Low frequency stimulation modifies receptor binding in rat brain

Epilepsy Research 59 (2004) 95–105

Low frequency stimulation modifies receptor binding in rat brain M.L. López-Meraz a,b , L. Neri-Bazán a , L. Rocha a,∗ a

b

Departamento de Farmacobiolog´ıa, Centro de Investigación y de, Estudios Avanzados del I.P.N., Sede Sur Tenorios 235, Col. Granjas Coapa, DF 14330, Mexico Subdirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatr´ıa “Ramón de la Fuente Muñiz”, DF, Mexico Received 18 August 2003; received in revised form 18 December 2003; accepted 2 April 2004 Available online 10 June 2004

Abstract Experiments were designed to reproduce the antiepileptic effects of low frequency stimulation (LFS) during the amygdala kindling process and to examine LFS-induced changes in receptor binding levels of different neurotransmitters in normal brain. Male Wistar rats were stereotactically implanted in the right amygdala with a bipolar electrode. Rats (n = 14) received twice daily LFS (15 min train of 1 Hz, 0.1 ms at an intensity of 100 to 400 ␮A) immediately after amygdala kindling stimulation (1 s train of 60 Hz biphasic square waves, each 1 ms at amplitude of 200–500 ␮A) during 20 days. The LFS suppressed epileptogenesis (full attainment of stage V kindling) but not the presence of partial seizures (lower stages of kindling) in 85.7% of the rats. Thereafter, normal rats (n = 7) received amygdala LFS twice daily for 40 trials. Animals were sacrificed 24 h after last stimulation and their brain used for labeling ␮ opioid, benzodiazepine (BZD), ␣1 -adrenergic, and adenylyl cyclase binding. Autoradiography experiments revealed increased BZD receptor binding in basolateral amygdala (20.5%) and thalamus (29.3%) ipsilateral to the place of stimulation and in contralateral temporal cortex (18%) as well as decreased values in ipsilateral frontal cortex (24.2%). Concerning ␮ receptors, LFS decreased binding values in ipsilateral sensorimotor (7.2%) and temporal (5.6%) cortices, dentate gyrus (5.8% ipsi and 6.8% contralateral, respectively), and contralateral CA1 area of dorsal hippocampus (5.5%). LFS did not modify ␣1 receptor and adenylyl cyclase binding values. These findings suggest that the antiepileptic effects of LFS may involve activation of GABA–BZD and endogenous opioid systems. © 2004 Elsevier B.V. All rights reserved. Keywords: Epilepsy; Low frequency stimulation; ␮ Receptors; Benzodiazepine receptors; ␣1 -Adrenergic receptors; Adenylyl cyclase

1. Introduction The application of focal deep brain stimulation has gained attention as a potential treatment for several neurological conditions including pain, Parkinson’s disease, and pharmacoresistant epilepsies. Previous studies have demonstrated that in adult and immature ∗ Corresponding author. Tel.: +52-55-50-61-28-59; fax: +52-55-50-61-28-63. E-mail address: [email protected] (L. Rocha).

animals as well as in patients with mesial temporal lobe epilepsy, the low frequency stimulation (LFS, 0.9–3 Hz) could produce inhibitory effects in epileptic activity (Vélisek et al., 2002; Weiss et al., 1995; Yamamoto et al., 2002). LFS raises the afterdischarge and seizures thresholds, effects that persist from weeks to months even after this type of stimulation is discontinued (Gaito, 1980a,b; Gaito and Gaito, 1980; Weiss et al., 1995, 1998). Additionally, LFS depotentiates the long-term potentiation in the amygdala efferent transmission induced by partial kindling and

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avoids the changes in affect resulting from the kindling phenomenon in cats and rats (Adamec, 1999; Adamec and Young, 2000). However, the mechanisms by which the LFS induces antiepileptic effects are unknown. The present study was aimed to investigate the consequences of LFS on various receptor systems (benzodiazepine (BZD), ␮ opioid, and ␣1 receptors) and adenylyl cyclase binding in normal conditions. Studies from other authors support an inhibitory effect of BDZ, ␮ opioid, and ␣1 receptors in convulsions and epileptogenesis (Albertson et al., 1984, 1990; Böhme et al., 1987; Gundlach et al., 1995; Henrinksen, 1998; Weinshenker et al., 2001), whereas the activation of Gs–AC–cAMP system facilitates epileptic seizures (Higashima et al., 2002; Iwasa et al., 2000; Ludvig et al., 1992; Williams et al., 1993). The present study supports that the LFS is capable of avoiding generalized kindled seizures, and it can also alter the BDZ and ␮ receptor levels in specific brain areas of non-epileptic rats.

