signal transduction in astrocytes is of fundamental , but largely unrecognized , importance for brain function under normal and abnormal conditions . due to the extreme difficulty in studying specifically astrocytic signaling in the brain in vivo , many studies have been carried out in isolated preparations of astrocytes , that is , freshly isolated cells obtained directly from the brain or cultured astrocytes . claims by the kimelberg that astrocyte cultures are misleading   are unfortunately often correct . however , these researchers have unjustifiably ignored that many types of astrocyte cultures exist and that they are not similar in their characteristics . the fact that the cultures used by us are well suited to study drug - induced signaling changes is demonstrated in table 1 ( slightly modified from ) . it shows identical changes in gene expression and editing induced by chronic treatment with drugs used to treat mood disorders ( fluoxetine ; carbamazepine ) in cultured astrocytes and in astrocytes freshly dissociated from the brains of animals chronically treated with the same drugs . the freshly dissociated cells are probably not sufficiently intact to show the mechanisms involved in these gene changes or their functional consequences , which for this reason have been elucidated in the cultured cells . such studies have indicated important correlations between different effects on gene expression or editing shown in table 1 and they have enabled tentative functional interpretations . the prototypes of antidepressant drugs ( which also have anxiolytic effect ) are the serotonin - specific reuptake inhibitors ( ssris ) . when fluoxetine , the first of the presently used ssris , was approved for clinical use in 1987 , two effects of the drug had been established : inhibition of serotonin reuptake by the serotonin transporter ( sert )   and partial displacement of serotonin ( 5-ht ) binding to cultured astrocytes , which have no sert expression . however , at that time , astrocytes were supposed to be unimportant for brain function , and inhibition of sert has since then been regarded as the mechanism responsible for ssris effects . in 1987 , the 5-ht2b receptor was unknown , but it is now established to have the highest affinity between 5-ht receptors for ssris , with a ki for displacement of serotonin binding to cultured astrocytes of 70  nm . this is identical to its ki for inhibition of serotonin uptake via the human placental sert . this is a distinct difference from the large potency difference in their effect on sert , although the therapeutic doses are of roughly comparable magnitudes , a difference , which can only partly be explained by differences in drug kinetics and protein binding . the metabolic pathway activated in cultured astrocytes by fluoxetine an expanded version is shown in figure 1 . with increasing realization of the importance of glycogenolysis for signaling in astrocytes [ 1214 ] , it may be important that fluoxetine acutely stimulates glycogenolysis , an effect that is secondary to an increase in [ ca]i . fluoxetine might also affect glycogen synthesis , since it stimulates the akt pathway ( see below ) . the involvement of 5-ht2b receptor - stimulated glycogenolysis has been established during learning , where acute administration of serotonin can rescue long - term learning in a one trial aversive learning paradigm in day - old chickens under conditions when the aversive stimulus was otherwise too weak to establish more than transient long - term memory retention . fluoxetine and paroxetine have a similar effect and are equipotent , showing that the rescue was not due to inhibition of sert , and the rescuing effect was inhibited by an inhibitor of glycogenolysis ( gibbs and hertz , submitted , frontiers in pharmacology ( neuropharmacology ) after invitation to research topic ) . in accordance with figure 1 , fluoxetine acutely phosphorylates akt in cultured astrocytes . akt - mediated phosphorylation of glycogen synthase kinase-3 ( gsk-3 ) inhibits its enzymatic activity . akt phosphorylation may stimulate glycogen synthesis , since phosphorylation and activation of glycogen synthase by gsk-3 decreases the activity of glycogen synthase . , from the jope group , found in whole brain that fluoxetine administration increases the levels of phosphorylated gsk3. later , beurel et al .   from the same group confirmed that fluoxetine rapidly and robustly increases serine phosphorylation of gsk3 but that these responses in young mice are blunted or absent . this is consistent with an effect on astrocytes , since these glial cells are generated postnatally . an increased glycogen synthesis following 5-ht2b receptor activation has been directly shown in hepatocytes , whereas agonists of 5-ht1 and 5-ht2a receptors had the opposite effect . the effects of ssris that are important in connection with major depression are the chronic effects , since the effect of clinical treatment takes several weeks to appear . to - date , all gene effects caused by chronic treatment with fluoxetine ( the only ssri studied on astrocytes in vivo ) after mice had been treated for 14 days with i.p . injection of fluoxetine ( 10  mg / kg per day ) have been identical to those in cultured astrocytes chronically treated with fluoxetine or other ssris , as was shown in table 1 . they have also been similar to those found by other authors or ourselves in total brain with the exception of gene expression of spla2 and gluk4 , genes that were upregulated in neurons of the treated animals and accordingly also in whole brain . anhedonia , one of the components of major depression , caused oppositely directed changes in expression of some of the same genes . other gene expression changes established in the cultured cells