review article signal transduction in astrocytes during...

Review ArticleSignal Transduction in Astrocytes during Chronic or AcuteTreatment with Drugs (SSRIs Antibipolar Drugs GABA-ergicDrugs and Benzodiazepines) Ameliorating Mood Disorders

Leif Hertz Dan Song Baoman Li Ting Du Junnan Xu Li Gu Ye Chen and Liang Peng

Department of Clinical Pharmacology China Medical University No 92 Beier Road Heping District Shenyang China

Correspondence should be addressed to Liang Peng hkkid08yahoocom

Received 30 October 2013 Accepted 16 December 2013 Published 24 February 2014

Academic Editor Joseph I Shapiro

Copyright copy 2014 Leif Hertz et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chronic treatment with fluoxetine or other so-called serotonin-specific reuptake inhibitor antidepressants (SSRIs) or with a lithiumsalt ldquolithiumrdquo carbamazepine or valproic acid the three classical antibipolar drugs exerts amultitude of effects on astrocytes whichin turn modulate astrocyte-neuronal interactions and brain function In the case of the SSRIs they are to a large extent due to5-HT

2B-mediated upregulation and editing of genesThese alterations induce alteration in effects of cPLA2 GluK2 and the 5-HT

2Breceptor probably including increases in both glucose metabolism and glycogen turnover which in combination have therapeuticeffect onmajor depressionThe ability of increased levels of extracellular K+ to increase [Ca2+]i is increased as a sign of increasedK

+-induced excitability in astrocytes Acute anxiolytic drug treatmentwith benzodiazepines orGABAA receptor stimulation has similarglycogenolysis-enhancing effects The antibipolar drugs induce intracellular alkalinization in astrocytes with lithium acting on oneacid extruder and carbamazepine and valproic acid on a different acid extruder They inhibit K+-induced and transmitter-inducedincrease of astrocytic [Ca2+]i and thereby probably excitability In several cases they exert different changes in gene expression thanSSRIs determined both in cultured astrocytes and in freshly isolated astrocytes from drug-treated animals

1 Introduction

Signal transduction in astrocytes is of fundamental butlargely unrecognized importance for brain function undernormal and abnormal conditions Due to the extreme diffi-culty in studying specifically astrocytic signaling in the brainin vivo many studies have been carried out in isolated prepa-rations of astrocytes that is freshly isolated cells obtaineddirectly from the brain or cultured astrocytes Claims bythe Kimelberg that astrocyte cultures are misleading [1] areunfortunately often correct However these researchers haveunjustifiably ignored that many types of astrocyte culturesexist and that they are not similar in their characteristicsThefact that the cultures used by us are well suited to study drug-induced signaling changes is demonstrated in Table 1 (slightlymodified from [2]) It shows identical changes in gene expres-sion and editing induced by chronic treatment with drugsused to treat mood disorders (fluoxetine carbamazepine)in cultured astrocytes and in astrocytes freshly dissociatedfrom the brains of animals chronically treated with the same

drugs [3] Not a single gene was affected differently in thetwo situations The freshly dissociated cells are probably notsufficiently intact to show the mechanisms involved in thesegene changes or their functional consequences which forthis reason have been elucidated in the cultured cells Suchstudies have indicated important correlations between dif-ferent effects on gene expression or editing shown in Table 1and they have enabled tentative functional interpretations

2 Effects of SSRIs

21 Acute Effects

211 Pathway The prototypes of antidepressant drugs(which also have anxiolytic effect) are the serotonin-specificreuptake inhibitors (SSRIs) When fluoxetine the first ofthe presently used SSRIs was approved for clinical use in1987 two effects of the drug had been established inhibitionof serotonin reuptake by the serotonin transporter (SERT)[6] and partial displacement of serotonin (5-HT) binding

Hindawi Publishing CorporationJournal of Signal TransductionVolume 2014 Article ID 593934 21 pageshttpdxdoiorg1011552014593934

2 Journal of Signal Transduction

Table 1 Comparison between effects on gene expression and editing of chronic treatment with the SSRI fluoxetine or the antibipolar drugcarbamazepine in cultured mouse astrocytes and in astrocytes freshly isolated from drug-treated mice

Gene Drug FACS astrocytes Culture astrocytesADAR2 Fluoxetine Up Up5-HT2B receptor expression Fluoxetine Up Up5-HT2B editing Fluoxetine Up Up5-HT2c receptor expression Fluoxetine Unchanged UnchangedcPLA2a Fluoxetine Up UpsPLA2 Fluoxetine Unaltered UnalteredGluK2 expression Fluoxetine Up UpGluK2 editing Fluoxetine Up UpGluK4 expression Fluoxetine Unchanged Unchangedcfos expression Fluoxetine Up UpfosB expression Fluoxetine Up UpCav 12 Fluoxetine Up UpNBCe1 Carbamazepine Up UpGluK2 Carbamazepine Down DowncPLA2 Carbamazepine Up UpTable 1 shows all experiments in which drug effects were compared in cultured astrocytes and in astrocytes freshly obtained by FACS as described by Lovattet al [3] For FACS astrocytes had been obtained from mice stained with GFP based on expression of the astrocyte-specific GFAP in transgenic animalsand the stained cells were separated after cell dissociation by means of their fluorescent signal The cultures were treated chronically with either fluoxetine orcarbamazepine and the animals had been treated chronically for a similar length of time (2 weeks) Complete agreement was found From Peng et al [2] withthe exception of Cav12 which is from Du et al [4] using similar techniques

to cultured astrocytes [7] which have no SERT expression[8] However at that time astrocytes were supposed to beunimportant for brain function and inhibition of SERT hassince then been regarded as the mechanism responsible forSSRIs effects In 1987 the 5-HT

2B receptor was unknown butit is now established to have the highest affinity between 5-HTreceptors for SSRIs with a K

119894for displacement of serotonin

binding to cultured astrocytes of 70 nM [5] This is identicalto its K

119894for inhibition of serotonin uptake via the human

placental SERT [9] Moreover all five SSRIs are equipotentin their effect on astrocytes [10] This is a distinct differencefrom the large potency difference in their effect on SERTalthough the therapeutic doses are of roughly comparablemagnitudes a difference which can only partly be explainedby differences in drug kinetics and protein binding

The metabolic pathway activated in cultured astrocytesby fluoxetine was first established by Li et al [11] and anexpanded version is shown in Figure 1 With increasingrealization of the importance of glycogenolysis for signalingin astrocytes [12ndash14] it may be important that fluoxetineacutely stimulates glycogenolysis an effect that is secondaryto an increase in [Ca2+]

119894[15] Fluoxetine might also affect

glycogen synthesis since it stimulates the AKT pathway(see below) The involvement of 5-HT

2B receptor-stimulatedglycogenolysis has been established during learning whereacute administration of serotonin can rescue long-termlearning in a one trial aversive learning paradigm in day-oldchickens under conditions when the aversive stimulus wasotherwise too weak to establish more than transient long-term memory retention [16] Fluoxetine and paroxetine havea similar effect and are equipotent showing that the rescuewas not due to inhibition of SERT and the rescuing effect was

inhibited by an inhibitor of glycogenolysis (Gibbs and Hertzsubmitted Frontiers in Pharmacology (Neuropharmacology)after invitation to Research Topic)

In accordance with Figure 1 fluoxetine acutely phos-phorylates AKT in cultured astrocytes [5] AKT-mediatedphosphorylation of glycogen synthase kinase-3120573 (GSK-3120573)inhibits its enzymatic activity AKT phosphorylation maystimulate glycogen synthesis since phosphorylation andactivation of glycogen synthase by GSK-3 decreases theactivity of glycogen synthase [17] It is consistent with theseobservations that Li et al [18] from the Jope group foundin whole brain that fluoxetine administration increases thelevels of phosphorylated GSK3120573 Later Beurel et al [19]from the same group confirmed that fluoxetine rapidly androbustly increases serine phosphorylation of GSK3 but thatthese responses in young mice are blunted or absent Thisis consistent with an effect on astrocytes since these glialcells are generated postnatally [20] An increased glycogensynthesis following 5-HT

2B receptor activation has beendirectly shown in hepatocytes whereas agonists of 5-HT

1and

5-HT2A receptors had the opposite effect [21]

22 Chronic Effects

221 Affected Genes The effects of SSRIs that are importantin connection with major depression are the chronic effectssince the effect of clinical treatment takes several weeks toappear To-date all gene effects caused by chronic treatmentwith fluoxetine (the only SSRI studied on astrocytes in vivo)after mice had been treated for 14 days with ip injection offluoxetine (10mgkg per day) have been identical to thosein cultured astrocytes chronically treated with fluoxetine or

Journal of Signal Transduction 3

5 2B

U

3120573

2 gt 3

Fluoxetine

siRNA

SB204741

BAPTAAM

AG1478

LY294002

GM6001

GF 109203X

MMP

MEK Raf Ras

Gene expression

ERK

HB-EGF

EGFR

pEGFR

AKT

PKC

PI3K

Ca2+

Figure 1 Schematic illustration of pathways leading to stimulation of ERK and AKT phosphorylation by fluoxetine in astrocytes Fluoxetinebinds to 5-HT

2B receptors The activation of the receptors in turn induces an enhancement of protein kinase C (PKC) activity and ofintracellular Ca2+ concentration by Ca2+ release from intracellular stores The latter activates Zn-dependent metalloproteinases (MMPs) andleads to shedding of growth factor(s)The released epidermal growth factor receptor (EGFR) ligand stimulates phosphorylation of the EGFRThe downstream target of EGFR extracellular-regulated kinase (ERK) (shown in blue) is phosphorylated via the RasRafMEK pathway andAKT is phosphorylated via PI3K pathway During chronic fluoxetine administration inhibitors (shown in yellow) of the 5-HT

2B receptor(SB204741) or siRNA against this receptor of PKC (GF 109293X) of intracellular Ca2+ homeostasis (BAPTAAM an intracellular Ca2+chelator) of Zn-dependent metalloproteinases (GM6001) of the receptor-tyrosine kinase of the EGFR (AG1478) of ERK phosphorylation(U0126 amitogen-activated kinase (MEK) inhibitor) or of theAKTpathway (LY294002 a PI3K inhibitor) prevent changes in gene expressionand editing PIK3 catalyzes the formation of PIP

3from PIP

2 from Hertz et al [5]

other SSRIs as was shown in Table 1 They have also beensimilar to those found by other authors or ourselves in totalbrain with the exception of gene expression of sPLA

2and

GluK4 genes that were upregulated in neurons of the treatedanimals and accordingly also inwhole brain [22] Anhedoniaone of the components of major depression caused oppo-sitely directed changes in expression of some of the samegenes [22] Other gene expression changes established in thecultured cells did not occur but it should be rememberedthat anhedonia is only one component of major depressionFluoxetine can reverse both the anhedonia and the geneexpression changes (B Li and L Pengrsquos unpublished results)

222 ADAR2 The upregulation of ADAR2 by chronic treat-ment with fluoxetine (Table 1) depends on 5-HT

2B receptorstimulation since the 150ndash200 increase of mRNA and pro-tein expression of ADAR2was prevented in astrocytes treated

with 5-HT2B receptor siRNA [23] In contrast to the upregula-

tion of cPLA2(see below) it is not known if additional steps of

the fluoxetine-mediated pathways are also required ADARsconstitute a family of adenosine deaminases catalyzing deam-ination of adenosine to inosine in double-stranded regions ofmRNAs thus changing the translated protein sequence sinceinosine is read by the cells as guanosine [24] There are threemembers in the ADAR family ADAR1 ADAR2 and ADAR3[25] All three types of ADAR are expressed in the brain [26]ADAR2 upregulation in fluoxetine-treated mice was specificfor this subtype observed only in astrocytes and occurredwithin 3 days [23] In the brain ADAR2 has been shown inhippocampal pyramidal neurons and cerebellar Purkinje cellsand Bergmann glial cells with less expression of ADAR1 andADAR3 [27] ADAR2 and probably also its upregulation areessential for the editing changes shown in Table 1 this hasbeen directly demonstrated in cultured astrocytes by the useof cells treated with siRNA against ADAR2 [23]


223 cPLA2 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 thisphospholipase [29ndash31] Its activation specifically releasesarachidonic acid from the sn-2 position of membrane-bound phospholipid substrate in neural preparations [32ndash35] including glioma cells [36] Arachidonic acid stronglystimulates glucose metabolism in cultured astrocytes [37]So does treatment with 10 120583M fluoxetine for 24 h whichmight have sufficed to induce an increase in cPLA

2[38]

whereas acute exposure of astrocyte cultures to the sameconcentration of fluoxetine has no corresponding effect (LPeng and L Hertzrsquos unpublished experiments) Arachidonicacid also stimulates glycogenolysis [39 40]

Rapoport and coworkers [41ndash43] showed that chronicadministration of fluoxetine leads to stimulation andenhanced mRNA and protein expression in rat brain ofcPLA2 but not of the two other phospholipases A

2(secretory

PLA2(sPLA

2) and intracellular PLA

2(iPLA

2)) Li et al [28]

confirmed a slow and selective upregulation of mRNA andprotein expression of cPLA

2a the major isoform of cPLA2

in mouse astrocytes in primary cultures during chronicincubation with 1 or 10120583M fluoxetine The upregulation wasabrogated by the 5-HT

2B receptor antagonist SB 204741the metalloproteinase inhibitor GM6001 and the inhibitorof EGF receptor tyrosine phosphorylation AG1478 and byU0126 the inhibitor of ERK

12phosphorylation These are

all inhibitors of the signaling pathway that was shown inFigure 1 for fluoxetine showing that upregulation of mRNAand protein of cPLA

2were inhibited by the same drugs that

acutely inhibit ERK12

phosphorylation and by inhibitionof the phosphorylation itself As was shown in Table 1upregulation specifically of cPLA

2a has also been found infreshly dissociated astrocytes isolated by FACS after 2-weektreatment of rats with fluoxetine whereas no correspondingeffect was found in neurons [22] This finding stronglysuggests that the enhanced cPLA

2activity demonstrated

in whole brain after chronic fluoxetine treatment [42]selectively occurs in astrocytes

The stimulation of glucose metabolism may be impor-tant in the pathophysiology of depressive illness and itspharmacological treatment Glucose metabolism in brain isreduced in many regions primarily in the frontotemporalparts in patients suffering from unipolar depression [44ndash47]with a correlation between the degree of hypometabolismand severity of the illness [48] and normalization follow-ing treatment with an SSRI [49ndash51] Figure 2 shows thatarachidonic acid may play a role in determining rates ofcerebral glucose metabolism as seen from a rectilinear cor-relation in depressed patients between plasma concentrationof arachidonic acid and rate of cerebral glucose utilization inone of the regions affected metabolically by depression [52]Moreover genetic associations are found between cPLA

2

and major depression [53 54] Depending on the site(s)of impairment of glucose metabolism glycogen metabolismcould also be affected However the effect of arachidonicacid is not necessarily exerted on glucose metabolism itselfbut might also be exerted on energy-requiring reactionsThe importance of mitochondrial abnormalities in major

5 7 9 11 13 15 17Arachidonic acid ()