2. Materials and methods 2.1. Subjects and surgery Male Wistar rats initially weighing 250–350 g, individually housed at 22 ◦ C and maintained on 12 h light/dark cycle were used in the present study. The animals had free access to food and water. Rats were anesthetized with a combination of ketamine (100 mg/kg i.p.) and xylazine (20 mg/kg i.m.). Then, a bipolar electrode, consisting of two twisted strands of stainless steel wire, insulated except at the cross section of their tips, was stereotactically implanted in the right amygdala. Coordinates in millimeters were 2.5 caudal to bregma, 4.5 from the midsagittal plane, and 8.5 from the skull surface (Paxinos and Watson, 1998). Stainless steel screws were threaded into the cranium over the cortex to fix the electrode assembly. The electrodes were attached to male connector pins, which were inserted into an Amphenol connector strip. The electrode assembly was then fixed to the skull with dental acrylic, and the animals were allowed to recover for 7 days before any further manipulation.

2.2. Electrical stimulation Kindling stimulation consisted of 1 s train of 60 Hz biphasic square waves, each 1 ms in duration, at an amplitude from 100 to 400 ␮A, depending on the behavioral threshold. Behavioral seizure severity was rated according to Racine’s kindling scale (Racine, 1972). LFS was composed of 15 min train of 1 Hz biphasic square waves, each 0.1 ms in duration, at amplitude of 200–500 ␮A (100 ␮A over that used for kindling). Both, kindling and LFS, were produced by a Grass S-48 stimulator, and were applied through the same electrode. 2.3. Experimental groups 2.3.1. Kindling process associated with LFS (a) Stage V kindling group (K, n = 10). Rats received kindling stimulation twice daily until they attained the criterion of five consecutive stage V seizures (kindled state). (b) Kindling plus LFS group (K + LFS, n = 14). Animals received LFS immediately after each kindling stimulation, twice daily and for 40 trials. 2.3.2. Receptor binding following LFS (a) LFS group (LFS, n = 7). Rats received LFS twice daily for a total of 40 trials. (b) Control group (C, n = 7). Animals were manipulated as described above for the LFS group, except that they did not receive any electric stimulation. Animals from LFS and C groups were killed by decapitation 24 h after the last LFS or manipulation. The brains were quickly removed, frozen in pulverized dry ice, and stored at −70 ◦ C for subsequent autoradiography experiments. 2.3.3. Autoradiography Frozen coronal sections of 20 ␮m were cut on a cryostat, thaw-mounted onto gelatin-coated slides, and stored at −70 ◦ C until the day of incubation. In vitro autoradiography with 3 H-flunitrazepam (Amersham, Arlington Heights ILTM ), 3 H-Tyr-d-Ala-Gly-Me-PheGly-ol (DAMGO) (Amersham, Arlington Heights ILTM ), 3 H-prazosin (Amersham, Pharmacia BiotechTM ), and 3 H-forskolin (NENTM ) were performed

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Table 1 Experimental conditions for autoradiography experiments Binding site

3 H-ligand,

nM (SA, Ci/mmol)

Unlabelled ligand (␮M)

Buffer , pH 7.4 (mM)

Incubation in 3 H-ligand (min, ◦ C)

Exposure time (weeks)

Reference

BZD receptor ␮ Receptor ␣1 Receptor

Flunitrazepam, 2 (88) DAMGO, 2 (61) Prazosin, 2 (77.2)

Clonazepam, 1 Naloxone, 2 Phentolamine, 10

Tris–HCl, 170 Tris–HCl, 50 Krebs phosphate, 50

45, 4 60, 25 60, 25

3 8 12

Adenylyl cyclase

Forskolin, 10 (32)

Forskolin, 1

Tris–HCl, 50

20, 25

9

Rocha et al., 1994 Rocha et al., 1993 Giroux et al., 1999; Stephenson and Summers, 1986 Gartshore et al., 1996