did not occur , but it should be remembered that anhedonia is only one component of major depression . fluoxetine can reverse both the anhedonia and the gene expression changes ( b. li and l. peng 's unpublished results ) . the upregulation of adar2 by chronic treatment with fluoxetine ( table 1 ) depends on 5-ht2b receptor stimulation , since the 150200% increase of mrna and protein expression of adar2 was prevented in astrocytes treated with 5-ht2b receptor sirna . in contrast to the upregulation of cpla2 ( see below ) , it is not known if additional steps of the fluoxetine - mediated pathways are also required . adars constitute a family of adenosine deaminases catalyzing deamination of adenosine to inosine in double - stranded regions of mrnas , thus changing the translated protein sequence , since inosine is read by the cells as guanosine . there are three members in the adar family : adar1 , adar2 , and adar3 . adar2 upregulation in fluoxetine - treated mice was specific for this subtype , observed only in astrocytes and occurred within 3 days . in the brain adar2 has been shown in hippocampal pyramidal neurons and cerebellar purkinje cells and bergmann glial cells , with less expression of adar1 and adar3 . adar2 and probably also its upregulation are essential for the editing changes shown in table 1 ; this has been directly demonstrated in cultured astrocytes by the use of cells treated with sirna against adar2 . astrocytes are among the cells that express calcium - dependent phospholipase 2 ( cpla2 ) [ 27 , 28 ] , and in the brain in vivo , they may even be enriched in this phospholipase [ 2931 ] . its activation specifically releases arachidonic acid from the sn-2 position of membrane - bound phospholipid substrate in neural preparations [ 3235 ] , including glioma cells . so does treatment with 10  m fluoxetine for 24  h , which might have sufficed to induce an increase in cpla2 , whereas acute exposure of astrocyte cultures to the same concentration of fluoxetine has no corresponding effect ( l. peng and l. hertz 's unpublished experiments ) . rapoport and coworkers [ 4143 ] showed that chronic administration of fluoxetine leads to stimulation and enhanced mrna and protein expression in rat brain of cpla2 , but not of the two other phospholipases a2 ( secretory pla2 ( spla2 ) and intracellular pla2 ( ipla2 ) ) . confirmed a slow and selective upregulation of mrna and protein expression of cpla2a , the major isoform of cpla2 , in mouse astrocytes in primary cultures during chronic incubation with 1 or 10  m fluoxetine . the upregulation was abrogated by the 5-ht2b receptor antagonist sb 204741 , the metalloproteinase inhibitor gm6001 , and the inhibitor of egf receptor tyrosine phosphorylation ag1478 and by u0126 , the inhibitor of erk1/2 phosphorylation . these are all inhibitors of the signaling pathway that was shown in figure 1 for fluoxetine , showing that upregulation of mrna and protein of cpla2 were inhibited by the same drugs that acutely inhibit erk1/2 phosphorylation and by inhibition of the phosphorylation itself . as was shown in table 1 , upregulation , specifically of cpla2a , has also been found in freshly dissociated astrocytes isolated by facs after 2-week treatment of rats with fluoxetine , whereas no corresponding effect was found in neurons . this finding strongly suggests that the enhanced cpla2 activity demonstrated in whole brain after chronic fluoxetine treatment   selectively occurs in astrocytes . the stimulation of glucose metabolism may be important in the pathophysiology of depressive illness and its pharmacological treatment . glucose metabolism in brain is reduced in many regions , primarily in the frontotemporal parts , in patients suffering from unipolar depression [ 4447 ] , with a correlation between the degree of hypometabolism and severity of the illness , and normalization following treatment with an ssri [ 4951 ] . figure 2 shows that arachidonic acid may play a role in determining rates of cerebral glucose metabolism as seen from a rectilinear correlation in depressed patients between plasma concentration of arachidonic acid and rate of cerebral glucose utilization in one of the regions affected metabolically by depression . moreover , genetic associations are found between cpla2 and major depression [ 53 , 54 ] . depending on the site(s ) of impairment of glucose metabolism , glycogen metabolism could also be affected . however , the effect of arachidonic acid is not necessarily exerted on glucose metabolism itself but might also be exerted on energy - requiring reactions . the importance of mitochondrial abnormalities in major depression is shown by a significantly reduced number of mitochondria , but larger mean mitochondrial volume , in hippocampus in rats sensitive to stress - induced depression than in control rats . following treatment with the antidepressant imipramine , a significant increase in the number of mitochondria occurred in the stress - sensitive group . clinical studies also suggest that psychiatric features can be prominent features of mitochondrial disorders , but additional methodologically rigorous and adequately powered studies are needed before definitive conclusions can be drawn . arachidonic acid metabolites , including prostaglandins , may also exert other beneficial effects important for the amelioration of depression . prostaglandin synthesis inhibitors in doses used to treat pain [ 58 , 59 ] may cause fear , agitation , and affective lability , and one euthymic bipolar patient repeatedly developed depression during such exposure . the only glutamate receptors shown in table 1 are the kainate receptors gluk , among which gluk2 was upregulated and edited in astrocytes , whereas gluk4 is upregulated , but not edited , in neurons