Dopamine abnormalities in the neocortex of patients with temporal lobe epilepsy

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Neurobiology of Disease xxx (2011) xxx–xxx

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Neurobiology of Disease

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Dopamine abnormalities in the neocortex of patients with temporal lobe epilepsy

Luisa Rocha a,⁎, Mario Alonso-Vanegas b, Juana Villeda-Hernández b, Mario Mújica b,José Miguel Cisneros-Franco b, Mario López-Gómez b, Cecilia Zavala-Tecuapetla a,Christian Lizette Frías-Soria a, José Segovia-Vila c, Anna Borsodi d

a Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados, Mexicob Instituto Nacional de Neurología y Neurocirugía “Manuel Velasco Suárez”, Mexicoc Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados, Mexicod Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary

⁎ Corresponding autor at: Depto. Farmacobiología, Cin235, Col. Granjas Coapa, México, D.F. C.P. 14330, Mexico.

E-mail address: [email protected] (L. Rocha).Available online on ScienceDirect (www.scienced

0969-9961/$ – see front matter © 2011 Published by Eldoi:10.1016/j.nbd.2011.09.006

Please cite this article as: Rocha, L., et al., D(2011), doi:10.1016/j.nbd.2011.09.006

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Article history:Received 15 June 2011Revised 3 September 2011Accepted 13 September 2011Available online xxxx

Keywords:Temporal lobe epilepsyDopamineD1 receptorsD2 receptorsDopamine transporterNeuropsychiatric disorders

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RRECTED PExperiments were designed to evaluate different variables of the dopaminergic system in the temporal cortex
of surgically treated patients with temporal lobe epilepsy (TLE) associated with mesial sclerosis (MTLE,n=12) or with cerebral tumor or lesion (n=8). In addition, we sought to identify dopaminergic abnormal-ities in those patients with epilepsy that had comorbid anxiety and depression. Specifically, we investigatedchanges in dopamine and its metabolites, D1 and D2 receptors, tyrosine hydroxylase (TH) and dopaminetransporter. Results obtained from patients with epilepsy were compared with those found in experimentsusing autopsy material. The neocortex of patients with MTLE demonstrated high D1 expression (1680%,pb0.05) and binding (layers I–II, 31%, pb0.05; layers V–VI, 28%, pb0.05), and decreased D2 expression(77%, pb0.05). The neocortex of patients with TLE secondary to cerebral tumor or lesion showed high expres-sion of D1 receptors (1100%, pb0.05), and D2-like induced activation of G proteins (layers I–I, 503%; layersIII–IV, 557%; layers V–VI, 964%, pb0.05). Both epileptic groups presented elevated binding to the dopaminetransporter and low tissue content of dopamine and its metabolites. Analysis revealed the following correla-tions: a) D1 receptor binding correlated negatively with seizure onset age and seizure frequency, and posi-tively with duration of epilepsy; b) D2 receptor binding correlated positively with age of seizure onset andnegatively with duration of epilepsy; c) dopamine transporter binding correlated positively with durationof epilepsy and frequency of seizures; d) D2-like induced activation of G proteins correlated positivelywith the age of patients. When compared with autopsies and patients with anxiety and depression, patientswithout neuropsychiatric disorders showed high D2-like induced activation of G proteins, an effect that cor-related positively with age of patient and seizure onset age, and negatively with duration of epilepsy. Thepresent study suggests that alterations of the dopaminergic system result from epileptic activity and couldbe involved in the physiopathology of TLE and the comorbid anxiety and depression.

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UNCIntroduction

Epilepsy is the second most important neurologic disorder withdiverse etiology. Temporal lobe epilepsy (TLE) is the most frequentand becomes pharmacologically untreatable in a high percentage ofpatients (Engel, 1996). In TLE associated with mesial sclerosis (MTLE),the hippocampus represents the epileptic focus, while the temporalneocortex is involved in propagation of epileptic seizures to otherbrain areas (Chagnac-Amitai and Connors, 1989; Chervin et al., 1988;Sloviter, 1994). Neocortex has been proposed to be involved in

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secondary epileptogenesis as a consequence of progressive extensionof the primary epileptogenic zone with increased duration of epilep-sy (Morrell et al., 1987).

Epilepsy involves changes in several neurotransmitters (Fisherand Leppik, 2008; Olsen and Avoli, 1997). Concerning the dopaminer-gic system in temporal neocortex of patients with MTLE, studies usingPositron Emission Tomography (PET) indicate a reduction of D2/D3receptor binding in the pole and lateral areas, ipsilateral to the epilepticfocus (Werhahn et al., 2006). Regarding tissue content of dopamine andits metabolites, the results are controversial and it seems that changesdepend on the presence of epileptiform activity in the neocortex(Louw et al., 1989; Mori et al., 1987; Pacia et al., 2001; Pintor et al.,1990). In relation with tyrosine hydroxylase (TH), the enzyme respon-sible for DOPA synthesis (the precursor of dopamine), its activity isnot modified (Pintor et al., 1990). Despite the fact that D1 receptorsplay a modulating role in the synaptic activity of the neocortex

tients with temporal lobe epilepsy, Neurobiol. Dis.

http://dx.doi.org/10.1016/j.nbd.2011.09.006

mailto:[email protected]


http://www.sciencedirect.com/science/journal/09699961


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(Bandyopadhyay and Hablitz, 2007; Wu and Hablitz, 2005), it is cur-rently unknown if they are altered in the brain of patients with MTLE.

It is described that dopaminergic system is involved in anxiety anddepression (Lehto et al., 2008; Sarkisova et al., 2008). Although studiessupport a bidirectional relationship between depression and epilepsy(Kanner, 2011), at present no evidence exists to support that dopami-nergic system represents a common potential pathogenic mechanismsoperant in both disorders.