SA: specific activity and DAMGO: Tyr-d-Ala-Gly-Me-Phe-Gly-ol.

on parallel sections for labeling BZD, ␮, ␣1 receptors or adenylyl cyclase sites, respectively. Experimental conditions for autoradiography are summarized in Table 1. Brain sections were prewashed 30 min at room temperature in the respective buffer. Then, they were incubated in a solution containing the specific radioligand. Binding obtained in the presence of a unlabelled ligand was considered to be nonspecific. Incubation was completed with two consecutive buffer washes (1 min each at 4 ◦ C). Finally, the slides were rinsed (3 s) in distilled water at 4 ◦ C and the sections were then quickly dried under a gentle stream of cold air. The slides were arrayed in X-ray cassettes together with tritium standards (Amersham), and apposed to tritium-sensitive film (3 H-hyperfilm; Amersham) at room temperature (Table 1). Films were developed using developer D11 (Kodak) and fixer at room temperature. Optical densities of the autoradiograms were determined using a video-computer enhancement program (Jandel Video Analysis Sofware, JAVA). The optical density of the standards were used to determine tissue radioactivity values for the accompanying tissue sections and to convert them to fmol/mg protein. Brain regions were identified according to the rat atlas of Paxinos and Watson (1998). For each structure, 10 optical density readings were taken from at least two sections and were averaged. Binding was analyzed ipsilateral and contralateral to the place of electrical stimulation in caudate putamen; cingulated frontal, sensorimotor, piriform, temporal, and entorhinal cortices; amygdala complex (anterior, basolateral, central and medial nuclei); dentate gyrus, fields CA1-CA3 of dorsal and ventral hippocampus.

These specific structures were chosen to evaluate receptors and adenylyl cyclase binding because of their possible role in epileptogenesis (Fernandez-Guardiola et al., 1990) or in the expression of interictal and postictal behaviors in different models of epilepsy (Nobrega et al., 1989; Rocha et al., 1996a,b; Rocha and Ondarza-Rovira, 1999; Shin et al., 1985). 2.3.4. Histological verification of electrode placements Frozen coronal sections of 20 ␮m were stained with cresyl violet and examined with a light microscope to verify electrode implantation within the amygdala. 2.4. Statistical analysis The effect of LFS on the kindling evolution was analyzed using non-parametric chi-square test. Student’s t-test was used to compare the number of stimulations necessary to present the different kindling stages between K and K + LFS groups and to determine differences between the LFS and C groups on BZD, ␮, ␣1 receptor levels, and adenylyl cyclase binding. Values are represented as mean ± S.E.M. For all analysis, significance was assigned at the 0.05 level.

3. Results 3.1. LFS modifies amygdala kindling process The stimulation electrodes from all experimental groups were located in the central or basolateral amygdala nuclei. Animals from K group showed a

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B

STAGE

A V

V

IV

IV

III

III

II

II

I

I

0

10

20

30

40

0

10

STAGE

C V

IV

IV

III

III

II

II

I

I

10

20

30

40

30

40

D

V

0

20

30

40

0

10

20

No. STIMULATIONS

No. STIMULATIONS

Fig. 1. Evolution of kindling process according to Racine’s scale (Racine, 1972) in K and K + LFS groups (mean ± S.E.). Kindled state was established in all subjects of K group (A). Nevertheless, in K + LFS group, 50% of the animals remained in early kindling stages (I–III) (B); 36% achieved generalized seizures without attainment of the kindled state (C); and 14% reached kindled state (D).

progressive increase of the electrographic afterdischarge duration as well as the spike frequency during the kindling development. The kindled state was achieved after 28.2 ± 3 trials. The LFS applied immediately after each kindling stimulation significantly modified the development of amygdala kindling process of K + LFS group as follows: seven animals (50%) showed partial seizures (stage I–III) and they never attained stage IV or V (P < 0.001; Table 2). Table 2 Contingency table showing the number of rats from K and K+LFS groups staying in partial seizures (PS; stages I–III), generalized seizures (SVS; stage V) and kindled state (KS), following 40 kindling trials Group

PS

SVS

KS

K K + LFS

0 7∗

0 5∗

10 2∗

∗

P < 0.001, chi-square test.