following chronic fluoxetine treatment . mrna expression analysis has demonstrated that the human gluk2c splice variant in brain is mainly expressed in nonneuronal cells and barely expressed in neurons . gluk2 can operate not only in an inotropic , but also in a metabotropic mode . three positions can be edited in the genome - encoded gluk2 mrna at 3 sites , the i / v site , the y / c site , and the q / r site . this observation is consistent with another previous observation by the barbon group that editing of the q / r site in intact brain is slightly decreased by chronic fluoxetine treatment . in most cases , inclusion of subunits containing the edited r form of the q / r site lowers ca permeability , and in fluoxetine - treated cultures , a normally occurring increase in free cytosolic ca concentration of 300400% of control value in response to 100  m glutamate was abolished by the fluoxetine treatment . glutamate - mediated inhibition of a slow neuronal afterhyperpolarization current i ( sahp ) is blocked by kainate receptor(s ) in the metabotropic mode , and inhibition of sahp might be secondary to transmitter effects on astrocytes . inhibition of a slow neuronal afterhyperpolarization by glutamate would contribute to the importance of acute effects of glutamate in learning   and could also have a bearing on the mechanism by which gluk2 upregulation and editing may be therapeutically beneficial in major depression . nevertheless , it is often assumed that increase in [ ca]i in astrocytes is mediated by the metabotropic glutamate receptor mglur5 , but this is only in immature brain . moreover , mglur5 is at best minimally upregulated in either astrocytes or neurons after chronic in vivo treatment of adult mice with fluoxetine ( figure 3 ) . mice in which the gluk2 receptor is knocked - out exhibit less anxious or more risk - taking type behavior and less manifestation of despair . obsessive - compulsive disorder is also genetically linked to abnormalities in grik2 , the gene coding for gluk2 [ 69 , 70 ] . the role of glutamate in major depression and its drug treatment has repeatedly been discussed [ 5 , 22 , 71 , 72 ] , and some concepts are continuously changing . reviewed synaptic potentiation by glutamatergic stimulation of astrocytes , a topic which has been further mathematically analyzed by tewari and majumdar . correlations between drug effects on major depression and on glutamatergic activities have attracted special interest in connection with the rapid but short - lasting therapeutic effects of ketamine and riluzole in depressed patients . recently , the effects of riluzole and of ketamine have been reviewed by murrough and colleagues , lapidus et al . mainly discuss neuronal effects , although disregarding the gluk4 receptor ( which according to table 1 becomes upregulated in neurons after chronic fluoxetine treatment ) but mentioning mglurs ( of which we found at least mglur5 to be virtually unaffected by fluoxetine treatment ) . have shown that upregulation of the 5-ht2b receptor itself was specific for this 5-ht2 receptor , since neither 5-ht2a nor 5-ht2c receptors were upregulated . the lack of upregulation of the latter 5-ht2 receptor was confirmed in freshly isolated astrocytes from fluoxetine - treated mice   as shown in table 1 . in contrast to the changes in gene expression of adar , cpla2 and gluk2 described above and those in ca homeostasis to be discussed below , that of the 5-ht2b receptor occurred very slowly ( figures 4(a ) and 4(b ) ) , but as usual with the latency depending upon the fluoxetine concentration . 5-ht2b protein was not upregulated after 3 days , even at the highest fluoxetine concentration , whereas mrna expression occurred somewhat faster ( figures 4(a ) and 4(b ) ) . in contrast editing of the 5-ht2b receptor ( figure 4(c ) ) was obvious after 3 days of treatment , and after 7 days the edited receptor no longer responded to serotonin by an increase in activity measured as the ability of serotonin to evoke release of h - inositol phosphate ( ip ) from labeled ip3 ( figure 4(d ) ) or phosphorylation of erk1/2 ( not shown ) . to ascertain that the inhibition of 5-ht2b receptor activity was a direct result of receptor editing and not due to other effects by chronic fluoxetine administration , cos-7 cells were infected with receptor plasmids of either normal 5-ht2b receptors or receptors with 8 rna sites rna edited , and a similar inhibition was shown ( figure 4(e ) ) . in contrast to the ability of fluoxetine ( and many transmitters ) to acutely cause an increase in [ ca]i , chronic treatment with fluoxetine rapidly abolishes or reduces transmitter and fluoxetine - induced [ ca]i increase . however , a corresponding increase by elevation of extracellular concentrations of k above 15  mm   is not reduced , but rather increased , by chronic treatment with fluoxetine [ 4 , 23 ] . the reason for this is a fluoxetine - mediated upregulation of the l - channel gene cav1.2 , shown both in cultured cells and in astrocytes freshly obtained from fluoxetine - treated animals ( table 1 ) . this may compensate for a downregulation of capacitative ca uptake via store - operated channels , socs , as will be described below . in contrast , the cav1.3 gene , which shows higher expression in freshly isolated astrocytes than in corresponding neurons , is unaffected by treatment of mice with fluoxetine for 2 weeks . socs are very important for regulation of the amount of ca stored in the endoplasmic reticulum ( er ) and thus the amount of ca released by activation of inositol trisphosphate ( ip3 ) receptors ( ip3r ) or ryanodine receptors ( ryr ) ( figure 5 ) . after ip3r- or ryr - mediated unloading of er - bound ca , some ca may enter mitochondria , where it exerts a stimulatory