The present study was designed to determine the precise condi-tion of dopaminergic system in the temporal neocortex of patientswith MTLE. With the purpose to investigate the influence of clinicalfactors on dopaminergic alterations, the results obtained were corre-lated with subject's age, age of seizure onset, duration of epilepsy andfrequency of ictal events. Additionally, data were compared with anaged matched group of patients with TLE secondary to brain tumoror lesion that had shorter epilepsy duration and older age at seizureonset. Dopaminergic abnormalities were also investigated in thosepatients with epilepsy that had comorbid anxiety and depression.

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Table 1Summary of clinical data from patients with Temporal Lobe Epilepsy and Autopsies.

Patient Gender Age(years)

Seizureonset age(years)

Precipitatingfactors

Side offocus

Duration ofepilepsy(years)

Seizurefrequency(month)

P56 M 27 4 Hypoxia febrileseizures inchildhood

Right 23 3

P81 F 38 6 No Left 32 3

P88 F 29 8 No Right 21 30P93 M 25 7 Hypoxia Left 18 16P95 F 47 25 Cerebral

thrombosisRight 22 14

P98 M 32 8 Hypoxia Left 24 7.5

P104 M 60 6 No Left 54 9P105 M 24 6 Hypoxia febrile

seizures inchildhood

Right 18 4

P107 M 34 6 Cerebral injury Left 28 3

P123 M 35 13 Cerebral injury Left 22 12.5

P125 M 45 17 No Left 28 48

P127 F 38 3 Cerebral injuryfebrile seizuresin childhood

Left 35 15

P37 F 27 11 – Right 16 32

P54 F 34 1.33 – Right 32.6 38

P92 F 27 27 – Left 0.33 1

P99 F 48 46 – Left 2 18

P103 M 28 13 – Right 15 3

P112 M 40 40 – Right 0.16 1

P116 F 24 10 – Left 14 102

P119 M 32 28 – Left 4 1

C1 M 40 – – – – –

C2 M 29 – – – – –

C3 M 37 – – – – –

C4 M 33 – – – – –

C5 M 47 – – – – –

C6 M 51 – – – – –

AED, antiepileptic drugs; CBZ, carbamazepine; CLB, clobazam; CNZ, clonazepam; F, femaleepilepsy; OXCBZ, oxcarbazepine; PMI, POSTMORTEM INTERVAL; VAP, valproic acid; TOP, to

Please cite this article as: Rocha, L., et al., Dopamine abnormalities in th(2011), doi:10.1016/j.nbd.2011.09.006

F

Materials and methods

Patients and tissue collection

Temporal neocortex samples were collected from a group of 12patients with MTLE (7 men and 5 women) and from 8 patients withTLE secondary to brain tumor or lesion (4 men and 4 women).Table 1 includes a summary of relevant clinical data and location ofthe epileptic focus for each patient. Patients were submitted to theprotocol set out by the Epilepsy Surgery Program of the NationalInstitute of Neurology and Neurosurgery “Manuel Velasco Suarez” inMexico, which comprises an extensive pre-surgical clinical evalua-tion, video-electroencephalogram (EEG) record, magnetic resonanceimaging (MRI) and single photon emission computed tomography(SPECT). The EEG was used essentially to locate interictal epilepti-form activity. The video-EEG allowed at least the record of twocomplex partial seizures in each patient. Since ictal SPECT was not per-formed in every patient, interictal SPECT revealed valuable information

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AED beforesurgery

Psychiatriccomorbidity

Surgical outcome(Engel's classification)

Final diagnosis or cause ofdeath (PMI in hours)

CBZ, LMG,CNZ

No Class I MTLE

OXCBZ Depressionanxiety

Class II MTLE

VAP, CLB No Class I MTLECBZ, CNZ Depression Class I MTLELMG, CNZ,ZNS

Depression Class I MTLE

VAP, LMG,CLB

No Class IV Class IV

CBZ, LMG No Class I MTLEVAP, TOPCNZ

Depression Class I MTLE

VAP, CBZ,CLB

Depressionanxiety

Class II MTLE

VAP, CBZ,LMG

No Class I MTLE

OXCBZ, TOP,CLB

Anxiety Class I MTLE

VAP, LMGOXCBZ

Depressionanxiety

Class I MTLE

PHE, CBZ No Class III Astrocytoma inoccipitotemporal gyrus

CBZ, LMG No Class II Cavernoma inoccipitotemporal

DFH, VAP No Class I Oligoastrocitoma insuperior temporal gyrus

VAP, CNZ Depressionanxiety

Class I Cavernoma in hippocampusand parahippocampus

VAP, CBZ,CNZ

No Class I Glioma in hippocampusand parahippocampus

PHE No Class I Oligoastrocitoma in superiortemporal gyrus

CBZ No Class I Neuroectodermal tumorin temporal pole

PHE, CBZ Depression Class i Oligoastrocitoma insuperior temporal gyrus

– – – Asphyxia (5)– – – Hypovolemic shock (5)– – – Hypovolemic shock (10)– – – Hypovolemic shock (14)– – – Lymphoma (2)– – – Pulmonary cancer (4)

: HS, hipoccampal sclerosis; LMG, lamotrigine; M, male; MTLE, Mesial temporal lobepiramate; ZNS, zonizamida.

e neocortex of patients with temporal lobe epilepsy, Neurobiol. Dis.


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about the hypoperfusion area (Huberfeld et al., 2006; Tae et al., 2005).MRI was performed with a 1.5 or 3 T unit and revealed the presenceof mesial sclerosis and volume reduction in the temporal pole area,but no significant changes in T2 and T3 gyri in patients with MTLE.MRI findings showed a clear concordance with EEG records. Patientswith focal cortical dysplasia were specifically excluded.