0

2

4

6

8

rCM

Rglu

minus6

minus4

minus2

Figure 2 Correlation between cerebral metabolic rates of glucosemetabolism and plasma arachidonic acid levels Cerebral metabolicrates of glucose metabolism (rCMRglu) measured by NMR in vivoare shown to be correlated with plasma arachidonic acid expressedas a percentage of total phospholipid polyunsaturated fatty acidsThe correlation is statistically significant (119875 lt 00005) fromElizabeth Sublette et al [52]

depression is shown by a significantly reduced number ofmitochondria but larger mean mitochondrial volume inhippocampus in rats sensitive to stress-induced depressionthan in control rats [55] Following treatment with the antide-pressant imipramine a significant increase in the number ofmitochondria occurred in the stress-sensitive group Clinicalstudies also suggest that psychiatric features can be promi-nent features of mitochondrial disorders but additionalmethodologically rigorous and adequately powered studiesare needed before definitive conclusions can be drawn [56]

Arachidonic acid metabolites including prostaglandinsmay also exert other beneficial effects important for the amel-ioration of depression [57] Prostaglandin synthesis inhibi-tors in doses used to treat pain [58 59] may cause fear agita-tion and affective lability and one euthymic bipolar patientrepeatedly developed depression during such exposure [60]Thus the prostaglandins are likely to exert oppositely directedeffects

224 Kainate Receptors Theonly glutamate receptors showninTable 1 are thekainate receptorsGluK amongwhich GluK2was upregulated and edited in astrocytes whereas GluK4 isupregulated but not edited in neurons following chronicfluoxetine treatment [22] mRNA expression analysis hasdemonstrated that the human GluK2c splice variant in brainismainly expressed in nonneuronal cells and barely expressedin neurons [61] GluK2 can operate not only in an inotropicbut also in a metabotropic mode [62] Three positions canbe edited in the genome-encoded GluK2 mRNA at 3 sitesthe IV site the YC site and the QR site The editing wasincreased at all three sites by chronic treatment with fluoxe-tine [22]This observation is consistent with another previousobservation by the Barbon group that editing of the QR


site in intact brain is slightly decreased by chronic fluoxetinetreatment [63] In most cases inclusion of subunits contain-ing the edited R formof theQR site lowers Ca2+ permeability[64] and in fluoxetine-treated cultures a normally occurringincrease in free cytosolic Ca2+ concentration of 300ndash400 ofcontrol value in response to 100 120583M glutamate was abolishedby the fluoxetine treatment [23] This increase is evoked bythe GluK2 receptor operating in its metabotropic manner[5] Glutamate-mediated inhibition of a slow neuronal after-hyperpolarization current I (sAHP) is blocked by kainatereceptor(s) in the metabotropic mode [65] and inhibition ofsAHPmight be secondary to transmitter effects on astrocytes[66] Inhibition of a slow neuronal afterhyperpolarizationby glutamate would contribute to the importance of acuteeffects of glutamate in learning [16] and could also have abearing on themechanism by which GluK2 upregulation andeditingmay be therapeutically beneficial inmajor depressionNevertheless it is often assumed that increase in [Ca2+]

119894

in astrocytes is mediated by the metabotropic glutamatereceptor mGluR5 but this is only in immature brain [67]Moreover mGluR5 is at best minimally upregulated in eitherastrocytes or neurons after chronic in vivo treatment of adultmice with fluoxetine (Figure 3) Mice in which the GluK2receptor is knocked-out exhibit less anxious or more risk-taking type behavior and less manifestation of despair [68]Obsessive-compulsive disorder is also genetically linked toabnormalities in Grik2 the gene coding for GluK2 [69 70]

The role of glutamate in major depression and its drugtreatment has repeatedly been discussed [5 22 71 72] andsome concepts are continuously changing Hertz et al [5]reviewed synaptic potentiation by glutamatergic stimulationof astrocytes a topic which has been further mathematicallyanalyzed byTewari andMajumdar [73] Correlations betweendrug effects on major depression and on glutamatergicactivities have attracted special interest in connection withthe rapid but short-lasting therapeutic effects of ketamineand riluzole in depressed patients Recently the effects ofriluzole and of ketamine have been reviewed by Murroughand colleagues Lapidus et al [74] mainly discuss neuronaleffects although disregarding the GluK4 receptor (whichaccording to Table 1 becomes upregulated in neurons afterchronic fluoxetine treatment) but mentioning mGluRs (ofwhich we found at least mGluR5 to be virtually unaffectedby fluoxetine treatment) Murrough et al [75] note veryinteresting correlations between ketamine major depressionand cognitive function

225 The 5-HT2B Receptor Unpublished experiments incultured astrocytes by Li et al have shown that upregulationof the 5-HT

2B receptor itself was specific for this 5-HT2

receptor since neither 5-HT2A nor 5-HT

2c receptors wereupregulated The lack of upregulation of the latter 5-HT

2

receptor was confirmed in freshly isolated astrocytes fromfluoxetine-treated mice [22] as shown in Table 1 In contrastto the changes in gene expression of ADAR cPLA

2and

GluK2 described above and those in Ca2+ homeostasis to bediscussed below that of the 5-HT

2B receptor occurred veryslowly (Figures 4(a) and 4(b)) but as usual with the latency

depending upon the fluoxetine concentration 5-HT2B pro-

tein was not upregulated after 3 days even at the highest flu-oxetine concentration whereas mRNA expression occurredsomewhat faster (Figures 4(a) and 4(b)) In contrast editingof the 5-HT

2B receptor (Figure 4(c)) was obvious after 3days of treatment and after 7 days the edited receptor nolonger responded to serotonin by an increase in activitymeasured as the ability of serotonin to evoke release of3H-inositol phosphate (IP) from labeled IP

3(Figure 4(d))

or phosphorylation of ERK12

(not shown) To ascertainthat the inhibition of 5-HT

2B receptor activity was a directresult of receptor editing and not due to other effects bychronic fluoxetine administration COS-7 cells were infectedwith receptor plasmids of either normal 5-HT

2B receptorsor receptors with 8 RNA sites RNA edited and a similarinhibition was shown (Figure 4(e))

226 Ca2+ Homeostasis In contrast to the ability of fluox-etine (and many transmitters) to acutely cause an increasein [Ca2+]

119894 chronic treatment with fluoxetine rapidly abol-

ishes or reduces transmitter and fluoxetine-induced [Ca2+]119894

increase [23] However a corresponding increase by elevationof extracellular concentrations of K+ above 15mM [76] isnot reduced but rather increased by chronic treatment withfluoxetine [4 23]The reason for this is a fluoxetine-mediatedupregulation of the L-channel geneCav12 shownboth in cul-tured cells and in astrocytes freshly obtained from fluoxetine-treated animals (Table 1) This may compensate for a down-regulation of capacitative Ca2+ uptake via store-operatedchannels Socs [23] as will be described below In contrastthe Cav13 gene which shows higher expression in freshly iso-lated astrocytes than in corresponding neurons is unaffectedby treatment of mice with fluoxetine for 2 weeks [4]

Socs are very important for regulation of the amountof Ca2+ stored in the endoplasmic reticulum (ER) andthus the amount of Ca2+ released by activation of inositoltrisphosphate (IP

3) receptors (IP

3R) or ryanodine receptors

(RyR) (Figure 5) After IP3R- or RyR-mediated unloading of

ER-bound Ca2+ some Ca2+ may enter mitochondria [77]where it exerts a stimulatory effect on several tricarboxylicacid enzymes [78] and some Ca2+ exits via the Na+Ca2+exchanger Accordingly the cell suffers loss of intracellularCa2+ which under normal conditions is compensated for bycapacitativeCa2+ uptake via Socs Amajor component of Socsis the transient receptor potential channel (TRPC) proteinTRPC1 [79] In cells in which TRPC1 had been knockeddown by treatment with antisense oligonucleotides TRPC1antibody the capacitative Ca2+ uptake is also greatly reducedThe same occurs after short-lasting chronic treatment withfluoxetine and many other drugs (see below) and reducesor abrogates the ability of transmitters to increase astrocytic[Ca2+]

119894[80]Thus treatment with SSRIs inhibits the ability of

transmitters at least temporarily (see below) but on accountof the upregulation of Cav12 not that of elevated K

+ concen-trations to increase astrocytic [Ca2+]

119894 This is consistent with

the increased ability of elevated extracellular K+ concentra-tion to increase [Ca2+]

119894reported in [4 23] and it is a prereq-

uisite for transmitter effects for example on glycogenolysis


Control Fluoxetine

Control

Control Fluoxetine

CMS

Neu

ron

(YFP

H)

mGluR5

TBP

Astr

ocyt

es

(GFP

) mGluR5

TBP

(a)

0

05

1

15

Control Fluoxetine Control FluoxetineControl CMS

Neuron (YFPH)GFAP (GFP)

Ratio

of m

Glu

R5T

BP

(b)

Figure 3 mRNA of mGluR5 is not upregulated in either astrocytes or neurons freshly isolated from animals treated with fluoxetine (10mgfluoxetine hydrochloridekg ip) for 14 days In both cases astrocytes had been stained with GFP and neurons with YPHF in transgenicanimals and they were separated after cell dissociation bymeans of the different fluorescent signals (a) Blot showingmGluR5 RNAmeasuredby reverse transcription polymerase chain reaction (RT-PCR) in all experiments together with that of TATA-binding protein (TBP) used ashousekeeping gene (as a further check for application of similar amounts of total mRNA) mGluR5 PCR product is 513 bp Primer sequencefor mGlur5 is FWD 51015840GTCTCCTGATGTCAAGTGGTT31015840 REV 51015840GGACCACACTTCATCATCATC31015840 (b) mGlu5RTBP expression ratiois virtually unaffected by fluoxetine treatment regardless ofwhether normalmice (left part of (b)) ormice that showed some signs of depressionafter exposure to chronicmild stress (CMS) (right part of (b)) were studiedUnpublished experiments by B Li and L Peng usingmethodologysimilar to that used by Li et al [22]

227 Glycogenolysis and Glycogen Synthesis An increase in[Ca2+]

119894is a prerequisite for stimulation of glycogenolysis

which is consistent with the reduced or abrogated ability offluoxetine to increase [Ca2+]

119894after 1 week of treatment with

10 120583M fluoxetine However after treatment with this con-centration for 2-3 weeks acute administration of fluoxetinecauses a larger increase in glycogenolysis than in controlcultures [8] as shown in Figure 6This is a very long treatmentwith a very high concentration and it cannot be excludedthat this finding is an artifact However another possibility isthat it is a consequence of the late upregulation of the 5-HT

2Breceptor If this is the case the inability of transmitters toincrease [Ca2+]

119894might also be reversed after longer treatment

However the effects of the gene changes of ADAR2 cPLA2

and GluK2 are probably not further alteredAKT phosphorylation by serotonin or 10 120583M fluoxetine

is also impaired or abolished after 3 days of treatmentwith 10 120583M fluoxetine (B Li and L Pengrsquos unpublishedexperiments) but may similarly recover with an increasedresponse after 2 weeks [5] Since GSK3120573 is phosphorylatedby AKT (Figure 1) this might imply that glycogen synthesisis also increased after treatment with fluoxetine for a suffi-ciently long period The importance of GSK3120573 is shown byobservations that reduced immobility in the forced swim test(a sign a fear reduction) occur after administration of GSK3inhibitors to normal animals and in GSK3120573 haploinsufficientmice and that a selective GSK3120573 inhibitor can alter serotonin-associated behavioral phenotypes in tests evaluating 5-HT-related antidepressant and anxiolytic drug effects (reviewedin [81]) More recently Liu et al [82] found that rats exposedto chronic mild stress showed depression-like behaviors and

decreased levels of phosphorylated GSK3120573 in the hippocam-pus Chronic citalopram treatment alleviated the depression-like behaviors and reversed the disruptions of the phos-phorylated GSK3120573 in these animals (Figure 7) In contrastKarege et al [83] reported decrease in phosphorylatedGSK3120573protein and a reduced ratio between phosphorylated andtotal GSK3120573 in postmortem brain tissue from patients havingsuffered from major depression

228 Conclusion Chronic treatment with fluoxetine a5-HT2B agonist (confirmed by Diaz et al [84]) exerts a

multitude of effects on astrocytes which in turn modulateastrocyte-neuronal interactions and brain function Theseare to a large extent due to an upregulation and editing ofgenes with rapid editing but late upregulation of the 5-HT

2Breceptor gene itself These alterations induce alteration ineffects of cPLA

2 GluK2 and the 5-HT

2B receptor probablyincluding increases in both glucosemetabolism and glycogenturnover which in combination have therapeutic effect onmajor depression The fact that gene effects are involvedprobably contributes to the late manifestation of the ther-apeutic effect of SSRIs Moreover the ability of increasedlevels of extracellular K+ to increase [Ca2+]

119894is increased as

a sign of increased K+-induced excitability in astrocytes andtherefore probably also of an enhanced ability of elevated K+to stimulate glycogenolysis

3 Effects of Antibipolar Drugs

31 Effects Shared by Different Antibipolar Drugs The threeclassical drugs that have effect against bipolar disorder and


0

1

2

3

4

3 days 1 week 2 weeks

52B lowast

lowast

lowast

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(a)

0

1

2


52B

120573

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(b)

0

05

1

15

793 796 797 798 799 800 804 805RNA editing sites

Control con Control fluADAR2 siRNA(+) con ADAR2 siRNA(+) flu

Ratio

ofA

lowast lowast lowast lowast lowast lowast lowast lowast

G+

A

(c)

0

500

1000

1500

Control Fluoxetine

[3H] inositol phosphate production from [3H]IP3during 35min (cpmculture)

Control 5-HT 1nM5-HT 100nM 5-HT 10120583M

lowast

lowastlowast

lowast

(d)

Control 5-HT Control 5-HT0

2000

4000

6000

8000

10000

5-HT2BR 5-HT2BR edited


lowast

(e)

Figure 4 ((a) (b)) Time course for upregulation of 5-HT2B receptormRNA (a) and protein (b) during treatment of culturedmouse astrocytes

with different concentrations of fluoxetine (c) Editing of 5-HT2B receptor after 3 days of treatment with 10120583Mfluoxetine ((d) (e)) Reduction

of effect of 5-HT2B receptor stimulation after downregulation of cultured astrocytes and transfected COS-7 cells with 10120583M fluoxetine for 7

days Unpublished experiments by B Li and L Peng Methodologies for (a)ndash(c) were as in Li et al [22] Response of the receptor to serotoninwas measured as increase in the ability of serotonin to evoke release of 3H-inositol phosphate (IP) from labeled IP

3in cultured astrocytes (d)

and in cos-7 cells infected with receptor plasmids of either normal 5-HT2B receptors or receptors with 8 RNA sites RNA (e) Unpublished

experiments by B Li and L Peng

especially mania are the lithium ion ldquolithiumrdquo carbamaze-pine and valproic acid A common feature of these threedrugs is that they like SSRIs must be administered for acouple of weeks for the therapeutic action to become mani-fest In studies of mechanism(s) of action for antibipolardrugs it is therefore again important to determine effectsthat only appear after chronic administration Moreover themechanisms of drug actionmaywith advantage be elucidated

by studying shared effects of all three classical antibipolardrugs

A common effect of chronic treatment with these other-wise very different drugs is a gradual development of intra-cellular alkalinization in astrocytes caused by stimulationof acid extruders Lithium [85] increases intracellular pH(Figure 8(a1)) by directly stimulating the Na+H+ exchangerNHE1 which leads to a compensatory decrease in its gene