effect on several tricarboxylic acid enzymes , and some ca exits via the na / ca exchanger . accordingly , the cell suffers loss of intracellular ca , which under normal conditions is compensated for by capacitative ca uptake via socs . a major component of socs is the transient receptor potential channel ( trpc ) protein trpc1 . in cells in which trpc1 had been knocked down by treatment with antisense oligonucleotides trpc1 antibody the capacitative ca uptake is also greatly reduced . the same occurs after short - lasting chronic treatment with fluoxetine and many other drugs ( see below ) and reduces or abrogates the ability of transmitters to increase astrocytic [ ca]i . thus , treatment with ssris inhibits the ability of transmitters , at least temporarily ( see below ) , but on account of the upregulation of cav1.2 , not that of elevated k concentrations to increase astrocytic [ ca]i . this is consistent with the increased ability of elevated extracellular k concentration to increase [ ca]i reported in [ 4 , 23 ] , and it is a prerequisite for transmitter effects , for example , on glycogenolysis . an increase in [ ca]i is a prerequisite for stimulation of glycogenolysis , which is consistent with the reduced or abrogated ability of fluoxetine to increase [ ca]i after 1 week of treatment with 10  m fluoxetine . however , after treatment with this concentration for 2 - 3 weeks acute administration of fluoxetine causes a larger increase in glycogenolysis than in control cultures   as shown in figure 6 . this is a very long treatment with a very high concentration , and it can not be excluded that this finding is an artifact . however , another possibility is that it is a consequence of the late upregulation of the 5-ht2b receptor . if this is the case , the inability of transmitters to increase [ ca]i might also be reversed after longer treatment . however , the effects of the gene changes of adar2 , cpla2 , and gluk2 are probably not further altered . akt phosphorylation by serotonin or 10  m fluoxetine is also impaired or abolished after 3 days of treatment with 10  m fluoxetine ( b. li and l. peng 's unpublished experiments ) but may similarly recover with an increased response after 2 weeks . since gsk3 is phosphorylated by akt ( figure 1 ) , this might imply that glycogen synthesis is also increased after treatment with fluoxetine for a sufficiently long period . the importance of gsk3 is shown by observations that reduced immobility in the forced swim test ( a sign a fear reduction ) occur after administration of gsk3 inhibitors to normal animals and in gsk3 haploinsufficient mice and that a selective gsk3 inhibitor can alter serotonin - associated behavioral phenotypes in tests evaluating 5-ht - related antidepressant and anxiolytic drug effects ( reviewed in ) . more recently , liu et al .   found that rats exposed to chronic mild stress showed depression - like behaviors and decreased levels of phosphorylated gsk3 in the hippocampus . chronic citalopram treatment alleviated the depression - like behaviors and reversed the disruptions of the phosphorylated gsk3 in these animals ( figure 7 ) . in contrast , karege et al .   reported decrease in phosphorylated gsk3 protein and a reduced ratio between phosphorylated and total gsk3 in postmortem brain tissue from patients having suffered from major depression . chronic treatment with fluoxetine , a 5-ht2b agonist ( confirmed by diaz et al . ) , exerts a multitude of effects on astrocytes , which in turn modulate astrocyte - neuronal interactions and brain function . these are to a large extent due to an upregulation and editing of genes , with rapid editing but late upregulation of the 5-ht2b receptor gene itself . these alterations induce alteration in effects of cpla2 , gluk2 , and the 5-ht2b receptor , probably including increases in both glucose metabolism and glycogen turnover , which in combination have therapeutic effect on major depression . the fact that gene effects are involved probably contributes to the late manifestation of the therapeutic effect of ssris . moreover , the ability of increased levels of extracellular k to increase [ ca]i is increased as a sign of increased k - induced excitability in astrocytes and therefore probably also of an enhanced ability of elevated k to stimulate glycogenolysis . the three classical drugs that have effect against bipolar disorder , and especially mania , are the lithium ion  lithium  , carbamazepine , and valproic acid . a common feature of these three drugs is that they , like ssris , must be administered for a couple of weeks for the therapeutic action to become manifest . in studies of mechanism(s ) of action for antibipolar drugs , moreover , the mechanisms of drug action may with advantage be elucidated by studying shared effects of all three classical antibipolar drugs . a common effect of chronic treatment with these otherwise very different drugs is a gradual development of intracellular alkalinization in astrocytes , caused by stimulation of acid extruders . lithium   increases intracellular ph ( figure 8(a1 ) ) by directly stimulating the na / h exchanger nhe1 , which leads to a compensatory decrease in its gene expression ( figure 8(a2 ) ) . carbamazepine and valproic acid upregulate the expression and thus the function of the na / bicarbonate cotransporter nbce1 [ 85 , 86 , 89 ] in astrocytes but not in neurons , as seen from figure 8(b1 ) . however , the carbamazepine treatment increases nhe1 expression in neurons , but not in astrocytes figure 8(b2 ) . it is likely that ph becomes increased in extracellular fluid as a result of the increased astrocytic nbce1 function induced by carbamazepine . the neuronal increase in nhe1 expression may therefore be a defense against the development of