Patients were assessed with the Beck Depression Inventory (BDI),Montgomery-Asberg Depression Rating Scale (MADRS) and HamiltonAnxiety Scale (HAMA). They were classified as having a depressivesyndrome when BDI andMADRS scales indicated so by means of stan-dard cut-off points (14 points in the BDI and 20 points in the MADRS).Anxiety syndrome was established by means of a standard cut-offpoint (18 points) in the HAMA scale. The diagnosis of generalizedanxiety disorder was confirmed according to the results obtainedfrom the Structured Clinical Interview for DSM-IV Axis I Disorders(SCIDI), which diagnoses lifetime and current axis I psychiatric disor-ders (First et al., 1999; Lopez-Gomez et al., 2008). The scientific com-mittees of the institutions involved in this investigation approved thisstudy and informed consent was obtained from each patient.

An electrocorticographic (ECoG) record with 4×8-electrode grids(Ad-Tech, Racine, WI) was performed during epilepsy surgery to iden-tify neocortex with epileptiform activity for subsequent resection. Tis-sue (T2 and T3 gyri) was collected immediately after resection, frozenin milled dry ice and stored at−70 °C until processing. Tissue samplesused for the present study did not involve the tumor or any knownpathology.

Results obtained from patients with MTLE and TLE secondary tobrain tumor or lesion were compared to those obtained from temporalneocortex of 6 subjects (6 men) who died due to different causes, andhad no history of neurologic disease. T2 and T3 gyri were collected atthe time of the autopsy with a post-mortem interval (PMI) of 2 to14 h, and were immediately stored at −70 °C (Table 1).

Three fragments were obtained from each brain sample of patientswith epilepsy and from the autopsies. The fragmentation was donemaintaining the tissue frozen, a situation that allowed to preserve theproper conditions for the dopaminergic system (Nyberg et al., 1982).A fragment including only graymatterwas used to determine the tissuecontent of dopamine and its metabolites, 3,4-dihydroxyphenylaceticacid and homovanillic acid (DOPAC and HVA, respectively). A secondgray matter fragment was used for Western blot analysis to examineprotein expression of D1 and D2 receptors, and TH. The third fragmentthat included both, gray and white matter, was used for autoradiogra-phy experiments.

Autoradiography experiments

Preparation of tissue sectionsFrozen sections of 20m were cut in a cryostat, thaw-mounted on

gelatin-coated slides, and stored again at −70 °C. Serial and parallelsections were obtained from each sample for quantitative and func-tional autoradiography.

Quantitative autoradiographyTable 2 includes a summary of the experimental conditions for the

autoradiography experiments. Initially, brain sections were washed to

Table 2Conditions for autoradiography experiments.

Binding Ligand (nM) S.A. Buffer pH 7.6

D1 receptor 3H-SCH23390 (1.62 nM) 85 Ci/mmol Tris HCl (50 mM), 120 mM5 mM KCl 1 mM MgCl2 2 m

D2 receptor 3H-Raclopride (4 nM) 60 Ci/mmol Tris HCl (50 mM) 150 mMNaCl 0.1% Ascórbic Ac.

Dopamine transporter 3H-Mazindol (4 nM) 20.6 Ci/mmol Tris HCl (50 mM), 300 mMNaCl, 5 mM KCl

S.A., specific activity; RT, room temperature.


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remove endogenous ligands, and they were subsequently incubated ina solution with the specific ligand labeled with tritium (3H) in presenceor absence of a non-labeled specific ligand. The specific binding valueswere determined from the difference of values obtained from bothexperimental conditions. Incubation was completed with two consecu-tive washings in buffer solution and then rinsed with distilled water(2 s) at 4 °C. Sections were immediately dried in a gentle steam ofcold air.

Preparations were placed in X-ray cassettes together with 3H stan-dards (Amersham) and were exposed to 3H-sensitive film (Kodak MR)at room temperature. Films were developed with Kodak D19 developerand fast fixer at room temperature. Optical densities of cortical layers ofeach brain section were determined using image analysis software(JAVA Jandel). Optic density readings of the standardswere used to calcu-late radioactivity values of accompanying tissue sections. Finally, theywere converted into fmol/mg of protein based on the specific activity ofeach 3H-labeled ligand and tissue thickness (20 μm).

Functional autoradiographyAgonist-stimulated [35S]GTPγS autoradiography was performed

as described previously (Sim and Childers, 1997; Sim et al., 1996).Sections were soaked for 10 min at 25 °C in 50 mM Tris–HCl buffer(pH 7.4) to remove endogenous catecholamines. They were then pre-incubated for 15 min in the same buffer, to which 100 mM NaCl,3 mM MgCl2, 0.2 mM EGTA and 2 mM GDP were added. Forty pM[35S]GTPγS was then added to this solution, for a 2 h incubation at25 °C in basal conditions. Basal binding was measured in absence ofthe tested compound. To evaluate stimulated condition due to G pro-teins activity dependent of D2-like receptor activation it was useddopamine 100 μM in the presence of SCH23390 500 μM (Sigma, St.Louis, MO, USA), a non-selective D1-like DA receptor antagonist. Inother sections, the effects of dopamine receptor antagonist wereexamined on the activation evoked by dopamine 100 μM in the pres-ence of SCH23390 and sulpiride (non-selective D2-like receptor antago-nist), 500 μMeach (Sigma, St. Louis,MO, USA). Non-specific bindingwasdetermined in presence of 10 μMunlabelled GTPγS and subtracted frombasal binding. Finally, slides were washed twice in buffer and once indistilled water at 4 °C. The autoradiograms were produced on KodakBiomax MR™ film, exposed for 48–72 h along with 14C standards(Amersham) and processed in GBX™ (Kodak) developer. Optical densi-ties were obtained following the same procedure of autoradiographyand results were represented as mean values±SD in fmol/mg proteinafter subtracting non-specific value to the basal and stimulatedvalues. Net agonist-stimulated [35S]GTPγS binding was calculatedby subtracting basal binding (obtained in absence of agonist) fromagonist-stimulated binding, and expressed as percentage stimula-tion above basal.

