( gaba ) neurotransmitters . indeed , the perturbed flux through the complex was shown to affect the amino acid levels , which may explain the developmental impact of the ogdhc regulation , shown in our previous paper . in the present work , we apply the ogdhc inhibitor succinyl phosphonate to study the behavioral impact of the ogdhc function in adult rats . we show a compensatory response of cortex and striatum to the ogdhc inhibition , which correlates with behavioral changes . increasing the inhibition or combining the inhibition with hypoxia may abrogate the response . the antagonistic action on ogdhc of the hypoxic stress and synthetic inhibitor is of potential therapeutic significance . all experiments were performed with consent to helsinki declaration on the guide for the care and use of laboratory animals , defining the conduct of ethical research on laboratory and other animals . animals were kept at 21  2c on standard ration and 12/12  h light / dark cycle . wistar rats of about 200  g ( males ) or 250300  g ( females ) were used in the experiment . pregnant rats were exposed to hypobaric hypoxia at day 910 of pregnancy by placing in a decompression ( altitude ) chamber of 3.3  l volume , with a vacuum pump  mez mohelnice acute hypoxia was achieved by decreasing the atmospheric pressure in 1  min to 145  mm  hg , correspondent to 11500  m altitude . sp was introduced to animals at 5 and 25  mg / kg by intranasal application of the water solution of the trisodium salt , with the physiologic solution substituting for sp in all reference groups . in the study of the sp influence on hypoxic effects they were estimated in the standard tests : open - field , elevated plus maze ; light - dark chamber , and closed plus maze . video - recording and easy track program   were used to follow behavior . to increase the assessment power , different tests were also employed to exclude the animal adaptation to experimental conditions when the time dependence of the behavioral changes was studied . the parameters presented in figures correspond to those which showed statistically significant changes ( p < .05 ) and the changes at the level of trends ( p < .1 ) . the latter provided additional support for the statistically significant changes , thus increasing the conclusion accuracy . after physiological monitoring has been performed , the animals were sacrificed by decapitation ; cortex and striatum were excised on ice quickly , frozen in liquid nitrogen , and stored at 70c until assay . each sample corresponded to one animal , with the ogdhc assay in a sample repeated 3 - 4 times at three different protein concentrations . this was done to ensure that the activity is estimated in the interval where the dependence of the reaction rate on the catalyst concentration is linear . preservation of the linearity at low and high protein concentrations attests to the absence of the multienzyme complex dissociation upon dilution and of the interfering activities consuming the produced nadh , respectively . sigma ,  usa .   it was performed using statistica 6.0 software , inc .. values are expressed as means  sem . dispersion of the ratios of the affected to control values ( % ) was calculated by taking into account experimental errors in determination of both values as in . statistical significance of the differences in the parameter mean values was tested by one - way analysis of variance ( anova ) followed the student - newman - keuls post hoc test . statistical significance of the differences in the animal stratification by the resistance to hypoxia was assessed by the fisher 's exact test . behavior was assessed within 4  h ( figure 1 ) and the next day ( figure 2 ) after application of sp . all together , the results of the open field , elevated plus maze and closed plus maze tests pointed to increased exploratory activity and decreased anxiety , resulting from the sp application . an increase in exploratory activity was obvious from statistically significant elevations in the hole inspections ( figure 1(a ) ) , coming out into light ( figure 1(b ) ) , rearing and crossing the center ( figure 2 ) . visits and time per a round in the closed plus maze showed a trend to decrease ( figure 2 ) , suggesting improved orientation ability and inspection efficiency . along with the mentioned above increase in the coming out into light ( figure 1 ) , the shorter grooming times ( figures 1(a ) and 2 ) pointed to decreased anxiety . it is noteworthy , that these effects were not pronounced when the sp - treated animals were imposed to stress . that is , under stressful conditions of the light - dark chamber , no statistically significant changes in the exploratory activity and anxiety were detected 24  h after the treatment ( data not shown ) . thus , the sp - induced increases in exploratory activity and risk behavior do not persist under potentially dangerous conditions when animal is challenged with a bright light . the difference of the animal response to the sp treatment , revealed under comfortable and stressful conditions , is indicative of a certain adequacy of the response , supporting its behavioral rather than motoric origin . remarkably , increasing sp to 25  mg / kg did not always increase the behavioral effect of 5  mg / kg sp , often even alleviating effects of the low dose ( figures 1 and 2 ) . at the same time , elevating sp tended to decrease the moving episodes ( figure 1(a ) ) and muscle force ( data not shown ) . it thus appears that at 25  mg / kg the systemic action of sp increases , with the muscle ogdhc inhibition obviously causing energetic impairment and muscular weakness . thus , there is a specific window of the sp doze where the ogdhc inhibitor increases the exploratory activity and decreases anxiety of experimental animals . after the behavioral parameters were assessed , the animals were sacrificed and the ogdhc activity in the extracts of