When compared to the K group, this K + LFS subgroup required high number of kindling stimulations to induce the first stage II (7.8 ± 1 and 16.7 ± 6, respectively) and III (18 ± 3 and 32.3 ± 5, respectively, P < 0.05) kindling seizures. Five rats (36%) presented variable kindling evolution and eventually showed stage V seizures. However, they never attained the kindled state (P < 0.001; Table 2). Finally, two rats (14%) reached kindled state following 19.5 ± 8 electrical stimulations (P < 0.001; Table 2, Fig. 1). Animals from LFS group did not present behavioral changes during and after each LFS trial. In fact, they eventually slept during the stimulation. 3.2. Effects of LFS on receptor binding levels The autoradiography experiments using brains from LFS group revealed enhanced 3 H-flunitrazepam binding in basolateral amygdala (20%) and thalamus (29%)

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Fig. 2. 3 H-flunitrazepam autoradiograms at the level of ventral hippocampus from an animal of the control group (A) and LFS group (B). The arrow indicates the structure in which LFS increased the BZD receptor binding (temporal cortex).

ipsilateral to the place of stimulation as well as in the contralateral temporal cortex (18%) (Fig. 2). On the contrary, decreased BZD receptor levels were found in the ipsilateral frontal cortex (24%). A non-significant enhancement of BZD receptor levels were detected in the ipsilateral CA2 area of ventral hippocampus, contralateral basolateral amygdala and ipsilateral piriform, and temporal cortices (15–25%) (Table 3). On the other hand, rats from LFS group showed a low but significant decrease of ␮ receptor binding in the ipsilateral sensorimotor (7.2%) and temporal (5.6%) cortices, dentate gyrus (5.8% ipsi and 6.8%

contralateral, respectively), and contralateral CA1 area of dorsal hippocampus (5.4%). The other brain areas evaluated did not show significant differences when compared to C group (Table 4). Concerning ␣1 -adrenergic receptors, the animals from LFS group demonstrated increased receptor values in ipsilateral structures (anterior amygdala nucleus (26.5%), dentate gyrus (27.4%), CA1 field of dorsal hippocampus (24.7%), thalamus (27.8%), medial amyddala nucleus (22%), CA3 field of ventral hippocampus (26.4%), and both entorhinal cortices (39.3% ipsi and 20.5% contralateral, respectively).

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Table 3 3 H-flunitrazepam binding (fmol/mg protein) in rat brain areas of control and LFS groups

Table 3 (Continued ) Brain area

Control

LFS

Brain area

Control

LFS

Caudate putamen Ipsil Contral

VH CA1 area Ipsil Contral

173.7 ± 9.5 162.3 ± 7.7

163 ± 18 161.6 ± 21.1

341.2 ± 23.6 379.1 ± 22.8

322.2 ± 29.6 380.4 ± 28

Cingulate CX Ipsil Contral

VH CA2 area Ipsil Contral

425.9 ± 26 514.7 ± 52.6

459 ± 60.1 407.5 ± 43.1

346 ± 28.5 316.1 ± 24.6

296.6 ± 19.7 277.2 ± 19

Frontal CX Ipsil Contral

VH CA3 area Ipsil Contral

505.4 ± 56.7 456 ± 33.7

432.3 ± 51.1 345.5 ± 42.2∗

349.6 ± 33.6 338.2 ± 18.1

332.5 ± 22.6 300.2 ± 24.6

Anterior AMG Ipsil Contral

Entorhinal CX Ipsil Contral

422.4 ± 33.8 430.7 ± 35.5

395 ± 44.2 453.9 ± 37.8

466.4 ± 19.6 381.6 ± 21.7

430.8 ± 34.7 375.7 ± 31.8

Sensorimotor CX Ipsil Contral

388 ± 23.4 357.8 ± 33.3

398.8 ± 62 371.5 ± 45.4

Dentate gyrus Ipsil Contral

441.5 ± 26.16 445.2 ± 27.2

458.6 ± 55.3 433.7 ± 45.8

DH CA1 area Ipsil Contral

411.2 ± 27 389.8 ± 31.9

430.7 ± 40.4 405.2 ± 35

DH CA2 area Ipsil Contral

269.2 ± 8 237.6 ± 8.7

328.7 ± 34.3 256.3 ± 31.2

DH CA3 area Ipsil Contral

351.5 ± 26.7 313.8 ± 14

Thalamus Ipsil Contral

The values are the mean ± S.E. (n = 6–7). LFS: low frequency stimulation group. Structures: CX, cortex; AMG, amygdala; DH, dorsal hippocampus; VH, ventral hippocampus; ipsil, ipsilateral; and contral, contralateral to the place of stimulation. ∗ P < 0.05, Student’s t-test.