Tissue content of monoamines

Brain tissue was thawed and manually and individually homoge-nized into a mixture of perchloric acid (0.1 M, Baker), sodium meta-bisulphite (4 mM Na2S2O3, Sigma) and ethylenediaminetetraaceticacid (0.1 mM EDTA, Sigma). The homogenate was centrifuged at12,600 rpm for 20 min at 4 °C. Subsequently, 10 μL of the previously

Incubation conditions Exposition (R.T.) Non-labeled ligand

NaClM CaCl2

90 min+1 μM mianserin 22°C 12 weeks SCH23390 (1 μM)

45 min 22°C 8 weeks Butaclamol (1 μM)

40 min+0.3 μM desipramine 4°C 12 weeks GBR12935 (1 μM)



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filtered supernatant (Millex®-HV filters, 0.45 μm pore) were injectedinto the chromatographer. An isocratic system with a mobile phaseflow of 0.350 mL/min at 30 °C with +560 mV was used for the elutionof dopamine and its metabolites (DOPAC and HVA). A chromatographysystem coupled to an electrochemical detector (Waters® model 2465)with Atlantis dC18 precolumn (3.9×20 mm) and Atlantis dC18 reversephase column (3.9×50 mm), both with 3 μm pore, was used. Mobilephase was a combination of EDTA (0.054 mM Sigma), 10.5 g/L of citricacid (Sigma), octanesulfonic acid (21.61 mg/L Sigma) and methanol(3% Merck), pH 2.9 adjusted with sodium hydroxide. Quantification ofdopamine and its metabolites was performed with the Empower v.1software by Waters, from a calibration curve obtained with differentconcentrations of external standards.

Determination of proteins

Protein quantification was performed according to the protocoldescribed by Lowry et al. (1951) using the remnant pellet in theEppendorf tube after separating the brain homogenate supernatant(see above). The pellet was suspended in 250 μL of miliQ water andfrom this solution, a 1:40 dilution with water (1 μL of sample and39 μL of water) was prepared. An aliquot of 100 μL of this dilutionwas added with 500 μL of reagent A (100 μL of 1% CuSO4, 100 μL of2% potassium sodium tartrate and 10 mL of 2% Na2CO3). The mixturewas stirred and stored at room temperature for 10 min. Subsequently,reagent B (2 N Folin & Ciocalteu phenol reagent (Sigma) diluted 1:2 inmiliQ water) was added. The mixture was stirred and stored at roomtemperature in the dark for 30 min. Then, absorbance of the sampleswas determined using an UV–VIS spectrophotometer (Beckman DU640) at 700 nm. Protein concentration in samples was calculatedbased on a standard curve prepared with different concentrations ofbovine albumin fraction V (Gibco). The obtained values allowedexpressing the values resulting from the high performance liquidchromatography in ng/mg of proteins.

Western blot assay

Tissue was homogenized in 1 mL of “TriPure” reagent (RocheDiagnostics) in order to allow protein isolation. The homogenatewas added with 0.2 mL of chloroform, followed by isopropanol toprecipitate total proteins. Samples were centrifuged at 12,000 rpmfor 10 min at 4 °C, and washed 3 times with 0.3 M guanidine hydro-chloride in 95% ethanol and a final washing was performed with100% ethanol. Samples were centrifuged according to the previouslydescribed procedure (Perez-Severiano et al., 2002), and the resultingpellet was suspended in 1% sodium dodecyl sulfate (SDS). Volumesequivalent to 50 μg of proteins (determined by the bicinchoninic acidmethod) were transferred to 8% polyacrylamide gel, separated usingelectric current at 150 V and transferred again to a nitrocellulosemembrane (Immun-Blot PVD, Bio-Rad). Subsequently, membraneblockage was performed with 5% half-skimmed milk and 0.05%Tween-20 for 30 min at room temperature, followed by incubation for12 h at 4 °C with one of the following antibodies: polyclonal antibodyto TH (1:1000, Cell Signaling 2792), polyclonal antibody to D1 receptor(1:200, Santa Cruz, SC-14001) or monoclonal antibody to D2 receptor(1:200, Santa Cruz, SC-5303). After 24 h, membranes were washedand exposed to secondary antibodies labeled with peroxidase for THlabeling, and antibodies to rabbit and mouse for D1 and D2 receptors,respectively (1:3000; Invitrogen), in blocking solution for 1 h at roomtemperature. Membranes were washed and protein labeling was ana-lyzed using a Western chemiluminescent detection system (PerkingElmer LAS, Inc). Then, the aforementioned antibodies were removedfrom the membranes for incubation with a monoclonal antibody toβ-actin (Garcia-Tovar et al., 2001). The latter assay was used as controlto standardize the values of protein level expression. The procedure toremove antibodies from membranes was as follows: first, membranes


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were washed four times with phosphate buffer solution and salinesolution (0.015 M, 0.9% NaCl, pH 7.4). Then, they were immersed intoa solution containing 2-mercaptoethanol (100 mM), SDS (2%) andTris–HCl buffer solution (62.5 mM, pH 6.7) for 30 min at 60 °C withslow stirring. Membranes were washed five times in phosphate buffersolution and saline solution with 0.05% Tween-20. The obtained imageswere digitalized using the BioDoc-It system (UVP) and optical densitywas determined using image capture and analysis software (UVP),according to the previously stated description (Perez-Severiano et al.,2002).

Statistical analysis

Values are expressed as mean±standard deviation (SD) and wereexamined using ANOVA test and Dunnett's post-hoc test. Pearson'scorrelation coefficients were calculatedwith amultiple regression anal-ysis against the values obtained in different experiments performed todetermine the possible influence of subject's age, age of seizure onset,duration of epilepsy and frequency of ictal events.