cortex and striatum determined ( figure 3 ) . the cortex activity was increased at 5  mg / kg sp , returning to the control level at 25  mg / kg sp ( figure 3(a ) ) . sp also increased the ogdhc activity in striatum ( figure 3(b ) ) , with the maximal effect achieved already at a low doze . thus , increases in the ogdhc activity of cortex and striatum correlate with the increased exploratory activity and decreased anxiety of the animals , as observed under comfortable conditions in the open field , elevated plus maze and closed plus maze tests . in neurodegenerative diseases , the metabolic stress is often increased by hypoxia , in which damaging action is associated with the elevated ros and glutamate excitotoxicity . in cellular experiments , sp was shown to protect from the glutamate - induced ros   and excitotoxicity [ 16 , 17 , 21 ] . owing to this , we tested if sp would show a protective action on the behavioral and biochemical changes induced by hypoxia . we used the previously established model , in which acute hypoxia was created in a decompression chamber with female rats sensitized to the insult by pregnancy [ 21 , 24 , 31 ] . protective action of sp was observed already during the acute hypoxia in decompression chamber . within an animal group , the resistance to hypoxia assessed by both physiological and behavioral parameters is known to vary , defined as low , medium , or high when the time before collapsing under hypoxic conditions is less than 5 minutes , between 5 and 10 minutes , and 10 minutes and more , respectively [ 24 , 32 ] . figure 4 shows that pre - conditioning with a low doze of sp ( 5  mg / kg ) increased the percentage of the highly resistant animals at the expense of the medium resistant group . the sp - induced increase in the number of animals highly resistant to hypoxic conditions exhibits the protective effect of the pretreatment with the ogdhc effector upon acute hypoxia . the behavioral effects were determined in the most reactive low resistant rats 24 hours after recovery from the hypoxia - induced collapse . compared to the males assessed at the same time after the sp treatment ( figure 2 ) , pregnant females showed more resistance to the action of sp per se ( figures 5 and 6 ) . that is , sp alone did not significantly influence the behavioral parameters in the open field or elevated plus maze tests . only the grooming time was significantly decreased by the low sp doze in both females ( 2-fold , figure 6 ) and males ( 3-fold , figure 2 ) , indicative of the sex - independent anxiolytic effect of sp on animals . remarkably , not only sp , but also hypoxia per se showed an anxiolytic effect . however , the latter was expressed in the rat sensitivity to light rather than grooming . that is , hypoxia caused the statistically significant increase in the looking out into light ( figure 6 ) , with the freezing time tending to decrease ( figure 5 ) , but did not change the grooming time ( figure 6 ) . thus , a certain degree of similarity in the action of both sp and hypoxia was revealed , although the common anxiolytic effect was expressed in different behavioral parameters , such as grooming or light sensitivity in the sp or hypoxia treatments , respectively . noteworthy , the sp pre - treatment abrogated the hypoxia effects , exposing the protective action of sp upon hypoxia . that is , the hypoxia - induced changes in the locomotor activity and freezing time were abolished in the sp - treated animals ( figure 5 ) , as was the statistically significant increase in the looking out into light ( figure 6 ) . analysis of the ogdhc activity in cortex of pregnant females provided further evidence on the sp - induced protection from the hypoxic effects . as seen from figure 7 , hypoxia increased the ogdhc activity in cortex , whereas pre - treatment with sp returned the ogdhc activity to the control level . the data of figure 7 also support the conclusion that the ogdhc activity level correlates with behavioral parameters . that is , the hypoxia - induced behavioral effects ( figures 5 and 6 ) were accompanied by the up - regulation of the cortex ogdhc , with the combined action of hypoxia and sp returning both the ogdhc activity ( figure 7 ) and behavioral parameters ( figures 5 and 6 ) close to the norm . thus , the pre - treatment with a low sp doze ( 5  mg / kg ) normalized the hypoxia - induced changes in the cortex ogdhc activity ( figure 7 ) and behavioral parameters ( figures 5 and 6 ) , increasing the proportion of rats exhibiting the high resistance to hypoxia ( figure 4 ) . this means that the sp pre - treatment protected from hypoxia on biochemical , behavioral , and physiological levels . in male rats treated with the ogdhc inhibitor sp , we revealed increased exploratory activity and decreased anxiety ( figures 1 and 2 ) concomitant with increases in the ogdhc activity in cortex and striatum ( figure 3 ) . that is , the impaired cognition in patients with neurodegenerative diseases was observed along with the decreased ogdhc activity in their brains , whereas in animal model we observe increased exploratory activity with the orientation and inspection efficiency tending to improve ( figures 1 and 2 ) , when the brain ogdhc activity is elevated ( figure 3 ) . it should be kept in mind that the ogdhc activity assayed in brain extracts under standard test conditions is not equal to the flux through ogdhc inside brain cells . the flux may differ dependent on the cell type , being determined , in particular , by the substrate concentrations within the cellular mitochondria , which are most probably different from those in the standard assay system in vitro . however , the activity assays do show that ( i ) application of sp induces the brain response at the level of the ogdhc regulation , and ( ii ) the response is to up - regulate ogdhc , obviously compensating for the enzyme inhibition by sp . the compensatory response