MIT

IP3R RyR

Ca2+

Ca2+

Soc (TRPC 1)

Ca2+Na+

exchanger

Transmitter High K+ concentration

Figure 5Aspects ofCa2+ homeostasis in astrocytes discussed in thispaper Many transmitters increase [Ca2+] in astrocytes by triggeringrelease of intracellularly bound Ca2+ (brown) by stimulation ofinositol trisphosphate (IP

3) receptors Highly elevated extracellular

K+ concentrations (ge15mM) cause L-channel-mediated Ca2+ entryand additional Ca2+ is released by stimulation of Ca2+-activatedryanodine receptors (RyR) Free intracellular Ca2+ (red) is accu-mulated by ER or mitochondria or leaves the cell via a Ca2+Na+exchanger This creates a need for Ca2+ entry via store-operatedchannels (Socs) of which TRPC1 is an important component inastrocytes

expression (Figure 8(a2)) Carbamazepine and valproic acidupregulate the expression and thus the function of theNa+bicarbonate cotransporter NBCe1 [85 86 89] in astro-cytes but not in neurons as seen from Figure 8(b1) Howeverthe carbamazepine treatment increases NHE1 expressionin neurons but not in astrocytes Figure 8(b2) It is likelythat pH becomes increased in extracellular fluid as a resultof the increased astrocytic NBCe1 function induced bycarbamazepine The neuronal increase in NHE1 expressionmay therefore be a defense against the development ofa resulting neuronal acidosis The intracellular astrocyticalkalinization may be the cause of an upregulation of cPLA

2

in astrocytes shown in Table 1 since the activity of thisenzyme increases with rising pH [90] This effect is similarto that seen after chronic fluoxetine administration and mayhave similar consequences including stimulation of glucosemetabolism However cPLA

2is downregulated in neurons

[86] and in whole brain [91] after carbamazepine treatmentwhich has contributed to the probably exaggerated conceptof a role in neuroinflammation in bipolar disorder [57]In contrast to the effect of fluoxetine GluK2 expression isdownregulated Some potential effects of this downregulationare discussed by Song et al [86] and it would be importantto know the detailed differences between the consequencesof the downregulated GluK2 function and the function of theupregulated but edited Gluk2 observed after chronic treat-ment with fluoxetine The most important consequence ofthe intracellular alkalosis is probably the effect it has onmyo-inositol uptake and thereby on phosphatidylinositide signal-ing Moreover antibipolar drugs have effects on ion home-ostasis Na+ andK+ by altering expression of Na+ K+-ATPase

subtype expression and Ca2+ by downregulating TRPC1apparently with no concomitant upregulation of Cav12 sinceK+ effects are also reduced or abrogated as shown below

32 Inositol Uptake and Metabolism Transportersand pH Effects

321 Depletion of myo-Inositol A classical proposition theBerridge hypothesis for lithiumrsquos therapeutic effects in bipo-lar disorder is that myo-inositol is depleted by lithium[92] Transmitter-mediated phospholipase C (PLC) stimula-tion hydrolyzes phosphatidyl-45-bisphosphate (PIP

2) in the

cell membrane This produces two second messengers thecytosolic IP

3and themembrane-associated 12-diacylglycerol

(DAG) IP3triggers Ca2+ release from intracellular Ca2+

stores via stimulation of the IP3receptor and DAG activates

protein kinase C Subsequently IP3is stepwise dephospho-

rylated eventually to myo-inositol by inositol phosphatasesmyo-Inositol cannot resynthesize PIP

2without contribution

from a DAG metabolite Initially DAG is converted tophosphatidic acid (PA) an ester between a diglyceride andphosphoric acid which condenses with cytidine triphos-phate to cytidine monophosphoryl-phosphatidate (CMP-PA) which combines with myo-inositol to form PIP

2via

phosphatidyl-4-monophosphate (PIP) [93 94] Accordingto the ldquoBerridge hypothesisrdquo inhibition of the regenerationof PIP

2by lithium-induced reduction of inositol formation

from IP decreases renewed responses to PLC-linked receptoragonists It is consistent with this concept that a normallyoccurring noradrenaline-induced [Ca2+]

119894increase in cul-

tured astrocytes is inhibited by chronic exposure to lithium[87] (Figure 9) Moreover in nonmedicated bipolar patientsa significant increase in localmyo-inositol concentration wasrecently shown by NMR [95] However the lithium effecton inositol phosphate hydrolysis to free myo-inositol is notshared by either carbamazepine or valproic acidThe originalBerridge hypothesis is therefore insufficient to explain effectsby anti-bipolar drugs but amodified and expanded Berridge-like hypothesis will be presented as follows

322 Cellular Contents of myo-Inositol The concentration ofinositol in plasma is 30ndash60 120583M [96 97] and the intracellularconcentration is at a low millimolar level [98] Accordinglythere is a steep gradient between extracellular and intracel-lularmyo-inositol levels necessitating transport mechanismsfor continuous resupply of myo-inositol since myo-inositoland many of the PIP

2metabolites are further degraded

This is partly effectuated by dietary uptake following a slowtransfer across the blood-brain barrier [99] and partly bysynthesis in brain which occurs only in the vasculature [100]Therefore inhibition of cellular uptake would also depletemyo-inositol in astrocytes and neurons

323 Inositol Transporters Wolfson et al [101] showed thattreatment of cultured mouse astrocytes with 1mM LiClfor 8 days reduced the cellular content of myo-inositolafter exposure to 50120583M myo-inositol for 24 hours Acuteadministration of lithium had no similar effect Lubrich andvanCalker [102] expanded this observation by demonstrating


0

30

20

10Gly

coge

noly

sis (

)

(a)

0

30

20

10Gly

coge

noly

sis (

)

(b)

Figure 6 Effects on glycogenolysis (as percent of total glycogen) by 10min acute exposure to 10120583M fluoxetine in cultured mouse astrocyteschronically treated (filled columns) with 10 120583Mfluoxetine for either 1 week (a) or for 2-3 weeks (b) compared to the effect of acute fluoxetineadministration to similar untreated cultures from the same batches measured in the same experiments (open cloumns) In both (a) and (b)the treatment effect is significant (119875 lt 005 or better) Glycogenolytic rates in (a) and (b) for the treated cultures were different at 119875 lt 00005whereas there was no significant difference in the untreated cultures from Kong et al [8]

GSK3120573

p-GSK3120573

GAPDH

(a)

00

05

10

15

20 Relative total GSK3120573

(b)

1

2

3

4 Relative phosphorylated

GSK3120573totalGSK3120573

lowastlowastlowast

(c)

Figure 7 Treatment with citalopram (15mgkg) for 14 days (brown columns) has no effect on total GSK3120573 expression in hippocampus (ab)compared to that in untreated control animals (white columns) but dramatically increases its phosphorylation (c) This effect is abolished bysulindac an inhibitor of the stimulated pathway (green columns) from Liu et al [82] with permission


7

71

72

73

74

75

76

77

78

1 2 3 4Time (weeks)

0

05

1

15

1 week 2 weeks 4 weeks1 day 3 days 3 weeks

Ratio

ofN

HE1T

BP

lowast lowast lowast lowast lowast lowast lowast lowast lowastlowast

(a1) (a2)

pHi

(a)

0

05

1

15

Astrocytes (GFP) Neuron (YFPH)

Control CBZ

Ratio

ofN

BCe1T

BP

lowast

0

05

1

15

2


Control CBZ

Ratio

ofN

HE1T

BP lowast

(b1) (b2)

(b)

Figure 8 (a1) Effects of chronic treatment with different concentrations of lithium on intracellular pH measured as a function of the lengthof the treatment and the lithium concentration (diamonds control circles 05mM Li+ triangles 1mM Li+ or squares 2mM Li+) (a2) mRNAexpression of NHE1 in control cultures (white) cultures treated with 05mM Li+ (light gray) 1mM Li+ (darker gray) or 2mM Li+ (black)lowastindicates statistically significant difference (119875 lt 005 from control cultures and cultures treated with 05mm Li+ for the same length of time(b) mRNA expression of the acid extruders NBCe1 and NHE1 in astrocytes and neurons from astrocytes obtained from untreated animalsor animals treated for 14 days with carbamazepine (CBZ) In both cases astrocytes had been stained with GFP and neurons with YPHF intransgenic animals and they were separated after cell dissociation by means of the different fluorescent signals The size of the PCR productsof NBCe1 is 298 bp of NHE1 is 422 bp and of TBP is 236 bpThe primers for NBCe1 were (FWD) 51015840CTCACTTCTCCTGTGCTTGCCT31015840 and(REV) 51015840GTGGTTGGAAAATAGCGGCTGG31015840 and those for NHE1 and TBP the same as used by Song et al [85] (a1) and (a2) from Songet al [86] ((b1) and (b2)) Unpublished experiments by B Li D Song and L Peng using methodology similar to that used by Li et al [22]

that chronic treatment of cultured rat astrocytes with lithiumcarbamazepine or valproic inhibited myo-inositol uptakeWolfson et al [88] confirmed this observation in culturedhuman astrocytoma cells at a myo-inositol concentration of50120583M but at 25 120583Mmyo-inositol the uptake was stimulated(Figure 10) Such an effect can be explained by the combinedeffects of (i) an induced alkalinization (ii) the presenceof two different myo-inositol transporters in astrocytes thehigh-affinity Na+-dependent transporter (SMIT) and thelower-affinity H+-dependent (HMIT) and (iii) stimulationof SMIT but inhibition of HMIT at increased pH [103 104]We have recently confirmed that uptake of myo-inositolin cultured astrocytes at normal extracellular myo-inositol

levels mainly is catalyzed by a lower-affinity higher-capacityHMIT-mediated uptake (K

119898143 120583M and Vmax 358 pmolmg

protein) whereas a higher-affinity lower-capacity SMIT-mediated uptake (K

119898167 120583M and Vmax 602 pmolmg pro-

tein) plays a minor role [105] Moreover the uptake of100 120583M myo-inositol was inhibited at increased intracellularpH whereas that at 10 120583M myo-inositol was enhancedThe observed increase in uptake at 25120583M myo-inositol butreduction of uptake at 50 120583M reported by Wolfson et al [88]can therefore be explained by changes in relative contributionof the two myo-inositol transporters to total uptake onaccount of the induced intracellular alkalinization [85 86]Since di Daniel et al [106] reported that HMIT in neurons


0

250

500

750

lowast

lowast

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(a)

0

400

200

600

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(b)

Figure 9 [Ca2+]119894in mouse astrocytes during basal conditions control (C) and during exposure to 1120583M noradrenaline (Na) in untreated

control cultures and in sister cultures which had been treated for either 7ndash14 days (a) or 30ndash45min (b) with 1mM lithium chloride (Li) Thechronic lithium treatment decreased [Ca2+]

119894significantly ( lowast119875 lt 005) both under basal condition and during exposure to noradrenaline (a)

whereas the short-lasting exposure to Li+ had no effect from Chen and Hertz [87]

0

10

30

40

50

60

Inos

itol u

ptak

e (nm

olm

g)

25 40 50

lowast

lowast

(120583M)

Figure 10 Uptake of [3H]myo-inositol at concentrations of 25 40and 50 120583Mduring 60min inU251MGastrocytoma cells treatedwith1mM lithium chloride for 2 weeks before the uptake experiment aswell as during the uptake (filled columns) and in untreated controlcultures (open columns) Note lithium-induced decrease of uptakeat 50120583M myo-inositol versus increase at 25 120583M SEM values areshown by vertical bars lowastStatistically significant difference betweenlithium-treated and control cultures (119875 lt 005) fromWolfson et al[88]

can transport IP3and Gossman and Zhao [107] and Wang

et al [108] found in the cochlea that IP3can be released

from nonneuronal cells through hemichannels it cannot beexcluded that astrocytically generated IP

3might be trans-

ferred to neurons In that case a decrease in astrocytic IP3

formation in response to antibipolar drug treatment mightalso affect neuronal IP

3 Effects of transmitters operating via

the protein kinase C (PKC) and the phosphatidylinositidesignaling system might therefore become reduced primarilyin astrocytes but possibly also in neurons by antibipolar

drug treatment Effects exerted via protein kinase A (PKA)like those of adrenaline acting on 120573-adrenergic receptorsmay also be affected in astrocytes on account of a GsGishift in their pathway [109] This might include 120573-adrenergicglycogenolysis which is dependent on the increase in [Ca2+]

119894

occurring after this shift Since 120573-adrenergic glycogenolysis isa prerequisite for neuronal glutamatergic signaling [12 110]reduced glycogenolysis might have considerable dampeningeffect on neuronal excitability Although GSK3120573 is very oftendiscussed in connection with this disease and its treatmentpossible enhancement of glycogen synthesis seems not tohave been considered or tested

33 Ion Homeostasis Like fluoxetine chronic treatment withany of the three antibipolar drugs downregulates TRPC1and therefore interferes with Ca2+ homeostasis Howeveras shown in Figure 11 the ability of elevated extracellularK+ concentrations to increase [Ca2+]

119894is also inhibited [76]

Thus both transmitter-induced and depolarization-mediatedchanges in astrocytic [Ca2+]

119894are reduced This represents an

important difference from astrocytes treated with an SSRIwhere only the transmitter-mediated responses are inhibited[80] and the inhibition of the transmitter-induced effectmight even be transient

An effect onTRPC1may affect not onlyCa2+ homeostasisbut also Na+ homeostasis in astrocytes because the TRPC1channel has equal permeability for Ca2+ and Na+ [111]This may be of major importance in astrocytes since thesenonexcitable cells may become deficient in intracellular Na+required for Na+ K+-ATPase activity [14] Paradoxicallyantibody-mediated partial inactivation of TRPC1rsquos Ca2+ per-meability is accompanied by an increase of the intracellularconcentration of Na+ suggesting that the binding of theantibody to the channel decreases Ca2+ flux but increasesNa+flux [112] Findings of decreased Na+ K+-ATPase activity in


Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl

lowast lowast lowast lowast lowast

(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)

Figure 11 [Ca2+]119894in astrocytes in arbitrary units (fluorescence ratio) in response to addition of 45mmKCl in control cultures and in cultures

treated during 14 days with 1mM lithium (a) 50mM carbamazepine (CBZ) (b) or 1mM valproic acid (VPA) (c) from Yan et al [76]

bipolar disorder are robust (i) in brain tissue from bipolardisorder a significantly lower Na+ K+-ATPase density wasfound in tissues from patients having suffered from majordepression or schizophrenia [113] (ii) expression of the 1205722gene is decreased in temporal cortex from bipolar disorderpatients [114] (iii) Na+-K+-ATPase activity is significantlyreduced in erythrocytes from patients with bipolar disorder[115] and (iv) lymphoblastoid cell lines originating frombipolar patients express less Na+ K+-ATPase in responseto an increase in intracellular Na+ concentration than sim-ilar cell lines from unaffected siblings A possible genomicinvolvement of Na+ K+-ATPase dysfunction in bipolar dis-order is also suggested by a genetic association betweenbipolar disorder and variants of the genes encoding theNa+K+-ATPase subunits 1205721 1205722 and 1205723 [116] Brains inbipolar patients show increased brain acidity in vivo [117]This might have influenced Na+ K+-ATPase activity andexpression since Deigweiher et al [118] demonstrated thatin cuttlefish gill tissues increases in water CO