Results

The clinical data from patients with MTLE were as follows (mean±SD): age of subjects, 36.5±10.8 years (ranged from 24 to 60 years); ageat seizure onset, 9.18±6.6 years; years of epilepsy duration, 27.3±10.4; and seizures per month, 14.8±13.4. Patients with TLE secondaryto tumor or lesion presented similar age (32.6±8.1 years old, rangedfrom 24 to 48 years old, pb0.4) and seizure frequency (24.5±34seizures per month, pb0.409), but the age at seizure onset (22.1±15.8 years) and epilepsy duration (10.5±11.2 years)were significantlydifferent (pb0.01 and pb0.001, respectively), when compared withpatients with MTLE. The average age of patients with MTLE and TLEsecondary to tumor or lesion was not significantly different whencompared with subjects from autopsies (39.5±8.3 years, rangingfrom 21 to 51 years, pb0.51). Fifty-eight % (n=7) of patients withMTLE and 25% (n=2) of patients with TLE secondary to tumor or lesionpresented comorbid anxiety and depression. Postoperative outcomeevaluated 1 year after epilepsy surgery was similar for both epilepsygroups: nine patients (75%) from the MTLE group and 6 patients(75%) from the lesional TLE group achieved seizure freedom (Engelclass I); two patients (16.6%) from MTLE group and one patient(12.5%) with TLE secondary to tumor or lesion showed rare disablingseizures (Engel class II); one patient (12.5%) from TLE secondary totumor or lesion demonstrated worthwhile improvement (Engel classIII); and one patient (8.3%) from the MTLE group exhibited no worth-while improvement (Engel class IV) (Table 1).

Autopsies

Tissue levels of dopamine, DOPAC and HVA (mean±SD) from theautopsies were 2.9±2.2, 13.9±10.38 and 18.7±13.97 nM/mg ofprotein, respectively (Fig. 1). The Western blot experiments allowedthe evaluation of protein expression in D1 and D2 receptors, as wellas in TH (Fig. 2). Binding of 3H-SCH23390, 3H-Raclopride and 3H-Mazindol to D1 and D2 receptors, and to the dopamine transporter,respectively, was widely distributed in the various temporal neocor-tex layers. We detected high values for binding to 3H-Mazindol (ap-proximately 400 fmol/mg protein), while values for binding to 3H-SCH23390 and 3H-Raclopride were low (around 50 fmol/mg protein)(Fig. 3). Functional autoradiography revealed that in the presence ofSCH23390, dopamine induced a [35S]GTPγS incorporation of 14.3%in layers I–II, 7.6% in layers III–IV and 4.4% in layers V–VI (Fig. 4).

In order to determine the post-mortem stability of tissue, valuesobtained from the different experiments were subjected to correla-tion analysis with PMI. Data obtained from this examination indicatedthat, under our experimental conditions, post-mortem delay did not



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Fig. 1. Tissue levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homo-vanillic acid (HVA) in neocortex obtained from autopsies and patients with MTLE orTLE secondary to brain tumor or lesion. The values represent the mean±SD expressedin nM/mg of protein. *pb0.05; **pb0.01, regarding the autopsy values.

Fig. 3. Binding of 3H-SCH23390 (D1), 3H-Raclopride (D2) and 3H-Mazindol (Transporter)in external (I–II), middle (III–IV) and deep (V–VI) cortical layers in samples of autopsiesand patients with MTLE or TLE secondary to brain tumor or lesion. The values representthemean±SDexpressed in fmol/mg of protein. *pb0.05; **pb0.01, regarding the autopsyvalues.


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affect the different components of the dopaminergic system evaluat-ed in the present study (Table 3).

Patients with epilepsy

The diagnosis of each patient with epilepsy was performed accordingto clinical, MRI and electrophysiological criteria. Every patient presentedinterictal epileptiformactivity in the temporal cortex ipsilateral to the ep-ileptogenic focus. Nomorphological alterationwas identified in the later-al temporal neocortex. SPECT revealed interictal hypoperfusion inmesialstructures in patients with MTLE. ECoG performed intraoperatively con-firmed the presence of epileptiform activity in T2 and T3 gyri.

Patients with MTLEThe values for tissue content of dopamine, DOPAC and HVA

were significantly lower (60%, pb0.05; 96%, pb0.01; and 79%, pb0.01,respectively) than those obtained in autopsies' tissue (Fig. 1). The tem-poral neocortex obtained from patients with MTLE presented high(1680%, pb0.05) and low (77%, pb0.05) protein expression of D1 andD2 receptors, respectively, while the expression of TH showed no signifi-cant changes (Fig. 2). Likewise, an increasewas detected in binding of 3H-SCH23390 (layers I-II, 31%, pb0.05; layers V-VI, 28%, pb0.05), as well asof 3H-Mazindol (layers I–II, 89%, pb0.05; layers III–IV, 141%, pb0.01;layers V–VI, 114%, pb0.01) (Fig. 3). Functional autoradiography demon-strated a D2-like induced [35S]GTPγS incorporation similar to thatdetected in autopsy samples (Fig. 4).

Correlations between these results and clinical data suggest the fol-lowing: the younger the age of seizure onset, the higher the binding toD1 receptors in all temporal cortex layers and the lower the binding to

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Fig. 2. Protein expression of D1 and D2 receptors, and tyrosine hydroxylase (TH) in neocor-tex obtained from autopsies and patients with MTLE or TLE secondary to brain tumor or le-sion. Western blot analyses representative of each experiment are shown in the upper partof the graphs. Values obtained in the image densitometric analysis are shown in bars; theywere corrected for β-actin values. The graphs show themean±SD of the ratio between op-tical density (OD) and β-actin expression. *pb0.05, regarding the autopsy values.


D2 receptors in layers I–II and V–VI; the higher the frequency of sei-zures, the lower the binding to D1 receptor in all temporal cortexlayers; the older the age of the patient, the higher the D2-like in-duced Gi stimulation in layers V-VI (Table 4). Results from high per-formance liquid chromatography and Western blot revealed nosignificant correlations with clinical data.