a resulting neuronal acidosis . the intracellular astrocytic alkalinization may be the cause of an upregulation of cpla2 in astrocytes shown in table 1 , since the activity of this enzyme increases with rising ph . this effect is similar to that seen after chronic fluoxetine administration and may have similar consequences , including stimulation of glucose metabolism . however , cpla2 is downregulated in neurons   and in whole brain   after carbamazepine treatment , which has contributed to the probably exaggerated concept of a role in neuroinflammation in bipolar disorder . some potential effects of this downregulation are discussed by song et al . , and it would be important to know the detailed differences between the consequences of the downregulated gluk2 function and the function of the upregulated , but edited gluk2 observed after chronic treatment with fluoxetine . the most important consequence of the intracellular alkalosis is probably the effect it has on myo - inositol uptake and thereby on phosphatidylinositide signaling . moreover , antibipolar drugs have effects on ion homeostasis : na and k by altering expression of na , k - atpase subtype expression , and ca by downregulating trpc1 , apparently with no concomitant upregulation of cav1.2 , since k effects are also reduced or abrogated as shown below . a classical proposition , the berridge hypothesis for lithium 's therapeutic effects in bipolar disorder is that myo - inositol is depleted by lithium . transmitter - mediated phospholipase c ( plc ) stimulation hydrolyzes phosphatidyl-4,5-bisphosphate ( pip2 ) in the cell membrane . this produces two second messengers , the cytosolic ip3 and the membrane - associated 1,2-diacylglycerol ( dag ) . ip3 triggers ca release from intracellular ca stores via stimulation of the ip3 receptor , and dag activates protein kinase c. subsequently , ip3 is stepwise dephosphorylated , eventually to myo - inositol by inositol phosphatases . initially , dag is converted to phosphatidic acid ( pa ) , an ester between a diglyceride and phosphoric acid , which condenses with cytidine triphosphate to cytidine monophosphoryl - phosphatidate ( cmp - pa ) , which combines with myo - inositol to form pip2 via phosphatidyl-4-monophosphate ( pip ) [ 93 , 94 ] . according to the  berridge hypothesis , inhibition of the regeneration of pip2 by lithium - induced reduction of inositol formation from ip decreases renewed responses to plc - linked receptor agonists . it is consistent with this concept that a normally occurring noradrenaline - induced [ ca]i increase in cultured astrocytes is inhibited by chronic exposure to lithium   ( figure 9 ) . moreover , in nonmedicated bipolar patients a significant increase in local myo - inositol concentration was recently shown by nmr . however , the lithium effect on inositol phosphate hydrolysis to free myo - inositol is not shared by either carbamazepine or valproic acid . the original berridge hypothesis is therefore insufficient to explain effects by anti - bipolar drugs , but a modified and expanded berridge - like hypothesis will be presented as follows . the concentration of inositol in plasma is 3060  m [ 96 , 97 ] , and the intracellular concentration is at a low millimolar level . accordingly , there is a steep gradient between extracellular and intracellular myo - inositol levels , necessitating transport mechanisms for continuous resupply of myo - inositol , since myo - inositol and many of the pip2 metabolites are further degraded . this is partly effectuated by dietary uptake following a slow transfer across the blood - brain barrier   and partly by synthesis in brain , which occurs only in the vasculature . therefore , inhibition of cellular uptake would also deplete myo - inositol in astrocytes and neurons . wolfson et al .   showed that treatment of cultured mouse astrocytes with 1  mm licl for 8 days reduced the cellular content of myo - inositol after exposure to 50  m myo - inositol for 24 hours . lubrich and van calker   expanded this observation by demonstrating that chronic treatment of cultured rat astrocytes with lithium , carbamazepine , or valproic inhibited myo - inositol uptake . wolfson et al .   confirmed this observation in cultured human astrocytoma cells at a myo - inositol concentration of 50  m , but at 25  m myo - inositol , the uptake was stimulated ( figure 10 ) . such an effect can be explained by the combined effects of ( i ) an induced alkalinization , ( ii ) the presence of two different myo - inositol transporters in astrocytes , the high - affinity na - dependent transporter ( smit ) , and the lower - affinity h - dependent ( hmit ) , and ( iii ) stimulation of smit but inhibition of hmit at increased ph [ 103 , 104 ] . we have recently confirmed that uptake of myo - inositol in cultured astrocytes at normal extracellular myo - inositol levels mainly is catalyzed by a lower - affinity , higher - capacity hmit - mediated uptake ( km 143  m and vmax 358  pmol / mg protein ) , whereas a higher - affinity , lower - capacity smit - mediated uptake ( km 16.7  m and vmax 60.2  pmol / mg protein ) plays a minor role . moreover , the uptake of 100  m myo - inositol was inhibited at increased intracellular ph , whereas that at 10  m myo - inositol was enhanced . the observed increase in uptake at 25  m myo - inositol but reduction of uptake at 50  m reported by wolfson et al .   