of brain to the ogdhc inhibition by sp ( figure 3 ) may occur through activation of internal mechanisms of the ogdhc stimulation , for example , by protein - protein interactions , posttranslational modifications , increased synthesis . in particular , increased synthesis of the first component of ogdhc , e1o , was observed in response to the acute ethanol - induced stress . besides , in addition to its action as the reversible ogdhc inhibitor , sp is a protector of ogdhc from an irreversible inactivation occurring in the course of catalysis [ 16 , 17 ] . during a certain time period , this protective effect may manifest itself as an apparent increase in the ogdhc activity compared to the control value with the unprotected enzyme ( figure 3 ) . it can not , however , be the only reason for the increase , as the latter may be observed in the absence of sp as well . that is , the ogdhc activity of cortex increases also as a result of acute hypoxia in pregnant rats ( figure 7 ) . worth noting , also in this case the elevation of the ogdhc activity in cortex is accompanied by increased exploratory activity and decreased anxiety ( figures 5 and 6 ) .   in our earlier work on the developmental impact of the brain ogdhc activity , we observed the sex - determined differences in the brain ogdhc expression and reactivity to sp . the present study confirms dependence of the behavioral impact of the ogdhc regulation on the animal physiology , as behavior of pregnant females is less responsive to sp than that of males . nevertheless , the sex - independent anxiolytic effect of sp was also observed , with the low doze of sp decreasing the grooming time in both males ( figures 1(a ) and 2 ) and females ( figure 6 ) . remarkably , both earlier   and here , the sp action exhibited an interplay with different stresses . for instance , in males , the sp - induced increase in exploratory activity and decrease in anxiety were pronounced only under comfortable test conditions ( figures 1 and 2 ) , but not under the stress conditions of the light - dark chamber ( data not shown ) . likewise , in pregnant rats exposed to hypoxia , the sp pre - treatment abrogated most of the hypoxia - induced behavioral changes ( figures 5 and 6 ) . combination of the hypoxic stress with sp also reduced the brain ogdhc activity elevated by hypoxia ( figure 7 ) . our data thus point to the antagonistic action of stress and sp at both biochemical and behavioral levels . this antagonism could be used to protect from the negative effects of the metabolic stress . in particular , we show the protection in the hypoxic model , where sp not only normalized the behavioral parameters ( figures 5 and 6 ) and ogdhc activity in cortex ( figure 7 ) , but also increased the proportion of rats highly resistant to hypoxia ( figure 4 ) . earlier , we suggested that metabolic stress accompanying the onset of neurodegeneration increases an irreversible inactivation of ogdhc [ 34 , 35 ] , which may also lead in appearance of aberrant enzyme forms with an elevated ability to catalyze hazardous side reactions . this irreversible damage of the ogdhc function occurring in neurodegenerative diseases [ 7 , 36 ] may be alleviated through the reversible inhibition of ogdhc by sp due to several reasons . first of all , the inhibition by sp may induce compensatory effects up - regulating ogdhc ( figure 3 ) . furthermore , as shown earlier , sp binding to the active site protects the essential groups of enzyme from irreversible modifications causing inactivation [ 16 , 17 ] . the sp binding also prevents the enzyme from catalysis of the side reactions , including production of ros and enzyme - bound radicals irreversibly damaging ogdhc . the work presented here extends our previous in vitro and in situ studies to animal experiments , showing that reversible inhibition of ogdhc under conditions of metabolic stress may indeed normalize both the behavior ( figures 5 and 6 ) and brain ogdhc activity level ( figure 7 ) . it is probable that up - regulation of ogdhc occurs also in response to its irreversible inactivation upon initial stages of the neurodegenerative diseases . however , when the inactivatory conditions persist with the up - regulated ogdhc not protected , the cellular compensatory ability is exhausted , eventually resulting in the increasing damage of the brain mitochondrial metabolism . in contrast , the protective effects of sp described above would allow cells to combine the ogdhc up - regulation with the enzyme protection , which may underlie cellular ability to overcome metabolic stress . up - regulation of the brain ogdhc activity correlates with increased exploratory activity and decreased anxiety . the up - regulation under metabolic stress may be adjusted by reversible inhibition of ogdhc , normalizing the biochemical and behavioral deviations .
decreased activity of the mitochondrial 2-oxoglutarate dehydrogenase complex ( ogdhc ) in brain accompanies neurodegenerative diseases . to reveal molecular mechanisms of this association , we treated rats with a specific inhibitor of ogdhc , succinyl phosphonate , or exposed them to hypoxic stress . in males treated with succinyl phosphonate and in pregnancy - sensitized females experiencing acute hypobaric hypoxia 
 , we revealed upregulation of brain ogdhc ( within 24 hours ) , with the activity increase presumably representing the compensatory response of brain to the ogdhc inhibition . 
 this up - regulation of brain ogdhc was accompanied by an increase in exploratory activity and a decrease in anxiety of the experimental animals . remarkably , the hypoxia - induced elevation of brain ogdhc and most of the associated behavioral changes were abrogated by succinyl phosphonate . 
 the antagonistic action of hypoxia and succinyl phosphonate demonstrates potential therapeutic significance of the ogdhc regulation by the phosphonate analogs of 2-oxoglutarate .