Table 4 3 H-DAMGO binding (fmol/mg protein) in rat brain areas of control and LFS groups Brain area

Control

LFS

384 ± 41.6 332.8 ± 40.7

Caudate putamen Ipsil Contral

116.8 ± 3.7 111.52 ± 1.9

110.6 ± 6.6 106.5 ± 6.2

128.8 ± 9.5 148.8 ± 14.5

166.5 ± 13.6∗ 161.9 ± 9.3

Cingulate CX Ipsil Contral

116.1 ± 4.6 117.8 ± 5.9

109.13 ± 4.5 107.5 ± 5.1

Basolateral AMG Ipsil Contral

398.4 ± 38 454.3 ± 31.7

480 ± 23.8∗ 522.3 ± 77.4

Frontal CX Ipsil Contral

115 ± 2.8 110.1 ± 1.3

108 ± 5.7 109.9 ± 0.9

Central AMG Ipsil Contral

227.9 ± 19.3 231.9 ± 18.7

248.5 ± 19.9 268.3 ± 26.6

Anterior AMG Ipsil Contral

117.6 ± 1.6 114.2 ± 1.1

113.5 ± 0.9 111.9 ± 1.6

Medial AMG Ipsil Contral

304.2 ± 18.1 383.6 ± 25.5

341.4 ± 26.5 386.6 ± 19.4

Sensorimotor CX Ipsil Contral

113.2 ± 2.4 110.3 ± 1.7

105.1 ± 2∗ 109 ± 1.5

Piriform CX Ipsil Contral

365.9 ± 30.1 339.2 ± 24.1

422.7 ± 11.8 370.1 ± 23.7

Dentate GYRUS Ipsil Contral

115.7 ± 1.8 114.1 ± 1.5

106.4 ± 4∗ 107.1 ± 2.3∗

Temporal CX Ipsil Contral

401.9 ± 30.7 362.3 ± 25.5

461.9 ± 2 427.6 ± 9.6∗

DH CA1 area Ipsil Contral

105.6 ± 1.4 102.9 ± 1.5

102 ± 2.2 97.3 ± 0.8∗

M.L. L´opez-Meraz et al. / Epilepsy Research 59 (2004) 95–105

Table 5 3 H-prazosin binding (fmol/mg protein) in rat brain areas of control and LFS groups

Table 4 (Continued ) Brain area DH CA2 area Ipsil Contral

Control

LFS

112.8 ± 2 110.3 ± 2.2

110.2 ± 2.2 107 ± 1.4

113.8 ± 1.1 110.1 ± 2.2

110.7 ± 1.7 105.8 ± 1.1

Thalamus Ipsil Contral

153.8 ± 27.5 150.3 ± 26.6

147.8 ± 22.7 136.2 ± 20.3

Basolateral AMG Ipsil Contral

263.5 ± 39.4 194.2 ± 15.9

340.3 ± 46.4 206.6 ± 10.5

89 ± 1.8 85.8 ± 1.2

94.1 ± 2.3 88 ± 3.5

Medial AMG Ipsil Contral

124.4 ± 1.4 117 ± 1.9

123.2 ± 4 115.4 ± 2.7

Piriform CX Ipsil Contral

123.3 ± 3 109.16 ± 1.7

122.7 ± 1.8 111.6 ± 2.6

DH CA3 area Ipsil Contral

Central AMG Ipsil Contral

Temporal CX Ipsil Contral

1.1∗

112.2 ± 1.9 114.7 ± 2.6

106 ± 112.3 ± 1.1

129.7 ± 6.6 130.3 ± 3.8

128.4 ± 3.8 135.2 ± 5.2

VH CA2 area Ipsil Contral

112.2 ± 1.5 113.13 ± 1.9

112.4 ± 1.7 111.77 ± 5

VH CA3 area Ipsil Contral

119 ± 2 115.4 ± 2.4

116.6 ± 1.7 114.8 ± 1.1

118.9 ± 2.1 115.2 ± 1.7

117.8 ± 1.4 115.9 ± 1.6

VH CA1 area Ipsil Contral

Entorhinal CX Ipsil Contral

101

Notes as in Table 3. ∗ P < 0.05, Student’s t-test.