2tension

which must decrease pH led to a decline in Na+ K+-ATPaseactivity Previously Cummins and Hyden [119] had shown apH maximum around 80 for ATP hydrolysis in microdis-sected astrocytes Based on all this evidence we investigatedwhether chronic treatment with carbamazepine could alterexpression of Na+ K+-ATPase subtypes and of its auxiliaryprotein FXYD in neurons and astrocytes of normal mice[120] Mice which coexpress one fluorescent marker with aneuron-specific gene and another marker with an astrocyte-specific gene [86] were either treated with carbamazepine

for 2 weeks or received no treatment but were used ascontrols FromFigure 12 it can be seen that1205723 expressionwasunchanged and 1205722 increased in the treated animals both inastrocytes and in neurons where it is normally not expressedwhereas 1205721 increased only in neurons FXYD expressionwas reduced by the carbamazepine treatment but only inneurons and 1205731 was upregulated in astrocytes but not inneurons (results not shown) These changes should facilitateK+ uptake in neurons (see discussion in [120]) withoutcompromising preferential uptake in astrocytes at increasedextracellular K+ concentrations a process which is importantfor K+ homeostasis of the cellular level of the brain [14]Although an overall increase in Na+ K+-ATPase expressionand presumably activity could be related to pH changes theinduced isoform expressions are not a direct consequenceof the intracellular pH changes since 1205722 was upregulated inboth neurons and astrocytes and 1205721 which shows a slightincrease in activity at increased pH [121] showed increasedexpression in neurons but not in astrocytes However Na+K+-ATPase activity and thus expression is regulated by amultitude of different factors many of which may have beenaltered in the carbamazepine-treated mice [122]

34 Conclusion The intracellular alkalinization induced inastrocytes by the three otherwise very dissimilar drugslithium carbamazepine and valproic acid suggests that thiseffect is therapeutically important If anything this conceptis strengthened by the finding that lithium acts on one acidextruder and carbamazepine and valproic acid on a different


0

05

1

Astrocyte (GFP) Con Astrocyte (GFP) CBZNeuron (YFPH) Con Neuron (YFPH) CBZ

Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast

Figure 12 Expression of mRNA for the Na+ K+-ATPase subunits1205721 1205722 and 1205723 expressed as ratios between mRNA of the subtypegene and TATA-binding protein (TBP) used as a housekeepinggene in astrocytes and neurons of mice treated for 14 days withcarbamazepine (25mgkg per day) As in Figures 3 and 8(b) theastrocytes had been stained with GFP and the neurons with YPHFin transgenic animals and they were separated after cell dissociationby means of the different fluorescent signals from Li et al [120]

acid extruder and by the finding of increased intracellularacidity in the brains of bipolar patients In contrast to theability of chronic treatment with antidepressant medica-tion to enhance the increase of [Ca2+]

119894and thus probably

glycogenolysis by exposure to elevated extracellular K+ con-centrations (and after sufficiently long treatment perhaps alsoto transmitters) chronic treatment with antibipolar drugsinhibit both K+-induced and transmitter-induced increaseof astrocytic [Ca2+]

119894and thereby probably excitability The

effect on astrocytic cPLA2expression is similar to that after

antidepressant treatment and may have similar beneficialeffects but the effect on the neuronal enzyme is the oppo-site The expression of GluK2 which is upregulated (andedited) by antidepressant therapy is downregulated but thetherapeutic consequences have not been determined withcertainty Further studies of GluK2 function in brain anddrug effects on this kainate receptor under normal andpathological conditions are urgently needed Together withthe opposite effect of SSRIs and antibipolar drugs on theability of elevated extracellular K+ concentrations to affectastrocytic [Ca2+]

119894 they may constitute important differences

in the directions of changes caused by the antidepressantSSRIs and the mainly antimanic antibipolar drugs

4 Acute Anxiolytic Drug Effects

41 GABA119860Receptor Stimulation Because of a high intra-

cellular Clminus concentration in astrocytes [123 124] GABAAreceptor-mediated increase of Clminus fluxes causes a depo-larization resulting in GABAA-induced [Ca

2+]119894increases

[124 125] and in glycogenolysis (Figure 13) These effectsare strikingly similar to those seen after chronic treatmentwith fluoxetine and are consistent with the observation thatGABAA but notGABAB receptor stimulation has antianxiety-like effects in rats tested in the elevated-plus-maze [126]

Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)

Figure 13 Glycogenolytic effect of GABA andor addition of5mMK+ indicated as reduction of glycogen content in astrocytesCultured astrocytes were incubated for 20min in DMEM (con-taining 75mM glucose) without any addition (control) addition of5mMK+ to a final extracellular concentration of 10mM (+5K+)of 120574-aminobutyric acid (GABA) to a final concentration of 100120583M(GABA) or of GABA and K+ (GABA plus +5K+) After theincubation the astrocytes were washed three times with ice-coldphosphate-buffered saline (PBS) and sonicated in 30mM HCl Thesuspension was used to measure nonhydrolyzed glycosyl units ofglycogen Three 50 120583L aliquots were sampled In the first aliquot150 120583L of acetate buffer (01M pH 465) was added In the second150 120583L of a solution containing 1 amyloglucosidase (10mgmL) inthe acetate buffer was added in order to degrade remaining glycogento glucose and the mixture was incubated at room temperaturefor 30min Subsequently the two aliquots were treated identicallyTwo mL of Tris-HCl buffer (01M pH 81) containing 33mMMgCl

2 02mM ATP 25 120583gmL NADP 4120583gmL hexokinase and

2 120583gmL glucose-6-phosphate dehydrogenase was added to eachand the mixture was incubated at room temperature for 30minThe fluorescence of the NADPH formed in amounts equivalentto glucose metabolized by hexokinase was then read (excitation340 nm emission 450 nm) The first aliquot measures the sum ofglucose and glucose-6-phosphate in the tissue whereas the secondaliquot in addition to those also measures the glycosyl units fromglycogen remaining in the tissue Determination of the differencebetween these two aliquots provides a measurement of the amountof the latter The third aliquot was used to measure the proteincontent by the Lowry method to normalize the glycogen contents(nmol) per mg protein Average glycogen contents were indicatedas percentages of those under control conditions All values wereexpressed asmeansplusmn SEM indicated by vertical bars andwere fromthree-five individual cultures lowastStatistically significant (119875 lt 005)difference from control Results are unpublished experiments by JXu D Song L Hertz and L Peng

Recent publications suggest that the role of GABA effects onastrocytes is currently greatly underestimated [124 127] Thisis in spite of astrocytic subunit composition of the GABAAreceptor indicative of an anxiolytic role

GABAA receptors are made up of 120572 120573 and 120574 subunitseach of which can be further subdivided In the presentcontext the 1205741 subunit which is densely expressed in basalganglia [130] but scarcely in cortex may be of special interestA comparison between mRNA expression levels of differentGABAA receptor subunits in freshly isolated astrocytes andneurons [131] showed that gt90 percent of 1205731 cortical mRNA


300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100

0Control + +PK + + PK

lowast

2+] i

()

(b)

Figure 14 (a) Effects of 500 nM diazepam (+Diaz) of 1120583M of the ldquoneuronal-type benzodiazepine antagonistrdquo flumazenil (+Flum) and ofdiazepam plus flumazenil (+Diaz +Flum) on the increase in [Ca2+]

119894evoked by an increase in the extracellular K+ concentration to 20mM

The control value (100) represents the increase by the elevated K+ concentration alone which approximately doubled resting [Ca2+]119894(about

100 nM) Diazepam more than doubles the response to the increase in extracellular K+ and this effect is abrogated by flumazenil The valuein the presence of diazepam is statistically significantly different from all other values none of which differs from any of the other (b) Effectsof 10 nM midazolam (+Mid) of 1 120583M of the ldquomitochondrial benzodiazepine antagonistrdquo PK 11195 (+PK) and of midazolam plus PK 11195(+Mid +PK) on the increase in [Ca2+]

119894evoked by an increase in the extracellular K+ concentration to 20mMMidazolam almost quadruples

the response to the increase in extracellular K+ and this effect is abrogated by PK 11195The value in the presence of midazolam is statisticallysignificantly different from all other values none of which differs from any of the other from Hertz et al [128]

Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50

Figure 15 Release of previously incorporated label in glycogen dur-ing exposure to 10mMextracellularK+ for 30 sec in the presence andabsence of 20 nMmidazolam and to 20mMK+ without midazolamfor 60 sec In the presence of midazolam glycogenolysis duringexposure to 10mMK+ for 30 sec is significantly increased and itsmagnitude becomes similar to that seen after 1min of exposure to20mMK+ alone from Subbarao et al [129]

was astrocytic and astrocytes also accounted for sim70 percentof 1205722 expression sim60 of 1205731 and sim40 of 1205724 (Table 2) Sincelimbic areas are enriched in the 1205741 subunit GABAA receptorscontaining this subtype might serve affective functions [132]and the 1205722 subunit is known to mediate anxiolysis [133]Future attempts to develop anxiolytics acting on GABA

119860

receptors [133] should include potential effects on astrocytes

42 Effects of Benzodiazepines Emphasis on a MembraneReceptor It was initially believed that all anxiolytic effects

Table 2 Percentage astrocytic expression (astrocytic expression(astrocytic + neuronal expression)) of GABAA receptor subunits asfound by Cahoy et al [131] in astrocytic and neuronal cell fractionsobtained from murine brainlowast

GABAA receptor subunit Percentage astrocytic expression1205721 01205722 696 plusmn 3651205724 439 plusmn 2151205725 01205731 614 plusmn 8731205733 01205741 926 plusmn 0501205742 0lowastNo information was provided about 1205732

of benzodiazepines were exerted on the neuronal ldquocentralrdquobenzodiazepine receptor [134] which facilitated GABAergictransmission by binding to its receptor Since this effect wasabrogated by the benzodiazepine antagonist flumazenil itwas often concluded that all benzodiazepine effects that wereinhibited by this antagonist were exerted on the ldquocentralrdquobenzodiazepine receptor However the peripheral-type ben-zodiazepine receptor localized in mitochondria the translo-cator protein (18 kDa) [135] is also important in anxiolysis[136] It is stimulated not only by exogenously administeredbenzodiazepines but also by derivatives of the 86-aminoacid polypeptide diazepam-binding inhibitor (DBI) which isfound in brain [137] It can be cleaved into several biologicallyactive peptides including the triakontatetraneuropeptideTTN and the octadecaneuropeptide ODN [138] Gandolfo etal [139] showed (i) that binding of the peripheral-type benzo-diazepine receptor ligand [3H]Ro5-4864 to intact astrocytes


cultures was displaced by TTN whereas ODN did not com-pete for [3H]Ro5-4864 binding and (ii) that TTN provoked aconcentration-dependent increase in [Ca2+]

119894in the cultured

astrocytes which was blocked by chelation of extracellularCa2+ by EGTA or blockage of Ca2+ channels with Ni2+ andsignificantly reduced by the L-type calcium channel blockernifedipine Patch-clamp studies showed that TTN induced asustained depolarization and the authors suggested that TTNacted through the peripheral benzodiazepine binding site andopening of Clminus channels that is a mechanism similar to thatof stimulation of GABAA receptors

It has long been knownmdashand virtually ignoredmdashthatperipheral benzodiazepine receptors are present not only inmitochondria but also at the plasma membrane of astro-cytes [140ndash142] In addition the peripheral benzodiazepinebinding site shows multiplicity and has also been demon-strated on a plasma membrane-enriched preparation fromrat astrocytes [143] Backus et al [140] suggested the samemechanism for Ca2+ entry as Gandolfo et al [139] that is adepolarizing effect In contrast Bender and Hertz [141] andZhao et al [142] using cultures which had been differenti-ated by treatment with dibutyryl cyclic AMP and thereforeexpress L-channels suggested a direct interaction betweenthe benzodiazepine and this channel It seems consistentwith this interpretation that midazolam had no significanteffect at nonelevated extracellular K+ concentrations in theexperiments by Zhao et al [142] More conclusive evidencehas been reached in cardiac cells where direct activation ofthe channel by the Ca2+ channel activator BAY K 8644 wasblocked by the benzodiazepine antagonist PK11195 [144 145]Nevertheless the most important finding an increase inastrocytic [Ca2+]

119894 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+ con-centrations is selectively prevented in the presence of 1120583Mof the ldquoperipheral-typerdquo benzodiazepine antagonist PK 11195and notably also by the supposedly ldquocentral-typerdquo antagonistflumazenil [128 142 146] The inhibition by flumazenil con-firms previous observation by Backus et al [140] and empha-sizes again that inhibition of a benzodiazepine response byflumazenil does not necessarily indicate that the responseis exerted on neuronal receptors The dihydropyridine L-channel blocker nimodipine abolishes both the effect ofelevated K+ and that of the benzodiazepine [142] althoughit should have no effect on the latter if only an L-channel-independent depolarization was involved Figure 15 showsthatmidazolam also increases the glycogenolytic effect of ele-vated extracellular K+ concentrations [129]The high potencyof benzodiazepines in increasing L-channel opening in astro-cytes (500 nM diazepam and 10 nM midazolam in Figure 14)is an indication that the response may be relevant for clin-ically observed benzodiazepine effects because the effectiveconcentrations are similar to those obtained clinically [147148] In contrast stimulation ofGABA-mediatedClminus influx incortical neurons by 100ndash1000 nM diazepam causes only a 5ndash40 increase [149] Peripheral-type receptors are also presenton several additional nonneural cell types as discussed byGandolfo et al [139] and mature erythrocytes which have

no mitochondria express peripheral-type benzodiazepinereceptors [150] These sites should not be identified astranslocator proteins and have been referred to as the ldquoJokerrdquoreceptor by Hertz et al [128] and Hertz and Chen [146]

Yet another indication that drugs increasing [Ca2+]119894and

causing glycogenolysis can provide acute anxiolytic effectsis that acute treatment with the 5-HT

2B receptor agonistBW 723C86 has been found to exert anxiolytic effects inanimal experiments [151ndash153] This is the same receptoras that activated by fluoxetine and other SSRIs and acuteadministration of fluoxetine increases [Ca2+]

119894and causes

glycogenolysis [154] Why some degree of anxiolysis can beobtained by acute 5-HT

2B activation but the antidepressanteffects require weeks of treatment and gene editing and up-regulation remains to be shown

43 Conclusion Acute anxiolytic drug therapy induces Ca2+-dependent anxiolysis and enhances glycogenolysis evokedby exposure to elevated extracellular K+ concentrations aneffect which is strikingly similar to that of chronic treatmentwith an SSRI which also has anxiolytic effect

The response to GABAA receptor stimulation is sec-ondary to depolarization and that to benzodiazepines to adirect effect on the L-channel One might wonder about theimportance of L-channel opening since it in experimentswhere [K+]

119890is the only factor changed requires [K+]

119890values

above those usually seen in response to normal stimulationHowever it should be remembered that the [K+]

119890is probably

normally not changed alone but that the change might beaccompanied by changes in transmitters likeGABA that alsocontribute to the depolarization as seen by the tendency toa more pronounced glycogenolysis in Figure 13 Accordinglyactivation of Ca2+ uptake by elevated [K+]

119890may occur much

more frequently than should be anticipated based on theexperiments in which [K+]

119890alone is increased This may

also be of importance for the oppositely directed changes ineffects of increase in [K+]