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Patients with TLE secondary to brain tumor or lesionAs compared with the results obtained from biopsies, neocortex of

patients with TLE secondary to tumor or brain lesion presented signifi-cant lower values for tissue content of dopamine, DOPAC and HVA(81%, pb0.01; 98%, pb0.01; and 89%, pb0.01, respectively) (Fig. 1).This tissue presented an increase in the protein expression of D1 recep-tors (1100%, pb0.05), while expression of D2 receptors and TH showedno significant changes (Fig. 2). Likewise, an increase in binding of3H-Mazindol (layers I–II, 89%, pb0.01; layers III–IV, 127%, pb0.01;layers V–VI, 98%, pb0.01) was detected, in contrast with binding of3H-SCH23390 and 3H-Raclopride, which presented no alterations(Fig. 3). In contrast with autopsies and MTLE patients, tissue obtainedfrom patients with TLE secondary to tumor or lesion demonstrated asignificant increase of [35S]GTPγS incorporation in all cortical layers(layers I–II, 503%, pb0.05; layers III–IV, 557%, pb0.05; layers V–VI,964%, pb0.01) (Fig. 4).



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Fig. 4. Binding of [35S]GTPγS in external (I–II), middle (III–IV) and deep (V–VI) corticallayers in A) samples of autopsies and patients with MTLE or TLE secondary to braintumor or lesion; and B) samples of autopsies and patientswith epilepsy that had comorbiddepression and anxiety (D/A), and without psychiatric disorders (NO D/A). The values areexpressed as average of percentage stimulation above basal±SD. *pb0.05; **pb0.01,regarding the autopsy values.

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Table 4 t4:1

Correlations between clinical data and binding in temporal neocortex of patients withMTLE.

t4:2t4:3Binding and

ligandAge ofpatient

Seizure onsetage

Duration ofepilepsy

Frequency ofseizures

t4:4D1 3H-SCH23390t4:5Layers I–II −0.2768 −0.5261⁎ 0.2568 −0.5503⁎

t4:6Layers III–IV −0.3412 −0.5047⁎ −0.1467 −0.7180⁎⁎

t4:7Layers V–VI −0.1627 −0.6614⁎⁎ −0.1718 −0.6740⁎

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t4:9D2 3H-Raclopridet4:10Layers I–II 0.2799 0.6639⁎ −0.1467 −0.0951t4:11Layers III–IV 0.0552 0.3602 −0.1718 −0.1580t4:12Layers V–VI 0.2303 0.5718⁎ −0.0743 0.0232t4:13

t4:14Transporter 3H-Mazindolt4:15Layers I–II 0.0566 0.1976 −0.0689 −0.3277t4:16Layers III–IV 0.2226 0.2743 0.0422 −0.1705t4:17Layers V–VI 0.2888 0.3485 −0.0539 0.1998t4:18

t4:19Gi Protein 35S-GTPγSt4:20Layers I–II −0.2336 −0.0205 −0.2116 0.1823t4:21Layers III–IV 0.2861 0.2579 0.1143 0.1324t4:22Layers V–VI 0.5683⁎ 0.2599 0.3841 0.0693

Values represent the Pearson Correlation Coefficients.t4:23⁎ pb0.05. t4:24

⁎⁎ pb0.01. t4:25

Table 5 t5:1

Correlations between clinical data and binding in temporal neocortex of patients withTLE secondary to tumor or brain lesion.

t5:2t5:3Binding and

ligandAge ofpatient

Seizureonset age

Durationof epilepsy

Frequencyof seizures

t5:4D1 3H-SCH23390t5:5Layers I–II 0.3209 −0.2379 0.5674⁎ 0.1575t5:6Layers III–IV 0.3558 −0.0158 0.2359 −0.0957


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Correlations of clinical data with the obtained results suggest thatthe longer the duration of epilepsy, the higher the D1 (layers I–II) andthe lower the D2 (layers III–IV and V–VI) receptor binding; the olderthe age of epilepsy onset, the higher the binding to D2 receptors inlayers III–IV and V–Vl; the higher the frequency of seizures, the higherthe binding to dopamine transporter in layers I–II and III–IV; the olderthe age of the patient, the higher the D2-like induced [35S]GTPγSincorporation in layers III–IV and V–VI. No other significant correlationswere detected (Table 5).

Patients with epilepsy and psychiatric comorbidity

Concerning dopaminergic changes in patients with psychiatric co-morbidity, patients with MTLE and with TLE secondary to tumor or le-sion were combined because no significant differences were detectedbetween both epilepsy groups (data not shown). Analysis revealedthat neocortex of patients with anxiety and depression showed 35S-GTPγS binding values similar to autopsy samples, whereas neocortexof patients without psychiatric comorbidity presented higher 35S-GTPγS binding incorporation in layers I–II (494%, pb0.05), III–IV(228%, pb0.01) and layers V–VI (112%, pN0.05) (Fig. 4). No furtherchanges were detected.

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Table 3Correlations between the values obtained in autopsy samples and the interval betweendeath and tissue collection (post-mortem interval).

D13H-SCH23390

D23H-Raclopride

Transporter3H-Mazindol

Gi35S-GTPγS

Layers I–II −0.3719 −0.4328 0.6119 −0.1057Layers III–IV −0.1273 −0.1755 −0.4409 0.1599Layers V–VI −0.1854 0.2989 −0.5325 0.1148

Western blot HPLC

D1 Dopamine −0.5861 −0.2678D2 DOPAC 0.2044 0.5326TH HVA 0.3736 0.3466

DOPAC, 3,4-dihydroxyphenylacetic acid;HVA,homovanillic acid; TH, tyrosine hydroxylase.Values represent Pearson Correlation Coefficients.