However, values were not significantly different from the C group (Table 5). Regarding adenylyl cyclase binding, the LFS group presented a non-significant enhancement of 3 H-forskolin binding in the ipsilateral caudate putamen (14%), CA1 field of ventral hippocampus (28%), contralateral basolateral (13%), central (31.3%) and medial (18.1%) amygdaloid nu-

Brain area

Control

Caudate putamen Ipsil Contral

120.7 ± 9.9 85.8 ± 5.6

114.4 ± 12.5 101.7 ± 6.3

Cingulate CX Ipsil Contral

447.4 ± 60.5 451.3 ± 66

483 ± 104.2 468.8 ± 107

Frontal CX Ipsil Contral

593.7 ± 63.7 290.8 ± 35.7

519.9 ± 82.9 347.2 ± 55.3

153 ± 19.1 118 ± 15

193.6 ± 20.6 133 ± 14.6

Sensorimotor CX Ipsil Contral

350.4 ± 45.4 227.5 ± 14.9

382.5 ± 85 246.6 ± 39.2

Dentate GYRUS Ipsil Contral

263 ± 30.3 203.2 ± 13.8

335.1 ± 60.1 217.8 ± 27.7

DH CA1 area Ipsil Contral

176.1 ± 15 142.4 ± 10.9

219.6 ± 58 154.5 ± 18.3

DH CA2 area Ipsil Contral

131.3 ± 22.5 89.6 ± 7.2

119.7 ± 17.4 97.7 ± 8.2

DH CA3 area Ipsil Contral

126.9 ± 15.8 91.4 ± 8.2

130.7 ± 16.4 103.6 ± 6.6

Thalamus Ipsil Contral

850.4 ± 172.9 789.7 ± 70.9

1086.9 ± 239.2 800 ± 108.3

Basolateral AMG Ipsil Contral

212.3 ± 14 188.7 ± 14.1

238.4 ± 48.1 179.6 ± 26.6

Central AMG Ipsil Contral

221.4 ± 30.7 213.4 ± 31.1

176.7 ± 20.4 198.8 ± 34.2

Medial AMG Ipsil Contral

297.4 ± 26 260 ± 26

362.7 ± 69 282.6 ± 59.5

Piriform CX Ipsil Contral

211.7 ± 26.8 148.1 ± 6.3

293.2 ± 58.6 160.2 ± 23.5

Temporal CX Ipsil Contral

573.4 ± 112.3 493.5 ± 48.7

606.2 ± 112.2 513.1 ± 75.2

Anterior AMG Ipsil Contral

LFS

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Table 5 (Continued ) Brain area

Control

VH CA1 area Ipsil Contral

307.1 ± 56.5 319.4 ± 58

314.5 ± 53.9 338.9 ± 79.6

240.2 ± 34.1 226.9 ± 38.3

260.2 ± 49.9 217.7 ± 40.6

VH CA3 area Ipsil Contral

225.1 ± 45.2 230.5 ± 40.2

284.6 ± 71.7 220 ± 39

Entorhinal CX Ipsil Contral

257.6 ± 34.8 276.6 ± 42.7

358.8 ± 57.3 333.2 ± 65.9

VH CA2 area Ipsil Contral

LFS

Notes as in Table 3.

clei, piriform cortex (14.6%), and CA2 area of ventral hippocampus (35.1%) (Table 6).