119890after treatment with SSRIs and

with antibipolar drugs

5 Concluding Remarks

Astrocytic increase in [Ca2+]119894and in glycogenolysis after

chronic treatment with SSRIs (which also have antianx-iolytic effects) and during acute GABAA stimulation oradministration of benzodiazepines is probably importantfor antidepressant-anxiolytic drug effects and astrocyticglycogenolysis is essential to support astrocytic signalingTheglycogenolytic effect is unlikely to be shared by antibipolardrugs which all cause intracellular alkalinization in astro-cytes although by stimulation of different acid extrudersThese drugs inhibit effects of both elevated K+ concentrationsand transmitters on astrocytic [Ca2+]

119894 Carbamazepine was

also shown to have important effects on Na+ K+-ATPase inboth astrocytes and neurons Both antidepressant drugs andantibipolar drugs affect GluK2 but the effects are differentand further studies of this kainate receptor are warranted inboth major depression and bipolar disease So is that of theastrocytic upregulation of cPLA

2


Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Mrs Elna Hertz is cordially thanked for her very carefulreading of the paper and correction of a multitude of typosand other errors

References

[1] H K Kimelberg ldquoFunctions of mature mammalian astrocytesa current viewrdquo Neuroscientist vol 16 no 1 pp 79ndash106 2010

[2] L Peng C Guo T Wang B Li L Gu and Z Wang ldquoMethod-ological limitations in determining astrocytic gene expressionrdquoFrontiers in Endocrinology vol 4 article 176 2013

[3] D Lovatt U Sonnewald H S Waagepetersen et al ldquoThetranscriptome and metabolic gene signature of protoplasmicastrocytes in the adult murine cortexrdquo The Journal of Neuro-science vol 27 no 45 pp 12255ndash12266 2007

[4] T Du C Liang B Li L Hertz and L Peng ldquoChronic fluoxetineadministration increases expression of the L-channel gene Ca

1199071

2 in astrocytes from the brain of treated mice and in cultureand augments K+-induced increase in [Ca2+]

119894rdquo Cell Calcium In

press[5] L Hertz B Li D Song et al ldquoAstrocytes as a 5-HT

2B-mediatedSERT-independent SSRI target slowly altering depression-associated genes and functionsrdquo Current Signal TransductionTherapy vol 7 no 1 pp 65ndash80 2012

[6] D T Wong J S Horng F P Bymaster K L Hauser and B BMolloy ldquoA selective inhibitor of serotonin uptake Lilly 1101403 (p trifluoromethylphenoxy) n methyl 3 phenylpropylaminerdquoLife Sciences vol 15 no 3 pp 471ndash479 1974

[7] L Hertz F Baldwin and A Schousboe ldquoSerotonin receptorson astrocytes in primary cultures effects of methysergide andfluoxetinerdquo Canadian Journal of Physiology and Pharmacologyvol 57 no 2 pp 223ndash226 1979

[8] E K C Kong L Peng Y Chen A C H Yu and L Hertz ldquoUp-regulation of 5-HT

2B receptor density and receptor-mediatedglycogenolysis in mouse astrocytes by long-term fluoxetineadministrationrdquo Neurochemical Research vol 27 no 1-2 pp113ndash120 2002

[9] D R Cool F H Liebach and V Ganapathy ldquoInteraction offluoxetine with the human placental serotonin transporterrdquoBiochemical Pharmacology vol 40 no 9 pp 2161ndash2167 1990

[10] S Zhang B Li D Lovatt et al ldquo5-HT2B receptors are expressed

on astrocytes from brain and in culture and are a chronic targetfor all five conventional serotonin-specific reuptake inhibitorsrdquoNeuron Glia Biology vol 6 no 2 pp 113ndash125 2010

[11] B Li S Zhang H Zhang et al ldquoFluoxetine-mediated 5-HT2B

receptor stimulation in astrocytes causes EGF receptor transac-tivation and ERK phosphorylationrdquo Psychopharmacology vol201 no 3 pp 443ndash458 2008

[12] M E Gibbs D G Anderson and L Hertz ldquoInhibition ofglycogenolysis in astrocytes interrupts memory consolidationin young chickensrdquo Glia vol 54 no 3 pp 214ndash222 2006

[13] L F Obel M S Muller A B Walls et al ldquoBrain glycogen-new perspectives on itsmetabolic function and regulation at thesubcellular levelrdquo Front Neuroenergetics vol 4 no 3 2012

[14] J Xu D Song Z Xue L Gu L Hertz and L Peng ldquoRequire-ment of glycogenolysis for uptake of increased extracellular K+in astrocytes potential implications for K+ homeostasis andglycogen usage in brainrdquo Neurochemical Research vol 38 no3 pp 472ndash485 2013

[15] Y Chen L Peng X Zhang J-U Stolzenburg and L HertzldquoFurther evidence that fluoxetine interacts with a 5-HT

2Creceptor in glial cellsrdquo Brain Research Bulletin vol 38 no 2 pp153ndash159 1995

[16] M E Gibbs D Hutchinson and L Hertz ldquoAstrocytic involve-ment in learning and memory consolidationrdquo Neuroscience ampBiobehavioral Reviews vol 32 no 5 pp 927ndash944 2008

[17] N Embi D B Rylatt and P Cohen ldquoGlycogen synthase kinase-3 from rabbit skeletal muscle Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinaserdquo EuropeanJournal of Biochemistry vol 107 no 2 pp 519ndash527 1980

[18] X Li W Zhu M-S Roh A B Friedman K Rosboroughand R S Jope ldquoIn vivo regulation of glycogen synthasekinase-3120573 (GSK3120573) by serotonergic activity in mouse brainrdquoNeuropsychopharmacology vol 29 no 8 pp 1426ndash1431 2004

[19] E Beurel M A Mines L Song and R S Jope ldquoGlycogensynthase kinase-3 levels and phosphorylation undergo largefluctuations in mouse brain during developmentrdquo Bipolar Dis-orders vol 14 no 8 pp 822ndash830 2012

[20] A Schousboe ldquoDevelopment of potassium effects on ionconcentrations and indicator spaces in rat brain-cortex slicesduring postnatal ontogenesisrdquo Experimental Brain Researchvol 15 no 5 pp 521ndash531 1972

[21] S J Tudhope C-CWang J L Petrie et al ldquoA novelmechanismfor regulating hepatic glycogen synthesis involving serotoninand cyclin-dependent kinase-5rdquo Diabetes vol 61 no 1 pp 49ndash60 2012

[22] B Li L Dong B Wang L Cai N Jiang and L PengldquoCell type-specific gene expression and editing responses tochronic fluoxetine treatment in the in vivo mouse brain andtheir relevance for stress-induced anhedoniardquo NeurochemicalResearch vol 37 no 11 pp 2480ndash2495 2012

[23] B Li S Zhang H Zhang L Hertz and L Peng ldquoFluoxe-tine affects GluK2 editing glutamate-evoked Ca2+ influx andextracellular signal-regulated kinase phosphorylation in mouseastrocytesrdquo Journal of Psychiatry and Neuroscience vol 36 no5 pp 322ndash338 2011

[24] B L Bass ldquoRNA editing by adenosine deaminases that act onRNArdquoAnnual Review of Biochemistry vol 71 pp 817ndash846 2002

[25] L Valente and K Nishikura ldquoADAR gene family and A-to-I RNA editing diverse roles in posttranscriptional generegulationrdquo Progress in Nucleic Acid Research and MolecularBiology vol 79 pp 299ndash338 2005

[26] Y Kawahara K Ito H Sun M Ito I Kanazawa and S KwakldquoRegulation of glutamate receptor RNA editing and ADARmRNA expression in developing human normal and Downrsquossyndrome brainsrdquo Developmental Brain Research vol 148 no1 pp 151ndash155 2004

[27] G Y Sun J Xu M D Jensen and A Simonyi ldquoPhospholipaseA2 in the central nervous system implications for neurodegen-erative diseasesrdquo Journal of Lipid Research vol 45 no 2 pp205ndash213 2004

[28] B Li S ZhangM Li L Hertz and L Peng ldquoChronic treatmentof astrocytes with therapeutically relevant fluoxetine concen-trations enhances cPLA

2expression secondary to 5-HT

2B-induced transactivation-mediated ERK

12phosphorylationrdquo

Psychopharmacology vol 207 no 1 pp 1ndash12 2009


[29] L L Lautens X G Chiou J D Sharp et al ldquoCytosolicphospholipase A2 (cPLA

2) distribution in murine brain and

functional studies indicate that cPLA2does not participate

in muscarinic receptor-mediated signaling in neuronsrdquo BrainResearch vol 809 no 1 pp 18ndash30 1998

[30] M A Balboa and J Balsinde ldquoInvolvement of calcium-independent phospholipase A2 in hydrogen peroxide-inducedaccumulation of free fatty acids in human U937 cellsrdquo TheJournal of Biological Chemistry vol 277 no 43 pp 40384ndash40389 2002

[31] D T Stephenson J V Manetta D L White et al ldquoPhospho-lipase A2 in the central nervous system implication for neuro-degeneration diseasesrdquoThe Journal of Lipid Research vol 46 pp205ndash213 2004

[32] C C Felder R Y Kanterman A L Ma and J AxelrodldquoSerotonin stimulates phospholipase A2 and the release ofarachidonic acid in hippocampal neurons by a type 2 serotoninreceptor that is independent of inositolphospholipid hydroly-sisrdquoProceedings of theNationalAcademy of Sciences of theUnitedStates of America vol 87 no 6 pp 2187ndash2191 1990

[33] B D Stout W P Clarke and K A Berg ldquoRapid desensitizationof the serotonin2c receptor system effector pathway and agonistdependencerdquo Journal of Pharmacology and Experimental Ther-apeutics vol 302 no 3 pp 957ndash962 2002

[34] Y Qu L Chang J Klaff R Seemann and S I Rapoport ldquoImag-ing brain phospholipase A2-mediated signal transduction inresponse to acute fluoxetine administration in unanesthetizedratsrdquo Neuropsychopharmacology vol 28 no 7 pp 1219ndash12262003

[35] S I Rapoport ldquoBrain arachidonic and docosahexaenoic acidcascades are selectively altered by drugs diet and diseaserdquoProstaglandins Leukotrienes and Essential Fatty Acids vol 79no 3ndash5 pp 153ndash156 2008

[36] M C Garcia and H-Y Kim ldquoMobilization of arachidonate anddocosahexaenoate by stimulation of the 5-HT

2A receptor in ratC6 glioma cellsrdquo Brain Research vol 768 no 1-2 pp 43ndash481997

[37] N Yu J-L Martin N Stella and P J Magistretti ldquoArachidonicacid stimulates glucose uptake in cerebral cortical astrocytesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 90 no 9 pp 4042ndash4046 1993

[38] I Allaman H Fiumelli P J Magistretti and J-L Martin ldquoFlu-oxetine regulates the expression of neurotrophicgrowth factorsand glucose metabolism in astrocytesrdquo Psychopharmacologyvol 216 no 1 pp 75ndash84 2011

[39] O Sorg L Pellerin M Stolz S Beggah and P J Mag-istretti ldquoAdenosine triphosphate and arachidonic acid stimulateglycogenolysis in primary cultures of mouse cerebral corticalastrocytesrdquo Neuroscience Letters vol 188 no 2 pp 109ndash1121995

[40] L Hertz J Xu and L Peng ldquoGlycogenolysis and purinergic 19signalingrdquo in Glutamate and ATP at Interface of MetabolismandSignaling in the Brain V Parpura A Schousboe and AVerkhratsky Eds Advances ofNeurobiology Springer In press

[41] Y Qu L Chang J Klaff R Seemann D Greenstein and SI Rapoport ldquoChronic fluoxetine upregulates arachidonic acidincorporation into the brain of unanesthetized ratsrdquo EuropeanNeuropsychopharmacology vol 16 no 8 pp 561ndash571 2006

[42] J S Rao R N Ertley H J Lee S I Rapoport and R P BazinetldquoChronic fluoxetine upregulates activity protein and mRNAlevels of cytosolic phospholipase A2 in rat frontal cortexrdquo ThePharmacogenomics Journal vol 6 no 6 pp 413ndash420 2006

[43] H-J Lee J S Rao L Chang S I Rapoport and R P BazinetldquoChronic N-methyl-D-aspartate administration increases theturnover of arachidonic acid within brain phospholipids of theunanesthetized ratrdquoThe Journal of Lipid Research vol 49 no 1pp 162ndash168 2008

[44] J T Little T A Ketter T A Kimbrell et al ldquoVenlafaxine orbupropion responders but not nonresponders show baselineprefrontal and paralimbic hypometabolism compared withcontrolsrdquo Psychopharmacology Bulletin vol 32 no 4 pp 629ndash635 1996

[45] J T Little T A Ketter T A Kimbrell et al ldquoBupropion andvenlafaxine responders differ in pretreatment regional cerebralmetabolism in unipolar depressionrdquo Biological Psychiatry vol57 no 3 pp 220ndash228 2005

[46] P Videbech ldquoPET measurements of brain glucose metabolismand blood flow in major depressive disorder a critical reviewrdquoActa Psychiatrica Scandinavica vol 101 no 1 pp 11ndash20 2000

[47] N L Rasgon H A Kenna C Geist G Small andD SilvermanldquoCerebral metabolic patterns in untreated postmenopausalwomen with major depressive disorderrdquo Psychiatry Researchvol 164 no 1 pp 77ndash80 2008

[48] T A Kimbrell T A Ketter M S George et al ldquoRegionalcerebral glucose utilization in patients with a range of severitiesof unipolar depressionrdquo Biological Psychiatry vol 51 no 3 pp237ndash252 2002

[49] M S Buchsbaum W Joseph B V Siegel et al ldquoEffect ofsertraline on regional metabolic rate in patients with affectivedisorderrdquo Biological Psychiatry vol 41 no 1 pp 15ndash22 1997

[50] H S Mayberg S K Brannan J L Tekell et al ldquoRegionalmetabolic effects of fluoxetine in major depression serialchanges and relationship to clinical responserdquo Biological Psychi-atry vol 48 no 8 pp 830ndash843 2000

[51] S H Kennedy K R Evans S Kruger et al ldquoChanges in regionalbrain glucose metabolism measured with positron emissiontomography after paroxetine treatment of major depressionrdquoThe American Journal of Psychiatry vol 158 no 6 pp 899ndash9052001

[52] M Elizabeth Sublette M S Milak J R Hibbeln et al ldquoPlasmapolyunsaturated fatty acids and regional cerebral glucosemetabolism in major depressionrdquo Prostaglandins Leukotrienesand Essential Fatty Acids vol 80 no 1 pp 57ndash64 2009

[53] C-U Pae H-S Yu J-J Kim et al ldquoBanI polymorphism of thecytosolic phospholipase A2 gene and mood disorders in theKorean populationrdquoNeuropsychobiology vol 49 no 4 pp 185ndash188 2004

[54] K-P Su S-Y Huang C-Y Peng et al ldquoPhospholipase A2and cyclooxygenase 2 genes influence the risk of interferon-120572-induced depression by regulating polyunsaturated fatty acidslevelsrdquo Biological Psychiatry vol 67 no 6 pp 550ndash557 2010

[55] F Chen G Wegener T M Madsen and J R NyengaardldquoMitochondrial plasticity of the hippocampus in a genetic ratmodel of depression after antidepressant treatmentrdquo Synapsevol 67 no 3 pp 127ndash134 2013