ED PIn both groups of patients (with and without anxiety and depres-

sion), D1 receptor binding correlated positively with duration of epi-lepsy, whereas D2 receptor binding correlated positively with the ageof seizure onset. In patients with anxiety and depression, D2 receptorbinding correlated positively with age of patients, while transporterbinding correlated positively and negatively with duration of epilepsyand age of seizure onset, respectively. In patients without psychiatriccomorbidily, transporter binding correlated positively with seizurefrequency, whereas 35S-GTPγS binding correlated positively withage of patient and age of seizure onset, and negatively with durationof epilepsy. In contrast, patients with anxiety and depression did notshow significant correlations between clinical data and 35S-GTPγSbinding (Tables 6 and 7). No further correlations were detected.

t5:7Layers V–VI 0.3342 0.1148 0.0824 −0.3332t5:8

t5:9D2 3H-Raclopridet5:10Layers I–II −0.1660 0.2378 −0.4581 −0.0457t5:11Layer III–IV 0.5245 0.8194⁎⁎ −0.7782⁎ −0.1840t5:12Layer V–VI 0.5186 0.7526⁎ −0.6864⁎ −0.4172t5:13

t5:14Transporter 3H-Mazindolt5:15Layers I–II −0.5601 −0.6166 0.4683 0.6557⁎

t5:16Layers III–IV −0.4480 −0.5951 0.5186⁎ 0.6681⁎

t5:17Layers V–VI −0.3630 −0.5646 0.5380⁎ 0.4128t5:18

t5:19Gi Protein 35S-GTPγSt5:20Layers I–II 0.1810 0.2191 −0.4018 −0.3910t5:21Layers III–IV 0.5592⁎ −0.4327 −0.1290 −0.1368t5:22Layers V–VI 0.5372⁎ 0.4238 −0.2121 −0.2802


⁎⁎ pb0.01. t5:25



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Table 6t6:1

Correlations between clinical data and binding in temporal neocortex of patients withTLE and anxiety and depression.

t6:2t6:3 Binding and

ligandAge ofpatient

Seizureonset age

Durationof epilepsy

Frequencyof seizures

t6:4 D1 3H-SCH23390t6:5 Layers I–II −0.0709 −0.5382 0.6093⁎ 0.0290t6:6 Layers III–IV 0.0276 −0.2524 0.3335 −0.1158t6:7 Layers V–VI −0.0790 −0.4785 0.5292⁎ −0.0224t6:8

t6:9 D2 3H-Raclopridet6:10 Layers I–II 0.4765 0.2854 0.0195 −0.0458t6:11 Layers III–IV 0.6626⁎ 0.6148⁎ −0.0242 −0.0415t6:12 Layers V–VI 0.7156⁎ 0.3586 0.1159 −0.0065t6:13

t6:14 Transporter 3H-Mazindolt6:15 Layers I–II −0.0647 −0.3409 0.3705 −0.0906t6:16 Layers III–IV −0.0332 −0.5935⁎ 0.7070⁎ 0.0068t6:17 Layers V–VI −0.0715 −0.3649 0.3948 0.1058t6:18

t6:19 Gi Protein 35S-GTPγSt6:20 Layers I–II −0.0761 −0.0227 −0.0313 −0.1337t6:21 Layers III–IV 0.4203 0.0042 0.4791 0.4807t6:22 Layers V–VI −0.1026 0.3600 −0.4247 −0.3024

Values represent the Pearson Correlation Coefficients.t6:23 ⁎ pb0.05.t6:24

Table 7 t7:1

Correlations between clinical data and binding in temporal neocortex of patients withTLE without anxiety and depression.

t7:2t7:3Binding and

ligandAge ofpatient

Seizure onsetage

Duration ofepilepsy

Frequency ofseizures

t7:4D1 3H-SCH23390t7:5Layers I–II −0.0995 −0.2459 0.7333⁎ −0.0105t7:6Layers III–IV −0.3024 −0.0920 0.5810⁎ −0.1698t7:7Layers V–VI −0.0868 0.0070 0.4979⁎ −0.2982t7:8

t7:9D2 3H-Raclopridet7:10Layers I–II −0.0994 0.0080 0.1682 −0.0590t7:11Layers III–IV −0.0557 0.0174 0.0945 −0.0663t7:12Layers V–VI 0.0817 0.5085⁎ −0.2099 −0.2524t7:13

t7:14Transporter 3H-Mazindolt7:15Layers I–II 0.0885 −0.2371 −0.0536 0.4656⁎

t7:16Layers III–IV 0.0637 −0.3136 0.1346 0.6304⁎

t7:17Layers V–VI 0.1890 −0.2320 0.2188 0.3966t7:18

t7:19Gi Protein 35S-GTPγSt7:20Layers I–II 0.1388 0.5633⁎ −0.5465⁎ −0.2739t7:21Layers III–IV 0.5081⁎ −0.2191 −0.1876 −0.0840t7:22Layers V–VI 0.5581⁎ −0.2959 −0.2401 −0.1941



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Discussion

The present study supports the idea that dopaminergic neurotrans-mission is altered in the temporal neocortex of patients with MTLE orTLE secondary to tumor or lesion. Our experiments allowed us to eval-uate different issues of dopaminergic neurotransmission and analyzethe possible influence of clinical factors.

Values obtained from the surgical material of patients with MTLEor with TLE secondary to brain tumor or lesion were compared tovalues from autopsies of subjects of similar age in order to reduce var-iables such as subjects' age that normally influence the dopaminergicsystem (Zelnik et al., 1986). In the present study, correlation analysissupported that PMI up to 14 h did not exert a significant effect on thelevels of dopamine, its metabolites, transporter, TH and receptors. Al-though this information and results obtained from other authors(Gilmore et al., 1993; Nyberg et al., 1982; Staley et al., 1995; Tupalaet al., 2006; Wolf et al., 1991) led to suggest that tissue from autop-sies can serve as control, further experiments should be conductedin rodents to determine the precise impact of post-mortem delayon dopaminergic system.

Our data indicate that the temporal neocortex of patients withMTLEpresents changes in dopamine receptors and transporter, which are dif-ferent from those detected in the neocortex of patients with lesionalTLE. This situation can be explained because patientswithMTLEpresentlonger duration of epilepsy and younger age at seizure onset. Indeed,the increased duration of epilepsy, considered as predictor of poor out-come (Hennessy et al., 2001; Rodin, 1968), has been associatedwith re-cruitment of the adjacent cortex into the epileptogenic zone (Morrellet al., 1987), a situation that may involve progressive impairment ofthe dopaminergic system. An important aspect of the present study isthat many of the variables evaluated correlated with clinical data, lead-ing to suggest that dopaminergic changes result from epileptic activityand/or antiepileptic therapy.