can therefore be explained by changes in relative contribution of the two myo - inositol transporters to total uptake on account of the induced intracellular alkalinization [ 85 , 86 ] . since di daniel et al . reported that hmit in neurons can transport ip3 and gossman and zhao   and wang et al . found in the cochlea that ip3 can be released from nonneuronal cells through hemichannels , it can not be excluded that astrocytically generated ip3 might be transferred to neurons . in that case , a decrease in astrocytic ip3 formation in response to antibipolar drug treatment might also affect neuronal ip3 . effects of transmitters operating via the protein kinase c ( pkc ) and the phosphatidylinositide signaling system might therefore become reduced primarily in astrocytes but possibly also in neurons by antibipolar drug treatment . effects exerted via protein kinase a ( pka ) , like those of adrenaline acting on -adrenergic receptors , may also be affected in astrocytes on account of a gs / gi shift in their pathway . this might include -adrenergic glycogenolysis , which is dependent on the increase in [ ca]i occurring after this shift . since -adrenergic glycogenolysis is a prerequisite for neuronal glutamatergic signaling [ 12 , 110 ] , reduced glycogenolysis might have considerable dampening effect on neuronal excitability . although gsk3 is very often discussed in connection with this disease and its treatment , possible enhancement of glycogen synthesis seems not to have been considered or tested . like fluoxetine chronic treatment with any of the three antibipolar drugs downregulates trpc1 and therefore interferes with ca homeostasis . however , as shown in figure 11 , the ability of elevated extracellular k concentrations to increase [ ca]i is also inhibited . thus , both transmitter - induced and depolarization - mediated changes in astrocytic [ ca]i are reduced . this represents an important difference from astrocytes treated with an ssri , where only the transmitter - mediated responses are inhibited , and the inhibition of the transmitter - induced effect might even be transient . an effect on trpc1 may affect not only ca homeostasis , but also na homeostasis in astrocytes , because the trpc1 channel has equal permeability for ca and na . this may be of major importance in astrocytes , since these nonexcitable cells may become deficient in intracellular na required for na , k - atpase activity . paradoxically , antibody - mediated partial inactivation of trpc1 's ca permeability is accompanied by an increase of the intracellular concentration of na , suggesting that the binding of the antibody to the channel decreases ca flux but increases na flux . findings of decreased na , k - atpase activity in bipolar disorder are robust : ( i ) in brain tissue from bipolar disorder a significantly lower na , k - atpase density was found in tissues from patients having suffered from major depression or schizophrenia ; ( ii ) expression of the 2 gene is decreased in temporal cortex from bipolar disorder patients ; ( iii ) na - k - atpase activity is significantly reduced in erythrocytes from patients with bipolar disorder , and ( iv ) lymphoblastoid cell lines originating from bipolar patients express less na , k - atpase in response to an increase in intracellular na concentration than similar cell lines from unaffected siblings . a possible genomic involvement of na , k - atpase dysfunction in bipolar disorder is also suggested by a genetic association between bipolar disorder and variants of the genes encoding the na , k - atpase subunits 1 , 2 , and 3 . this might have influenced na , k - atpase activity and expression , since deigweiher et al . demonstrated that in cuttlefish gill tissues increases in water co2 tension , which must decrease ph , led to a decline in na , k - atpase activity . previously , cummins and hydn   had shown a ph maximum around 8.0 for atp hydrolysis in microdissected astrocytes . based on all this evidence , we investigated whether chronic treatment with carbamazepine could alter expression of na , k - atpase subtypes and of its auxiliary protein fxyd in neurons and astrocytes of normal mice . mice which coexpress one fluorescent marker with a neuron - specific gene and another marker with an astrocyte - specific gene   were either treated with carbamazepine for 2 weeks or received no treatment but were used as controls . from figure 12 , it can be seen that 3 expression was unchanged and 2 increased in the treated animals both in astrocytes and in neurons , where it is normally not expressed , whereas 1 increased only in neurons . fxyd expression was reduced by the carbamazepine treatment , but only in neurons , and 1 was upregulated in astrocytes , but not in neurons ( results not shown ) . these changes should facilitate k uptake in neurons ( see discussion in ) , without compromising preferential uptake in astrocytes at increased extracellular k concentrations , a process which is important for k homeostasis of the cellular level of the brain . although an overall increase in na , k - atpase expression and presumably activity could be related to ph changes , the induced isoform expressions are not a direct consequence of the intracellular ph changes , since 2 was upregulated in both neurons and astrocytes , and 1 , which shows a slight increase in activity at increased ph , showed increased expression in neurons but not in astrocytes . however , na , k - atpase activity , and thus expression , is regulated by a multitude of different factors , many of which may have been altered in the carbamazepine - treated mice . the intracellular alkalinization induced in astrocytes by the three otherwise very dissimilar drugs lithium , carbamazepine , and valproic acid suggests that this effect is therapeutically important . if anything , this concept is strengthened by the finding that lithium acts on one acid extruder and carbamazepine and valproic acid on a different acid extruder , and by the finding of increased intracellular acidity in the brains of bipolar patients . in contrast to the ability of chronic treatment with antidepressant medication to enhance