[56] R E Anglin M F Mazurek M A Tarnopolsky and P IRosebush ldquoThemitochondrial genome and psychiatric illnessrdquoThe American Journal of Medical Genetics B vol 159 no 7 pp749ndash759 2012

[57] L Hertz D Song B Li E Yan and L Peng ldquoImportance ofldquoinflammatory moleculesrdquo but not necessarily of inflammationin the pathophysiology of bipolar disorder and in the mecha-nisms of action of anti-bipolar drugsrdquoNeurology Psychiatry andBrain Reseach vol 19 no 4 pp 174ndash179 2013


[58] D Tharumaratnam S Bashford and S A Khan ldquoIndometh-acin induced psychosisrdquo Postgraduate Medical Journal vol 76no 901 pp 736ndash737 2000

[59] M Clunie L-A Crone L Klassen and R Yip ldquoPsychiatricside effects of indomethacin in parturientsrdquo Canadian Journalof Anesthesia vol 50 no 6 pp 586ndash588 2003

[60] H-K Jiang and D-M Chang ldquoNon-steroidal anti-inflamma-tory drugs with adverse psychiatric reactions five case reportsrdquoClinical Rheumatology vol 18 no 4 pp 339ndash345 1999

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[62] A Rodrıguez-Moreno and T S Sihra ldquoMetabotropic actions ofkainate receptors in the CNSrdquo Journal of Neurochemistry vol103 no 6 pp 2121ndash2135 2007

[63] A Barbon M Popoli L la Via et al ldquoRegulation ofediting and expression of glutamate 120572-amino-propionic-acid(AMPA)kainate receptors by antidepressant drugsrdquo BiologicalPsychiatry vol 59 no 8 pp 713ndash720 2006

[64] J Egebjerg and S F Heinemann ldquoCa2+ permeability of uneditedand edited versions of the kainate selective glutamate receptorGluR6rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 90 no 2 pp 755ndash759 1993

[65] Z Melyan B Lancaster and H V Wheal ldquoMetabotropicregulation of intrinsic excitability by synaptic activation ofkainate receptorsrdquo The Journal of Neuroscience vol 24 no 19pp 4530ndash4534 2004

[66] L Hertz J Xu D Song E Yan L Gu and L Peng ldquoAstrocyticand neuronal accumulation of elevated extracellular K+ witha 23 K+Na+ flux ratio-consequences for energy metabolismosmolarity and higher brain functionrdquo Frontiers in Computa-tional Neuroscience vol 7 article 114 2013

[67] W Sun E McConnell J F Pare et al ldquoGlutamate-dependentneuroglial calcium signaling differs between young and adultbrainrdquo Science vol 339 no 6116 pp 197ndash200 2013

[68] G Shaltiel S Maeng O Malkesman et al ldquoEvidence for theinvolvement of the kainate receptor subunit GluR6 (GRIK2) inmediating behavioral displays related to behavioral symptomsofmaniardquoMolecular Psychiatry vol 13 no 9 pp 858ndash872 2008

[69] R Delorme M-O Krebs N Chabane et al ldquoFrequencyand transmission of glutamate receptors GRIK2 and GRIK3polymorphisms in patients with obsessive compulsive disorderrdquoNeuroReport vol 15 no 4 pp 699ndash702 2004

[70] A S Sampaio J Fagerness J Crane et al ldquoAssociation betweenpolymorphisms in GRIK2 gene and obsessive-compulsive dis-order a family-based studyrdquo CNS Neuroscience ampTherapeuticsvol 17 no 3 pp 141ndash147 2011

[71] G Sanacora G Treccani and M Popoli ldquoTowards a glutamatehypothesis of depression an emerging frontier of neuropsy-chopharmacology for mood disordersrdquo Neuropharmacologyvol 62 no 1 pp 63ndash77 2012

[72] L Peng B Li T Du FWang and L Hertz ldquoDoes conventionalanti-bipolar and antidepressant drug therapy reduce NMDA-mediated neuronal excitation by downregulating astrocyticGluK2 functionrdquo Pharmacology Biochemistry and Behaviorvol 100 no 4 pp 712ndash725 2012

[73] S G Tewari and K K Majumdar ldquoA mathematical modelof the tripartite synapse astrocyte-induced synaptic plasticityrdquoJournal of Biological Physics vol 38 no 3 pp 465ndash496 2012

[74] K A Lapidus L Soleimani and J W Murrough ldquoNovelglutamatergic drugs for the treatment of mood disordersrdquoJournal of Neuropsychiatric Disease and Treatment vol 9 pp1101ndash1112 2013

[75] J W Murrough A M Perez S Pillemer et al ldquoRapidand longer-term antidepressant effects of repeated ketamineinfusions in treatment-resistant major depressionrdquo BiologicalPsychiatry vol 74 no 4 pp 250ndash256 2013

[76] E Yan B Li L Gu L Hertz and L Peng ldquoMechanisms for L-channel-mediated increase in [Ca2+]

119894and its reduction by anti-

bipolar drugs in cultured astrocytes combined with its mRNAexpression in freshly isolated cells support the importance ofastrocytic L-channelsrdquo Cell Calcium vol 54 no 5 pp 335ndash3422013

[77] G Szabadkai and M R Duchen ldquoMitochondria the hub ofcellular Ca2+ signalingrdquo Physiology vol 23 no 2 pp 84ndash942008

[78] RMDenton ldquoRegulation ofmitochondrial dehydrogenases bycalcium ionsrdquo Biochimica et Biophysica Acta vol 1787 no 11 pp1309ndash1316 2009

[79] S-Z Xu and D J Beech ldquoTrpC1 is a membrane-spanningsubunit of store-operated Ca2+ channels in native vascularsmoothmuscle cellsrdquoCirculation Research vol 88 no 1 pp 84ndash87 2001

[80] B Li L Dong H Fu B Wang L Hertz and L Peng ldquoEffectsof chronic treatment with fluoxetine on receptor-stimulatedincrease of [Ca2+]

119894in astrocytes mimic those of acute inhibition

of TRPC1 channel activityrdquo Cell Calcium vol 50 no 1 pp 42ndash53 2011

[81] J-M Beaulieu R R Gainetdinov andM G Caron ldquoAktGSK3signaling in the action of psychotropic drugsrdquo Annual Reviewof Pharmacology and Toxicology vol 49 pp 327ndash347 2009

[82] R Liu W Dang M Jianting et al ldquoCitalopram alleviateschronic stress induced depression-like behaviors in rats byactivating GSK3120573 signaling in dorsal hippocampusrdquo BrainResearch vol 1467 pp 10ndash17 2012

[83] F Karege N Perroud S Burkhardt et al ldquoProtein levels of 120573-catenin and activation state of glycogen synthase kinase-3120573 inmajor depression A study with postmortem prefrontal cortexrdquoJournal of Affective Disorders vol 136 no 1-2 pp 185ndash188 2012

[84] S L Diaz S Doly N Narboux-Nme et al ldquo5-HT2B receptors

are required for serotonin-selective antidepressant actionsrdquoMolecular Psychiatry vol 17 no 2 pp 154ndash163 2012

[85] D Song T Du B Li et al ldquoAstrocytic alkalinization bytherapeutically relevant lithium concentrations implicationsfor myo-inositol depletionrdquo Psychopharmacology vol 200 no2 pp 187ndash195 2008

[86] D Song B Li E Yan et al ldquoChronic treatment with anti-bipolar drugs causes intracellular alkalinization in astrocytesaltering their functionsrdquoNeurochemical Research vol 37 no 11pp 2524ndash2540 2012

[87] Y Chen and L Hertz ldquoInhibition of noradrenaline stimulatedincrease in [Ca2+]

119894in cultured astrocytes by chronic treatment

with a therapeutically relevant lithium concentrationrdquo BrainResearch vol 711 no 1-2 pp 245ndash248 1996

[88] MWolfson Y Bersudsky E ZingerM Simkin RH Belmakerand L Hertz ldquoChronic treatment of human astrocytoma cellswith lithium carbamazepine or valproic acid decreases inositoluptake at high inositol concentrations but increases it at lowinositol concentrationsrdquo Brain Research vol 855 no 1 pp 158ndash161 2000


[89] D Song YMan B Li J Xu L Hertz and L Peng ldquoComparisonbetween drug-induced and K+-induced changes in molar acidextrusion fluxes JH

+ and in energy consumption rates inastrocytesrdquo Neurochemical Research vol 38 no 11 pp 2364ndash2374 2013

[90] S Kol K Ruutiainen-Altman I Ben-Shlomo D W Payne MAndo and E Y Adashi ldquoThe rat ovarian phospholipase A2system gene expression cellular localization activity character-ization and interleukin-1 dependencerdquo Endocrinology vol 138no 1 pp 322ndash331 1997

[91] S Ghelardoni Y A Tomita J M Bell S I Rapoport andF Bosetti ldquoChronic carbamazepine selectively downregulatescytosolic phospholipase A2 expression and cyclooxygenaseactivity in rat brainrdquoBiological Psychiatry vol 56 no 4 pp 248ndash254 2004

[92] M J Berridge ldquoInositol trisphosphate and diacylglycerol assecond messengersrdquo Biochemical Journal vol 220 no 2 pp345ndash360 1984

[93] L Hertz Y Chen Y Bersudsky and M Wolfson ldquoSharedeffects of all three conventional anti-bipolar drugs on thephosphoinositide system in astrocytesrdquo in Non-Neuronal Cellsof the Nervous System Function and Dysfunction L Hertz Edpp 1033ndash1048 Elsevier 2004

[94] S K Fisher J ENovak andBWAgranoff ldquoInositol and higherinositol phosphates in neural tissues homeostasis metabolismand functional significancerdquo Journal of Neurochemistry vol 82no 4 pp 736ndash754 2002

[95] J D Port S S Unal DAMrazek and SMMarcus ldquoMetabolicalterations in medication-free patients with bipolar disorder a3T CSF-corrected magnetic resonance spectroscopic imagingstudyrdquo Psychiatry Research vol 162 no 2 pp 113ndash121 2008

[96] H M Aukema and B J Holub ldquoInositol and pyrroloquinolinequinone A inositolrdquo in Modern Nutrition in Health andDisease M E Shils J A Olson and M Shike Eds pp 466ndash473 Lea and Febiger 1994

[97] G Agam Y Shapiro Y Bersudsky O Kofman and R HBelmaker ldquoHigh-dose peripheral inositol raises brain inositollevels and reverses behavioral effects of inositol depletion bylithiumrdquo Pharmacology Biochemistry and Behavior vol 49 no2 pp 341ndash343 1994

[98] Y Patishi B Lubrich M Berger O Kofman D van Calker andR H Belmaker ldquoDifferential uptake ofmyo-inositol in vivo intorat brain areasrdquo European Neuropsychopharmacology vol 6 no1 pp 73ndash75 1996

[99] R Spector and A V Lorenzo ldquoMyo-inositol transport in thecentral nervous systemrdquo The American Journal of Physiologyvol 228 no 5 pp 1510ndash1518 1975

[100] Y-H H Wong S J Kalmbach B K Hartman and W RSherman ldquoImmunohistochemical staining and enzyme activitymeasurements show myo-inositol-1-phosphate synthase to belocalized in the vasculature of brainrdquo Journal of Neurochemistryvol 48 no 5 pp 1434ndash1442 1987

[101] M Wolfson E Hertz R H Belmaker and L Hertz ldquoChronictreatment with lithium and pretreatment with excess inositolreduce inositol pool size in astrocytes by differentmechanismsrdquoBrain Research vol 787 no 1 pp 34ndash40 1998

[102] B Lubrich and D van Calker ldquoInhibition of the high affinitymyo-inositol transport system a commonmechanism of actionof antibipolar drugsrdquo Neuropsychopharmacology vol 21 no 4pp 519ndash529 1999

[103] J Matskevitch C A Wagner T Risler et al ldquoEffect of extra-cellular pH on the myo-inositol transporter SMIT expressed in

Xenopus oocytesrdquo Pflugers Archiv vol 436 no 6 pp 854ndash8571998

[104] M Uldry M Ibberson J-D Horisberger J-Y Chatton B MRiederer and B Thorens ldquoIdentification of a mammalian H+-myo-inositol symporter expressed predominantly in the brainrdquoThe EMBO Journal vol 20 no 16 pp 4467ndash4477 2001

[105] H Fu B Li L Hertz and L Peng ldquoContributions in astrocytesof SMIT12 and HMIT to myo-inositol uptake at differentconcentrations and pHrdquo Neurochemistry International vol 61no 2 pp 187ndash194 2012

[106] E di Daniel M H S Mok E Mead et al ldquoEvaluation ofexpression and function of the H+myo-inositol transporterHMITrdquo BMC Cell Biology vol 10 article 54 pp 1ndash12 2009

[107] D G Gossman and H-B Zhao ldquoHemichannel-mediated inos-itol 145-trisphosphate (IP

3) release in the cochlea a novel

mechanism of IP3intercellular signalingrdquo Cell Communication

amp Adhesion vol 15 no 4 pp 305ndash315 2008[108] X-H Wang M Streeter Y-P Liu and H-B Zhao ldquoIden-

tification and characterization of pannexin expression in themammalian cochleardquo The Journal of Comparative Neurologyvol 512 no 3 pp 336ndash346 2009

[109] T Du B Li H Li M Li L Hertz and L Peng ldquoSignalingpathways of isoproterenol-induced ERK

12phosphorylation in

primary cultures of astrocytes are concentration-dependentrdquoJournal of Neurochemistry vol 115 no 4 pp 1007ndash1023 2010

[110] L Hertz J Xu D Song T Du E Yan and L Peng ldquoBrainglycogenolysis adrenoceptors pyruvate carboxylase Na+K+

minus

ATPase and Marie E Gibbsrsquo pioneering learning studiesrdquoFrontiers in Integrative Neuroscience vol 7 article 20 2013

[111] G Owsianik K Talavera T Voets and B Nilius ldquoPermeationand selectivity of TRP channelsrdquo Annual Review of Physiologyvol 68 pp 685ndash717 2006

[112] R C Reyes A Verkhratsky and V Parpura ldquoTRPC1-mediatedCa2+ and Na+ signalling in astroglia differential filtering ofextracellular cationsrdquo Cell Calcium vol 54 no 2 pp 120ndash1252013

[113] I Goldstein T Levy D Galili et al ldquoInvolvement of Na+ K+-ATPase and endogenous digitalis-like compounds in depressivedisordersrdquo Biological Psychiatry vol 60 no 5 pp 491ndash4992006

[114] A M Rose B J Mellett R Valdes Jr et al ldquoAlpha 2 isoformof the Na K-adenosine triphosphatase is reduced in temporalcortex of bipolar individualsrdquo Biological Psychiatry vol 44 no9 pp 892ndash897 1998

[115] U Banerjee A Dasgupta J K Rout and O P Singh ldquoEffects oflithium therapy on Na+-K+-ATPase activity and lipid peroxida-tion in bipolar disorderrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 37 no 1 pp 56ndash61 2012

[116] L Mynett-Johnson V Murphy J McCormack et al ldquoEvidencefor an allelic association between bipolar disorder and a Na+K+ adenosine triphosphatase alpha subunit gene (ATP1A3)rdquoBiological Psychiatry vol 44 no 1 pp 47ndash51 1998