Our experiments revealed a decrease in the tissue levels of dopamineand itsmetabolites in the temporal neocortex of patientswithMTLE andwith TLE secondary to tumor or lesion, and support the results obtainedbyMori et al. (1987) and Pacia et al. (2001). The reduction in tissue con-tent of DOPAC and HVA insinuates a decrease in metabolism and/orrelease of dopamine. Since there are no changes in TH expression (pre-sent study) and activity (Pintor et al., 1990), it is not possible to suggestalterations in the synthesis of DOPA, the dopamine precursor. Another


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possibility to explain the low levels of dopamine is an increase in theexpression of monoamine oxidase (MAO), enzyme involved in the deg-radation of this catecholamine. The increase of binding to B-typeMAO inthe cerebral cortex of patientswith TLE sustains this idea (Kumlien et al.,1995). Regarding alterations in reuptake, we found that dopaminetransporter binding was increased in the temporal cortex of patientswith MTLE and with TLE secondary to brain tumor or lesion, and in thelatter this change was positively correlated to frequency of seizures.These findings are similar to those reported by Del Sole et al. (2010) inpatients with epilepsy associated with ring chromosome 20 syndrome.Similarly, patients with generalized idiopathic epilepsy present anincrease in the expression of the geneencoding the dopamine transport-er (Sander et al., 2000). The positive correlation between seizure fre-quency and dopamine transporter binding observed in this study andin the study by Del Sole et al. (2010) can represent a compensatorymechanism to remove dopamine released as a result of ictal activity(Meurs et al., 2008) or prolonged administration of antiepileptic drugs(Clinckers et al., 2005; Murakami et al., 2001; Okada et al., 1997).However, further studies are necessary to support this idea.

Our results indicate an increase in protein expression and bindingof D1 receptors in the temporal neocortex of patients with MTLE. Animportant factor to be considered is that the increase in glutamatergicneurotransmission characterizing pharmacoresistant MTLE (Duringand Spencer, 1993; Luna-Munguia et al., 2011) has been associatedwith increased D1 receptors binding (Sarantis et al., 2009; Scott andAperia, 2009). In contrast, neocortex of patients with TLE secondaryto tumor or lesion did not present significant changes in D1 receptorbinding in spite of a higher protein expression. It is possible thathigher D1 receptor binding is evident after a long duration of epilepsyand/or in patients in whom the epileptic activity started at an earlierage, an idea supported by correlation analysis.

Although protein expression of D2 receptorswas reduced inpatientswith MTLE, its binding was not modified, which might be explained bya compensatory increase in receptor affinity. The absence of changes inD2 receptor binding found in our autoradiography experiments is indisagreement with the decrease in binding of 18F-Fallypride in corticalareas surrounding the epileptic foci of patients with MTLE detected byPET (Werhahn et al., 2006). Although 18F-Fallypride and 3H-Racloprideare both antagonists of the D2–D3 receptor family, diverse methodolo-gy differences between PET and in vitro autoradiography may be re-sponsible for the discrepancies between Werhahns's study and the



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results obtained in the present study. For example, the presence of en-dogenous competitors such as dopamine appears to alter themeasuredspecific binding of some ligands during PET studies (Morris and Yoder,2007).

In the present study, correlation analysis suggests high D2 recep-tor binding (patients with MTLE and lesional TLE) and elevated D2-like induced activation of G proteins (patients with epilepsy withoutpsychiatric comorbidity) when epilepsy initiates at later ages andwhen duration of illness is short. If activation of dopamine D2 sub-family receptors has an inhibitory role in the cerebral neocortex ofpatients with epilepsy (Barone et al., 1991), and alleviates symptomsproduced by depression (Bonci and Hopf, 2005; Breuer et al., 2009),these effects might be predominant during the early years followingepilepsy onset and might avoid psychiatric disorders. This idea is sup-ported by the increase in D2-like-induced activity on [35S]GTPgSbinding observed in the temporal neocortex of patients with TLE sec-ondary to tumor or lesion, who presented a shorter duration of epi-lepsy and less comorbid anxiety and depression. Further studiesshould be conducted to establish a relationship between alterationsin D2 receptor-induced neurotransmission and interictal psychiatricdisorders commonly occurring in patients with refractory epilepsyof longer duration (Briellmann et al., 2007; Hermann and Wyler,1989; Jackson and Turkington, 2005; Kanner and Balabanov, 2002).

Finally, we acknowledge that the number of surgically treated pa-tients involved in the present studywas not enough to obtain definitiveresults about the influence of dopaminergic systemon neuropsychiatricdisorders of patients with pharmacoresistant TLE. Another significantlimitation of the present study is that the impact of preoperative phar-macological treatment on alterations in the dopaminergic system couldnot be systematically assessed because of the high variability in antiepi-leptic medication (type and number of antiepileptic drugs, duration oftreatment, dosage, monotherapy, polytherapy, etc.) among patients. Inaddition, larger patient cohorts need to be evaluated to determinea) the impact of clinical factors on changes in the dopaminergic system,b) the relationship between dopaminergic alterations and surgical out-come, and c) if changes in the dopaminergic system represent the effector the cause of epilepsy. Finally, since dopamine could be important inthe comorbidity of epilepsy and neuropsychiatric disorders, furtherstudies are necessary to determine if dopaminergic abnormalities inthe brain of patients with TLE are bilateral.

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Acknowledgments

We thank Ms. Leticia Neri Bazan, Ms. Paula Vergara and Mr. HectorVazquez Espinosa for their excellent technical assistance and Dr. ErikaBrust Mascher for English improvement. This study was supported bythe National Council for Sciences and Technology of Mexico (grantsJ010.0170/2010 and 98386) andAcademy of Sciences of Hungary (grantsETT577/2006, RET67/2005).

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