the increase of [ ca]i and thus probably glycogenolysis by exposure to elevated extracellular k concentrations ( and after sufficiently long treatment perhaps also to transmitters ) , chronic treatment with antibipolar drugs inhibit both k - induced and transmitter - induced increase of astrocytic [ ca]i and thereby probably excitability . the effect on astrocytic cpla2 expression is similar to that after antidepressant treatment and may have similar beneficial effects , but the effect on the neuronal enzyme is the opposite . the expression of gluk2 , which is upregulated ( and edited ) by antidepressant therapy , is downregulated , but the therapeutic consequences have not been determined with certainty . further studies of gluk2 function in brain and drug effects on this kainate receptor under normal and pathological conditions are urgently needed . together with the opposite effect of ssris and antibipolar drugs on the ability of elevated extracellular k concentrations to affect astrocytic [ ca]i , they may constitute important differences in the directions of changes caused by the antidepressant ssris and the mainly antimanic antibipolar drugs . because of a high intracellular cl concentration in astrocytes [ 123 , 124 ] , gabaa receptor - mediated increase of cl fluxes causes a depolarization , resulting in gabaa - induced [ ca]i increases [ 124 , 125 ] and in glycogenolysis ( figure 13 ) . these effects are strikingly similar to those seen after chronic treatment with fluoxetine and are consistent with the observation that gabaa but not gabab receptor stimulation has antianxiety - like effects in rats tested in the elevated - plus - maze . recent publications suggest that the role of gaba effects on astrocytes is currently greatly underestimated [ 124 , 127 ] . this is in spite of astrocytic subunit composition of the gabaa receptor indicative of an anxiolytic role . gabaa receptors are made up of  ,  , and  subunits , each of which can be further subdivided . in the present context , the 1 subunit , which is densely expressed in basal ganglia   but scarcely in cortex , may be of special interest . a comparison between mrna expression levels of different gabaa receptor subunits in freshly isolated astrocytes and neurons   showed that > 90 percent of 1 cortical mrna was astrocytic , and astrocytes also accounted for ~70 percent of 2 expression , ~60% of 1 , and ~40% of 4 ( table 2 ) . since limbic areas are enriched in the 1 subunit , gabaa receptors containing this subtype might serve affective functions , and the 2 subunit is known to mediate anxiolysis . future attempts to develop anxiolytics acting on gabaa receptors   should include potential effects on astrocytes . it was initially believed that all anxiolytic effects of benzodiazepines were exerted on the neuronal  central  benzodiazepine receptor , which facilitated gabaergic transmission by binding to its receptor . since this effect was abrogated by the benzodiazepine antagonist flumazenil , it was often concluded that all benzodiazepine effects that were inhibited by this antagonist were exerted on the  central  benzodiazepine receptor . however , the peripheral - type benzodiazepine receptor localized in mitochondria , the translocator protein ( 18  kda ) , is also important in anxiolysis . it is stimulated not only by exogenously administered benzodiazepines but also by derivatives of the 86-amino acid polypeptide diazepam - binding inhibitor ( dbi ) , which is found in brain . it can be cleaved into several biologically active peptides , including the triakontatetraneuropeptide , ttn , and the octadecaneuropeptide odn . gandolfo et al .   showed ( i ) that binding of the peripheral - type benzodiazepine receptor ligand [ h]ro5 - 4864 to intact astrocytes cultures was displaced by ttn , whereas odn did not compete for [ h]ro5 - 4864 binding and ( ii ) that ttn provoked a concentration - dependent increase in [ ca]i in the cultured astrocytes , which was blocked by chelation of extracellular ca by egta or blockage of ca channels with ni and significantly reduced by the l - type calcium channel blocker nifedipine . patch - clamp studies showed that ttn induced a sustained depolarization , and the authors suggested that ttn acted through the peripheral benzodiazepine binding site and opening of cl channels , that is , a mechanism similar to that of stimulation of gabaa receptors . it has long been known  and virtually ignored  that peripheral benzodiazepine receptors are present not only in mitochondria , but also at the plasma membrane of astrocytes [ 140142 ] . in addition , the peripheral benzodiazepine binding site shows multiplicity and has also been demonstrated on a plasma membrane - enriched preparation from rat astrocytes . suggested the same mechanism for ca entry as gandolfo et al . , that is , a depolarizing effect . , using cultures which had been differentiated by treatment with dibutyryl cyclic amp and therefore express l - channels , suggested a direct interaction between the benzodiazepine and this channel . it seems consistent with this interpretation that midazolam had no significant effect at nonelevated extracellular k concentrations in the experiments by zhao et al . . more conclusive evidence has been reached in cardiac cells , where direct activation of the channel by the ca channel activator bay k 8644 was blocked by the benzodiazepine antagonist pk11195 [ 144 , 145 ] . nevertheless , the most important finding , an increase in astrocytic [ ca]i , is identical in all these studies . as shown in figures 14(a ) and 14(b ) , the benzodiazepine - mediated augmentation of the response to elevated k concentrations is selectively prevented in the presence of 1  m of the  peripheral - type  benzodiazepine antagonist pk 11195 and notably also by the supposedly  central - type  antagonist flumazenil [ 128 , 142 , 146 ] . the inhibition by flumazenil confirms previous observation by backus et al .   