[117] H Hamakawa J Murashita N Yamada T Inubushi N Katoand T Kato ldquoReduced intracellular pH in the basal gangliaand whole brain measured by 31P-MRS in bipolar disorderrdquoPsychiatry and Clinical Neurosciences vol 58 no 1 pp 82ndash882004

[118] K Deigweiher N Koschnick H-O Portner and M LucassenldquoAcclimation of ion regulatory capacities in gills of marine fishunder environmental hypercapniardquo The American Journal ofPhysiologymdashRegulatory Integrative and Comparative Physiologyvol 295 no 5 pp R1660ndashR1670 2008


[119] J Cummins and H Hyden ldquoAdenosine triphosphate levelsand adenosine triphosphatases in neurons glia and neuronalmembranes of the vestibular nucleusrdquo Biochimica et BiophysicaActa vol 60 no 2 pp 271ndash283 1962

[120] B Li L Hertz and L Peng ldquoCell-specific mRNA alterationsin Na+ K+-ATPase 120572 and 120573 isoforms and FXYD in micetreated chronically with carbamazepine an anti-bipolar drugrdquoNeurochemical Research vol 38 no 4 pp 834ndash841 2013

[121] L Segall S E Daly and R Blostein ldquoMechanistic basis forkinetic differences between the rat 1205721 1205722 and 1205723 isoforms ofthe NaK-ATPaserdquoThe Journal of Biological Chemistry vol 276no 34 pp 31535ndash31541 2001

[122] A G Therien and R Blostein ldquoMechanisms of sodium pumpregulationrdquo The American Journal of PhysiologymdashCell Physiol-ogy vol 279 no 3 pp C541ndashC566 2000

[123] L K Bekar and W Walz ldquoIntracellular chloride modulates a-type potassium currents in astrocytesrdquo Glia vol 39 no 3 pp207ndash216 2002

[124] K Egawa J Yamada T Furukawa Y Yanagawa and A FukadaldquoClminus homeodynamics in gap junction-coupled astrocytic net-works on activation of GABAergic synapsesrdquo The Journal ofPhysiology vol 591 pp 3901ndash3917 2013

[125] S D Meier K W Kafitz and C R Rose ldquoDevelopmentalprofile and mechanisms of GABA-induced calcium signalingin hippocampal astrocytesrdquo Glia vol 56 no 10 pp 1127ndash11372008

[126] M-R Zarrindast P Rostami and M Sadeghi-Hariri ldquoGABAAbut not GABAB receptor stimulation induces antianxiety profilein ratsrdquo Pharmacology Biochemistry and Behavior vol 69 no 1-2 pp 9ndash15 2001

[127] B E Yoon JWoo and C J Lee ldquoAstrocytes as GABA-ergic andGABA-ceptive cellsrdquoNeurochemical Research vol 37 no 11 pp22474ndash22479 2012

[128] L Hertz Z Zhao and Y Chen ldquoThe astrocytic GABAAbenzo-diazepine-like receptor the Joker receptor for benzodiazepine-mimetic drugsrdquo Recent Patents on CNS Drug Discovery vol 1no 1 pp 93ndash103 2006

[129] K V Subbarao J U Stolzenburg and L Hertz ldquoPharmaco-logical characteristics of potassium-induced glycogenolysis inastrocytesrdquo Neuroscience Letters vol 196 no 1-2 pp 45ndash481995

[130] T Goetz A Arslan W Wisden and P Wulff ldquoGABAA recep-tors structure and function in the basal gangliardquo Progress inBrain Research vol 160 pp 21ndash41 2007

[131] J D Cahoy B Emery A Kaushal et al ldquoA transcriptomedatabase for astrocytes neurons and oligodendrocytes a newresource for understanding brain development and functionrdquoThe Journal of Neuroscience vol 28 no 1 pp 264ndash278 2008

[132] W Wisden D J Laurie H Monyer and P H Seeburg ldquoThedistribution of 13 GABAA receptor subunit mRNAs in the ratbrain I Telencephalon diencephalon mesencephalonrdquo TheJournal of Neuroscience vol 12 no 3 pp 1040ndash1062 1992

[133] U Rudolph and H M Mohler ldquoGABAA receptor sub-typestherapeutic potential in down syndrome affective disor-ders schizophrenia and autismrdquo Annual Review of Pharmacol-ogyand Toxicology vol 54 pp 483ndash507 2014

[134] S A Bergman ldquoThe benzodiazepine receptorrdquo AnesthesiaProgress vol 33 no 5 pp 213ndash219 1986

[135] V Papadopoulos and L Lecanu ldquoTranslocator protein (18kDa) TSPO an emerging therapeutic target in neurotraumardquoExperimental Neurology vol 219 no 1 pp 53ndash57 2009

[136] B Costa E da Pozzo and C Martini ldquoTranslocator proteinas a promising target for novel anxiolyticsrdquo Current Topics inMedicinal Chemistry vol 12 no 4 pp 270ndash285 2012

[137] P Ferrero E Costa B Conti-Tronconi and A Guidotti ldquoAdiazepambinding inhibitor (DBI)-like neuropeptide is detectedin human brainrdquo Brain Research vol 399 no 1 pp 136ndash1421986

[138] E Slobodyansky A Guidotti C Wambebe A Berkovich andE Costa ldquoIsolation and characterization of a rat brain triakon-tatetraneuropeptide a posttranslational product of diazepambinding inhibitor specific action at the Ro 5-4864 recognitionsiterdquo Journal of Neurochemistry vol 53 no 4 pp 1276ndash12841989

[139] P Gandolfo E Louiset C Patte et al ldquoThe triakontatetra-neuropeptide TTN increases [Ca2+]

119894in rat astrocytes through

activation of peripheral-type benzodiazepine receptorsrdquo Gliavol 35 no 2 pp 90ndash100 2001

[140] K H Backus H Kettenmann and M Schachner ldquoEffectof benzodiazepines and pentobarbital on the GABA-induceddepolarization in cultured astrocytesrdquoGlia vol 1 no 2 pp 132ndash140 1988

[141] A S Bender and L Hertz ldquoPharmacological evidence that thenon-neuronal diazepam binding site in primary cultures of glialcells is associated with a calcium channelrdquo European Journal ofPharmacology vol 110 no 2 pp 287ndash288 1985

[142] Z Zhao L Hertz and W E Code ldquoEffects of benzodiazepineson potassium-induced increase in free cytosolic calcium con-centration in astrocytes interactions with nifedipine and theperipheral-type benzodiazepine antagonist PK 11195rdquoCanadianJournal of Physiology and Pharmacology vol 74 no 3 pp 273ndash277 1996

[143] Y Itzhak L Baker and M D Norenberg ldquoCharacterizationof the peripheral-type benzodiazepine receptors in culturedastrocytes evidence for multiplicityrdquo Glia vol 9 no 3 pp 211ndash218 1993

[144] G T Bolger A H Newman K C Rice H W M LueddensA S Basile and P Skolnick ldquoCharacterization of the effects ofAHN086 an irreversible ligand of ldquoperipheralrdquo benzodiazepinereceptors on contraction in guinea-pig atria and ileal longi-tudinal smooth musclerdquo Canadian Journal of Physiology andPharmacology vol 67 no 2 pp 126ndash134 1989

[145] G T Bolger S Abraham N Oz and B A Weissman ldquoInterac-tions between peripheral-type benzodiazepine receptor ligandsand an activator of voltage-operated calcium channelsrdquo Cana-dian Journal of Physiology and Pharmacology vol 68 no 1 pp40ndash45 1990

[146] L Hertz and Y Chen ldquoThe astroyctic GABAAbenxodiazepine-like receptor identifying the Joker receptor for benzodiazeping-mimetic drugsrdquo in Frontiers in CNS Drug Discovery Atta-ur-Rahman andM Iqbal Choudhary Eds pp 342ndash361 Bentham2010

[147] J Kanto L Kangas and T Siirtola ldquoCerebrospinal fluid con-centrations of diazepam and its metabolites in manrdquo ActaPharmacologica et Toxicologica vol 36 no 4 pp 328ndash334 1975

[148] P Polc ldquoElectrophysiology of benzodiazepine receptor ligandsmultiple mechanisms and sites of actionrdquo Progress in Neurobi-ology vol 31 no 5 pp 349ndash423 1988

[149] R Yu and M K Ticku ldquoChronic neurosteroid treatmentdecreases the efficacy of benzodiazepine ligands and neuros-teroids at the 120574-aminobutyric acid(A) receptor complex inmammalian cortical neuronsrdquoThe Journal of Pharmacology andExperimental Therapeutics vol 275 no 2 pp 784ndash789 1995


[150] J M M Olson B J Ciliax W R Mancini and A B YoungldquoPresence of peripheral-type benzodiazepine binding sites onhuman erythrocyte membranesrdquo European Journal of Pharma-cology vol 152 no 1-2 pp 47ndash53 1988

[151] G A Kennett F Bright B Trail G S Baxter and T PBlackburn ldquoEffects of the 5-HT

2B receptor agonist BW723C86on three rat models of anxietyrdquo British Journal of Pharmacologyvol 117 no 7 pp 1443ndash1448 1996

[152] M S Duxon G A Kennett S Lightowler T P Blackburn andK C F Fone ldquoActivation of 5-HT

2B receptors in the medialamygdala causes anxiolysis in the social interaction test in theratrdquo Neuropharmacology vol 36 no 4-5 pp 601ndash608 1997

[153] G A Kennett B Trail and F Bright ldquoAnxiolytic-like actions ofBW 723C86 in the rat Vogel conflict test are 5-HT

2B receptormediatedrdquo Neuropharmacology vol 37 no 12 pp 1603ndash16101998

[154] X Zhang L Peng Y Chen and L Hertz ldquoStimulation ofglycogenolysis in astrocytes byu fluoxetine an antidepressantacting like 5-HTrdquoNeuroReport vol 4 no 11 pp 1235ndash1238 1993

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Comparison between effects on gene expression and editing of chronic treatment with the SSRI fluoxetine or the antibipolar drugcarbamazepine in cultured mouse astrocytes and in astrocytes freshly isolated from drug-treated mice

Gene Drug FACS astrocytes Culture astrocytesADAR2 Fluoxetine Up Up5-HT2B receptor expression Fluoxetine Up Up5-HT2B editing Fluoxetine Up Up5-HT2c receptor expression Fluoxetine Unchanged UnchangedcPLA2a Fluoxetine Up UpsPLA2 Fluoxetine Unaltered UnalteredGluK2 expression Fluoxetine Up UpGluK2 editing Fluoxetine Up UpGluK4 expression Fluoxetine Unchanged Unchangedcfos expression Fluoxetine Up UpfosB expression Fluoxetine Up UpCav 12 Fluoxetine Up UpNBCe1 Carbamazepine Up UpGluK2 Carbamazepine Down DowncPLA2 Carbamazepine Up UpTable 1 shows all experiments in which drug effects were compared in cultured astrocytes and in astrocytes freshly obtained by FACS as described by Lovattet al [3] For FACS astrocytes had been obtained from mice stained with GFP based on expression of the astrocyte-specific GFAP in transgenic animalsand the stained cells were separated after cell dissociation by means of their fluorescent signal The cultures were treated chronically with either fluoxetine orcarbamazepine and the animals had been treated chronically for a similar length of time (2 weeks) Complete agreement was found From Peng et al [2] withthe exception of Cav12 which is from Du et al [4] using similar techniques

to cultured astrocytes [7] which have no SERT expression[8] However at that time astrocytes were supposed to beunimportant for brain function and inhibition of SERT hassince then been regarded as the mechanism responsible forSSRIs effects In 1987 the 5-HT

2B receptor was unknown butit is now established to have the highest affinity between 5-HTreceptors for SSRIs with a K

119894for displacement of serotonin

binding to cultured astrocytes of 70 nM [5] This is identicalto its K

119894for inhibition of serotonin uptake via the human

placental SERT [9] Moreover all five SSRIs are equipotentin their effect on astrocytes [10] This is a distinct differencefrom the large potency difference in their effect on SERTalthough the therapeutic doses are of roughly comparablemagnitudes a difference which can only partly be explainedby differences in drug kinetics and protein binding

The metabolic pathway activated in cultured astrocytesby fluoxetine was first established by Li et al [11] and anexpanded version is shown in Figure 1 With increasingrealization of the importance of glycogenolysis for signalingin astrocytes [12ndash14] it may be important that fluoxetineacutely stimulates glycogenolysis an effect that is secondaryto an increase in [Ca2+]

119894[15] Fluoxetine might also affect

glycogen synthesis since it stimulates the AKT pathway(see below) The involvement of 5-HT

2B receptor-stimulatedglycogenolysis has been established during learning whereacute administration of serotonin can rescue long-termlearning in a one trial aversive learning paradigm in day-oldchickens under conditions when the aversive stimulus wasotherwise too weak to establish more than transient long-term memory retention [16] Fluoxetine and paroxetine havea similar effect and are equipotent showing that the rescuewas not due to inhibition of SERT and the rescuing effect was

inhibited by an inhibitor of glycogenolysis (Gibbs and Hertzsubmitted Frontiers in Pharmacology (Neuropharmacology)after invitation to Research Topic)

In accordance with Figure 1 fluoxetine acutely phos-phorylates AKT in cultured astrocytes [5] AKT-mediatedphosphorylation of glycogen synthase kinase-3120573 (GSK-3120573)inhibits its enzymatic activity AKT phosphorylation maystimulate glycogen synthesis since phosphorylation andactivation of glycogen synthase by GSK-3 decreases theactivity of glycogen synthase [17] It is consistent with theseobservations that Li et al [18] from the Jope group foundin whole brain that fluoxetine administration increases thelevels of phosphorylated GSK3120573 Later Beurel et al [19]from the same group confirmed that fluoxetine rapidly androbustly increases serine phosphorylation of GSK3 but thatthese responses in young mice are blunted or absent Thisis consistent with an effect on astrocytes since these glialcells are generated postnatally [20] An increased glycogensynthesis following 5-HT

2B receptor activation has beendirectly shown in hepatocytes whereas agonists of 5-HT

1and

5-HT2A receptors had the opposite effect [21]

22 Chronic Effects

221 Affected Genes The effects of SSRIs that are importantin connection with major depression are the chronic effectssince the effect of clinical treatment takes several weeks toappear To-date all gene effects caused by chronic treatmentwith fluoxetine (the only SSRI studied on astrocytes in vivo)after mice had been treated for 14 days with ip injection offluoxetine (10mgkg per day) have been identical to thosein cultured astrocytes chronically treated with fluoxetine or


5 2B

U

3120573

2 gt 3

Fluoxetine

siRNA

SB204741

BAPTAAM

AG1478

LY294002

GM6001

GF 109203X

MMP

MEK Raf Ras

Gene expression

ERK

HB-EGF

EGFR

pEGFR

AKT

PKC

PI3K

Ca2+




3from PIP



2and











2[38]



2(secretory

PLA2(sPLA


2(iPLA

2)) Li et al [28]














2



0

2

4

6

8

rCM

Rglu

minus6

minus4

minus2







119894







2


2and






3(Figure 4(d))










3) receptors (IP












Control Fluoxetine

Control

Control Fluoxetine

CMS

Neu

ron

(YFP

H)

mGluR5

TBP

Astr

ocyt

es

(GFP

) mGluR5

TBP

(a)

0

05

1

15



Ratio

of m

Glu

R5T

BP

(b)























0

1

2

3

4


52B lowast

lowast

lowast

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(a)

0

1

2


52B

120573

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(b)

0

05

1

15



Ratio

ofA


G+

A

(c)

0

500

1000

1500

Control Fluoxetine



lowast

lowastlowast

lowast

(d)


2000

4000

6000

8000

10000



lowast

(e)












MIT

IP3R RyR

Ca2+

Ca2+

Soc (TRPC 1)

Ca2+Na+

exchanger






2







2) in the









2via











0

30

20

10Gly

coge

noly

sis (

)

(a)

0

30

20

10Gly

coge

noly

sis (

)

(b)


GSK3120573

p-GSK3120573

GAPDH

(a)

00

05

10

15


(b)

1

2

3



lowastlowastlowast

(c)



7

71

72

73

74

75

76

77

78

1 2 3 4Time (weeks)

0

05

1

15


Ratio

ofN

HE1T

BP


(a1) (a2)

pHi

(a)

0

05

1

15


Control CBZ

Ratio

ofN

BCe1T

BP

lowast

0

05

1

15

2


Control CBZ

Ratio

ofN

HE1T

BP lowast

(b1) (b2)

(b)




119898143 120583M and Vmax 358 pmolmg





0

250

500

750

lowast

lowast

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(a)

0

400

200

600

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(b)





0

10

30

40

50

60

Inos

itol u

ptak

e (nm

olm

g)

25 40 50

lowast

lowast

(120583M)





3might be trans-






119894









Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl


(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)




2tension





0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5 2B

U

3120573

2 gt 3

Fluoxetine

siRNA

SB204741

BAPTAAM

AG1478

LY294002

GM6001

GF 109203X

MMP

MEK Raf Ras

Gene expression

ERK

HB-EGF

EGFR

pEGFR

AKT

PKC

PI3K

Ca2+




3from PIP



2and











2[38]



2(secretory

PLA2(sPLA


2(iPLA

2)) Li et al [28]














2



0

2

4

6

8

rCM

Rglu

minus6

minus4

minus2







119894







2


2and






3(Figure 4(d))










3) receptors (IP












Control Fluoxetine

Control

Control Fluoxetine

CMS

Neu

ron

(YFP

H)

mGluR5

TBP

Astr

ocyt

es

(GFP

) mGluR5

TBP

(a)

0

05

1

15



Ratio

of m

Glu

R5T

BP

(b)























0

1

2

3

4


52B lowast

lowast

lowast

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(a)

0

1

2


52B

120573

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(b)

0

05

1

15



Ratio

ofA


G+

A

(c)

0

500

1000

1500

Control Fluoxetine



lowast

lowastlowast

lowast

(d)


2000

4000

6000

8000

10000



lowast

(e)












MIT

IP3R RyR

Ca2+

Ca2+

Soc (TRPC 1)

Ca2+Na+

exchanger






2







2) in the









2via











0

30

20

10Gly

coge

noly

sis (

)

(a)

0

30

20

10Gly

coge

noly

sis (

)

(b)


GSK3120573

p-GSK3120573

GAPDH

(a)

00

05

10

15


(b)

1

2

3



lowastlowastlowast

(c)



7

71

72

73

74

75

76

77

78

1 2 3 4Time (weeks)

0

05

1

15


Ratio

ofN

HE1T

BP


(a1) (a2)

pHi

(a)

0

05

1

15


Control CBZ

Ratio

ofN

BCe1T

BP

lowast

0

05

1

15

2


Control CBZ

Ratio

ofN

HE1T

BP lowast

(b1) (b2)

(b)




119898143 120583M and Vmax 358 pmolmg





0

250

500

750

lowast

lowast

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(a)

0

400

200

600

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(b)





0

10

30

40

50

60

Inos

itol u

ptak

e (nm

olm

g)

25 40 50

lowast

lowast

(120583M)





3might be trans-






119894









Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl


(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)




2tension





0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2[38]



2(secretory

PLA2(sPLA


2(iPLA

2)) Li et al [28]














2



0

2

4

6

8

rCM

Rglu

minus6

minus4

minus2







119894







2


2and






3(Figure 4(d))










3) receptors (IP












Control Fluoxetine

Control

Control Fluoxetine

CMS

Neu

ron

(YFP

H)

mGluR5

TBP

Astr

ocyt

es

(GFP

) mGluR5

TBP

(a)

0

05

1

15



Ratio

of m

Glu

R5T

BP

(b)























0

1

2

3

4


52B lowast

lowast

lowast

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(a)

0

1

2


52B

120573

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(b)

0

05

1

15



Ratio

ofA


G+

A

(c)

0

500

1000

1500

Control Fluoxetine



lowast

lowastlowast

lowast

(d)


2000

4000

6000

8000

10000



lowast

(e)












MIT

IP3R RyR

Ca2+

Ca2+

Soc (TRPC 1)

Ca2+Na+

exchanger






2







2) in the









2via











0

30

20

10Gly

coge

noly

sis (

)

(a)

0

30

20

10Gly

coge

noly

sis (

)

(b)


GSK3120573

p-GSK3120573

GAPDH

(a)

00

05

10

15


(b)

1

2

3



lowastlowastlowast

(c)



7

71

72

73

74

75

76

77

78

1 2 3 4Time (weeks)

0

05

1

15


Ratio

ofN

HE1T

BP


(a1) (a2)

pHi

(a)

0

05

1

15


Control CBZ

Ratio

ofN

BCe1T

BP

lowast

0

05

1

15

2


Control CBZ

Ratio

ofN

HE1T

BP lowast

(b1) (b2)

(b)




119898143 120583M and Vmax 358 pmolmg





0

250

500

750

lowast

lowast

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(a)

0

400

200

600

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(b)





0

10

30

40

50

60

Inos

itol u

ptak

e (nm

olm

g)

25 40 50

lowast

lowast

(120583M)





3might be trans-






119894









Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl


(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)




2tension





0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



119894







2


2and






3(Figure 4(d))










3) receptors (IP












Control Fluoxetine

Control

Control Fluoxetine

CMS

Neu

ron

(YFP

H)

mGluR5

TBP

Astr

ocyt

es

(GFP

) mGluR5

TBP

(a)

0

05

1

15



Ratio

of m

Glu

R5T

BP

(b)























0

1

2

3

4


52B lowast

lowast

lowast

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(a)

0

1

2


52B

120573

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(b)

0

05

1

15



Ratio

ofA


G+

A

(c)

0

500

1000

1500

Control Fluoxetine



lowast

lowastlowast

lowast

(d)


2000

4000

6000

8000

10000



lowast

(e)












MIT

IP3R RyR

Ca2+

Ca2+

Soc (TRPC 1)

Ca2+Na+

exchanger






2







2) in the









2via











0

30

20

10Gly

coge

noly

sis (

)

(a)

0

30

20

10Gly

coge

noly

sis (

)

(b)


GSK3120573

p-GSK3120573

GAPDH

(a)

00

05

10

15


(b)

1

2

3



lowastlowastlowast

(c)



7

71

72

73

74

75

76

77

78

1 2 3 4Time (weeks)

0

05

1

15


Ratio

ofN

HE1T

BP


(a1) (a2)

pHi

(a)

0

05

1

15


Control CBZ

Ratio

ofN

BCe1T

BP

lowast

0

05

1

15

2


Control CBZ

Ratio

ofN

HE1T

BP lowast

(b1) (b2)

(b)




119898143 120583M and Vmax 358 pmolmg





0

250

500

750

lowast

lowast

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(a)

0

400

200

600

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(b)





0

10

30

40

50

60

Inos

itol u

ptak

e (nm

olm

g)

25 40 50

lowast

lowast

(120583M)





3might be trans-






119894









Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl


(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)




2tension





0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Control Fluoxetine

Control

Control Fluoxetine

CMS

Neu

ron

(YFP

H)

mGluR5

TBP

Astr

ocyt

es

(GFP

) mGluR5

TBP

(a)

0

05

1

15



Ratio

of m

Glu

R5T

BP

(b)























0

1

2

3

4


52B lowast

lowast

lowast

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(a)

0

1

2


52B

120573

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(b)

0

05

1

15



Ratio

ofA


G+

A

(c)

0

500

1000

1500

Control Fluoxetine



lowast

lowastlowast

lowast

(d)


2000

4000

6000

8000

10000



lowast

(e)












MIT

IP3R RyR

Ca2+

Ca2+

Soc (TRPC 1)

Ca2+Na+

exchanger






2







2) in the









2via











0

30

20

10Gly

coge

noly

sis (

)

(a)

0

30

20

10Gly

coge

noly

sis (

)

(b)


GSK3120573

p-GSK3120573

GAPDH

(a)

00

05

10

15


(b)

1

2

3



lowastlowastlowast

(c)



7

71

72

73

74

75

76

77

78

1 2 3 4Time (weeks)

0

05

1

15


Ratio

ofN

HE1T

BP


(a1) (a2)

pHi

(a)

0

05

1

15


Control CBZ

Ratio

ofN

BCe1T

BP

lowast

0

05

1

15

2


Control CBZ

Ratio

ofN

HE1T

BP lowast

(b1) (b2)

(b)




119898143 120583M and Vmax 358 pmolmg





0

250

500

750

lowast

lowast

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(a)

0

400

200

600

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(b)





0

10

30

40

50

60

Inos

itol u

ptak

e (nm

olm

g)

25 40 50

lowast

lowast

(120583M)





3might be trans-






119894









Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl


(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)




2tension





0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1

2

3

4


52B lowast

lowast

lowast

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(a)

0

1

2


52B

120573

lowast

lowastlowast

Control120583M120583M120583M120583M

1120583M1120583M5120583M5120583M10120583M10120583M

(b)

0

05

1

15



Ratio

ofA


G+

A

(c)

0

500

1000

1500

Control Fluoxetine



lowast

lowastlowast

lowast

(d)


2000

4000

6000

8000

10000



lowast

(e)












MIT

IP3R RyR

Ca2+

Ca2+

Soc (TRPC 1)

Ca2+Na+

exchanger






2







2) in the









2via











0

30

20

10Gly

coge

noly

sis (

)

(a)

0

30

20

10Gly

coge

noly

sis (

)

(b)


GSK3120573

p-GSK3120573

GAPDH

(a)

00

05

10

15


(b)

1

2

3



lowastlowastlowast

(c)



7

71

72

73

74

75

76

77

78

1 2 3 4Time (weeks)

0

05

1

15


Ratio

ofN

HE1T

BP


(a1) (a2)

pHi

(a)

0

05

1

15


Control CBZ

Ratio

ofN

BCe1T

BP

lowast

0

05

1

15

2


Control CBZ

Ratio

ofN

HE1T

BP lowast

(b1) (b2)

(b)




119898143 120583M and Vmax 358 pmolmg





0

250

500

750

lowast

lowast

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(a)

0

400

200

600

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(b)





0

10

30

40

50

60

Inos

itol u

ptak

e (nm

olm

g)

25 40 50

lowast

lowast

(120583M)





3might be trans-






119894









Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl


(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)




2tension





0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


MIT

IP3R RyR

Ca2+

Ca2+

Soc (TRPC 1)

Ca2+Na+

exchanger






2







2) in the









2via











0

30

20

10Gly

coge

noly

sis (

)

(a)

0

30

20

10Gly

coge

noly

sis (

)

(b)


GSK3120573

p-GSK3120573

GAPDH

(a)

00

05

10

15


(b)

1

2

3



lowastlowastlowast

(c)



7

71

72

73

74

75

76

77

78

1 2 3 4Time (weeks)

0

05

1

15


Ratio

ofN

HE1T

BP


(a1) (a2)

pHi

(a)

0

05

1

15


Control CBZ

Ratio

ofN

BCe1T

BP

lowast

0

05

1

15

2


Control CBZ

Ratio

ofN

HE1T

BP lowast

(b1) (b2)

(b)




119898143 120583M and Vmax 358 pmolmg





0

250

500

750

lowast

lowast

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(a)

0

400

200

600

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(b)





0

10

30

40

50

60

Inos

itol u

ptak

e (nm

olm

g)

25 40 50

lowast

lowast

(120583M)





3might be trans-






119894









Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl


(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)




2tension





0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

30

20

10Gly

coge

noly

sis (

)

(a)

0

30

20

10Gly

coge

noly

sis (

)

(b)


GSK3120573

p-GSK3120573

GAPDH

(a)

00

05

10

15


(b)

1

2

3



lowastlowastlowast

(c)



7

71

72

73

74

75

76

77

78

1 2 3 4Time (weeks)

0

05

1

15


Ratio

ofN

HE1T

BP


(a1) (a2)

pHi

(a)

0

05

1

15


Control CBZ

Ratio

ofN

BCe1T

BP

lowast

0

05

1

15

2


Control CBZ

Ratio

ofN

HE1T

BP lowast

(b1) (b2)

(b)




119898143 120583M and Vmax 358 pmolmg





0

250

500

750

lowast

lowast

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(a)

0

400

200

600

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(b)





0

10

30

40

50

60

Inos

itol u

ptak

e (nm

olm

g)

25 40 50

lowast

lowast

(120583M)





3might be trans-






119894









Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl


(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)




2tension





0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


7

71

72

73

74

75

76

77

78

1 2 3 4Time (weeks)

0

05

1

15


Ratio

ofN

HE1T

BP


(a1) (a2)

pHi

(a)

0

05

1

15


Control CBZ

Ratio

ofN

BCe1T

BP

lowast

0

05

1

15

2


Control CBZ

Ratio

ofN

HE1T

BP lowast

(b1) (b2)

(b)




119898143 120583M and Vmax 358 pmolmg





0

250

500

750

lowast

lowast

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(a)

0

400

200

600

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(b)





0

10

30

40

50

60

Inos

itol u

ptak

e (nm

olm

g)

25 40 50

lowast

lowast

(120583M)





3might be trans-






119894









Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl


(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)




2tension





0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

250

500

750

lowast

lowast

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(a)

0

400

200

600

C Na C+Na Li+Na

[Ca2+

] i(n

m)

(b)





0

10

30

40

50

60

Inos

itol u

ptak

e (nm

olm

g)

25 40 50

lowast

lowast

(120583M)





3might be trans-






119894









Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl


(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)




2tension





0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13Ra

tio o

f340380

Time (min)

1mM Li+

45mM KCl


(a)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

50120583M CBZ

45mM KCl


Ratio

of3

40380

(b)

Control

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

09

10

11

12

13

Time (min)

45mM KCl

1mM VPA


Ratio

of3

40380

(c)




2tension





0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

05

1


Nor

mal

ized

by

TBP

ATP1205721 ATP1205722 ATP1205723

lowastlowast

lowast













2+]119894increases


Con GABA0

20

40

60

80

100

120

+5K+

+5K+

GABA+

lowastlowast

lowast

Gly

coge

n co

nten

t (

of c

ontro

l)







300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


300

250

200

150

100

50

0Control + + + +

2+

lowast

] i(

)

(a)

500

400

300

200

100


lowast

2+] i

()

(b)







Gly

coge

noly

sis (

)

10K+

30 s20K+

60 s10 K+ Mid

30 s

0

10

20

30

40

50



119860

















119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












119894and causes






119890values


119890is probably













2




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgment


References





1199071











































































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































































































minus




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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