and emphasizes again that inhibition of a benzodiazepine response by flumazenil does not necessarily indicate that the response is exerted on neuronal receptors . the dihydropyridine l - channel blocker nimodipine abolishes both the effect of elevated k and that of the benzodiazepine , although it should have no effect on the latter if only an l - channel - independent depolarization was involved . figure 15 shows that midazolam also increases the glycogenolytic effect of elevated extracellular k concentrations . the high potency of benzodiazepines in increasing l - channel opening in astrocytes ( 500  nm diazepam and 10  nm midazolam in figure 14 ) is an indication that the response may be relevant for clinically observed benzodiazepine effects , because the effective concentrations are similar to those obtained clinically [ 147 , 148 ] . in contrast , stimulation of gaba - mediated cl influx in cortical neurons by 1001000  nm diazepam causes only a 540% increase . peripheral - type receptors are also present on several additional , nonneural cell types as discussed by gandolfo et al . , and mature erythrocytes , which have no mitochondria , express peripheral - type benzodiazepine receptors . these sites should not be identified as translocator proteins and have been referred to as the  joker  receptor by hertz et al .   and yet another indication that drugs increasing [ ca]i and causing glycogenolysis can provide acute anxiolytic effects is that acute treatment with the 5-ht2b receptor agonist , bw 723c86 , has been found to exert anxiolytic effects in animal experiments [ 151153 ] . this is the same receptor as that activated by fluoxetine and other ssris , and acute administration of fluoxetine increases [ ca]i and causes glycogenolysis . why some degree of anxiolysis can be obtained by acute 5-ht2b activation , but the antidepressant effects require weeks of treatment and gene editing and up - regulation , remains to be shown . acute anxiolytic drug therapy induces ca - dependent anxiolysis and enhances glycogenolysis evoked by exposure to elevated extracellular k concentrations , an effect which is strikingly similar to that of chronic treatment with an ssri , which also has anxiolytic effect . the response to gabaa receptor stimulation is secondary to depolarization and that to benzodiazepines to a direct effect on the l - channel . one might wonder about the importance of l - channel opening , since it , in experiments where [ k]e is the only factor changed , requires [ k]e values above those usually seen in response to normal stimulation . however , it should be remembered that the [ k]e is probably normally not changed alone but that the change might be accompanied by changes in transmitters , like gaba , that also contribute to the depolarization , as seen by the tendency to a more pronounced glycogenolysis in figure 13 . accordingly , activation of ca uptake by elevated [ k]e may occur much more frequently than should be anticipated based on the experiments in which [ k]e alone is increased . this may also be of importance for the oppositely directed changes in effects of increase in [ k]e after treatment with ssris and with antibipolar drugs . astrocytic increase in [ ca]i and in glycogenolysis after chronic treatment with ssris ( which also have antianxiolytic effects ) and during acute gabaa stimulation or administration of benzodiazepines is probably important for antidepressant - anxiolytic drug effects , and astrocytic glycogenolysis is essential to support astrocytic signaling . the glycogenolytic effect is unlikely to be shared by antibipolar drugs , which all cause intracellular alkalinization in astrocytes , although by stimulation of different acid extruders . these drugs inhibit effects of both elevated k concentrations and transmitters on astrocytic [ ca]i . carbamazepine was also shown to have important effects on na , k - atpase in both astrocytes and neurons . both antidepressant drugs and antibipolar drugs affect gluk2 , but the effects are different , and further studies of this kainate receptor are warranted in both major depression and bipolar disease .
chronic treatment with fluoxetine or other so - called serotonin - specific reuptake inhibitor antidepressants ( ssris ) or with a lithium salt  lithium  , carbamazepine , or valproic 
 acid , the three classical antibipolar drugs , exerts a multitude of effects on astrocytes , which in turn modulate astrocyte - neuronal interactions and brain function . in the case of the ssris 
 , they are to a large extent due to 5-ht2b - mediated upregulation and editing of genes . these alterations induce alteration in effects of cpla2 , gluk2 , and the 5-ht2b receptor , probably including increases in both glucose metabolism and glycogen turnover , which in combination have therapeutic effect on major depression . 
 the ability of increased levels of extracellular k+ to increase [ ca2+]i is increased as a sign of increased k+-induced excitability in astrocytes . 
 acute anxiolytic drug treatment with benzodiazepines or gabaa receptor stimulation has similar glycogenolysis - enhancing effects . 
 the antibipolar drugs induce intracellular alkalinization in astrocytes with lithium acting on one acid extruder and carbamazepine and valproic acid on a different acid extruder . 
 they inhibit k+-induced and transmitter - induced increase of astrocytic [ ca2+]i and thereby probably excitability . 
 in several cases , they exert different changes in gene expression than ssris , determined both in cultured astrocytes and in freshly isolated astrocytes from drug - treated animals .