4. Discussion 4.1. LFS modifies amygdala kindling process Studies from Weiss et al. (1995) indicated that daily LFS using 1 Hz during 15 min immediately after each kindling stimulation blocked completely the development and progress of seizure and afterdischarges, an effect associated with increased seizure threshold. The present report supports that LFS applied twice daily is able to avoid the establishment of the kindled state in the rat but it does not prevent the induction of partial seizures during the kindling process. Several factors could explain the disagreement between Weiss’ study and the present report. One important aspect is the stimulation protocol used, i.e., Weiss et al. (1995) applied LFS daily while we applied it twice daily. Another important aspect is that Sprague Dawley rats, used by Weiss et al. (1995), present different susceptibility to the kindling process when compared to the Wistar rats used in the present report (Tsunoda et al., 1995). The blockage of generalized, but not partial, seizures is also produced by the vagal electrical stimulation during the kindling process in cats (Fernandez-Guardiola et al., 1999). Possibly, LFS and vagal electrical stimulation share a mutual anatomic

Table 6 3 H-forkolin binding (fmol/mg protein) in rat brain areas of control and LFS groups Brain area

Control

LFS

Caudate putamen Ipsil Contral

338.2 ± 16.2 368.5 ± 31

385.6 ± 36.6 383.7 ± 44.2

Cingulate CX Ipsil Contral

131.2 ± 4.3 131.6 ± 7

141 ± 5.3 141.3 ± 5.5

Frontal CX Ipsil Contral

117.7 ± 7.3 119.4 ± 5.5

122.7 ± 7.1 121.2 ± 5.8

Anterior AMG Ipsil Contral

91.5 ± 8.9 98.9 ± 8.7

90.5 ± 10.6 92.5 ± 10.3

Sensorimotor CX Ipsil Contral

118.1 ± 6 124.4 ± 5.4

116.6 ± 9.1 117.8 ± 8.6

Dentate GYRUS Ipsil Contral

152.5 ± 5.6 154 ± 4.8

150.2 ± 8.5 151.7 ± 7.4

DH CA1 area Ipsil Contral

85.7 ± 10.3 85.7 ± 9.7

86.8 ± 12.5 82.2 ± 9.1

DH CA2 area Ipsil Contral

49.9 ± 7.2 47.6 ± 7.2

64.1 ± 11.12 56.8 ± 6.6

DH CA3 area Ipsil Contral

61 ± 8.4 53.7 ± 5.3

71.5 ± 10.4 58 ± 9

Thalamus Ipsil Contral

76.5 ± 11 94.2 ± 11.2

75.8 ± 9.5 79.6 ± 8.7

Basolateral AMG Ipsil Contral

120.5 ± 9.4 122.5 ± 7.5

129.7 ± 5.2 138.4 ± 5.8

Central AMG Ipsil Contral

116.1 ± 12.8 98.1 ± 10.1

118.9 ± 7.4 128.8 ± 6.4

Medial AMG Ipsil Contral

119.2 ± 7.5 118.5 ± 7.7

129.9 ± 6.2 139.9 ± 7.4

Piriform CX Ipsil Contral

136.5 ± 5.3 126.3 ± 5.3

146.8 ± 4.3 144.7 ± 5.6

Temporal CX Ipsil Contral

122.4 ± 10.5 119.4 ± 9.8

139.2 ± 6.4 137.8 ± 7

M.L. L´opez-Meraz et al. / Epilepsy Research 59 (2004) 95–105 Table 6 (Continued ) Brain area

Control

LFS

VH CA1 area Ipsil Contral

88 ± 9.66 87.3 ± 10.4

112.7 ± 8.7 99 ± 5.6

VH CA2 area Ipsil Contral

75.8 ± 7.4 59.6 ± 8.8

81.1 ± 4.4 80.5 ± 5.4

VH CA3 area Ipsil Contral

75.4 ± 9.5 67 ± 9.9

76.1 ± 5.7 65 ± 3.8

Entorhinal CX Ipsil Contral

138.7 ± 5.5 141.5 ± 5.3

134.4 ± 7.6 135.2 ± 7.7

Notes as in Table 3.

substrate and/or common neuronal circuits that can be essential for the control of epilepsy. 4.2. Effects of LFS on the receptor binding levels Nowadays, the mechanisms associated with the antiepileptic effect of LFS are unknown. Our autoradiography experiments revealed that LFS applied twice daily in normal rats increased the BZD receptor levels in basolateral amygdala and thalamus ipsilateral to the place of stimulation and in contralateral temporal cortex. In contrast, LFS produced a decrease of BZD receptor levels in the contralateral frontal cortex. Another interesting finding from the present study was that repetitive LFS in normal rats produced a significant slight decrease of the ␮ receptor binding in the sensorimotor and temporal cortices ipsilateral to the place of stimulation, contralateral CA1 field of hippocampus, and bilateral dentate gyrus. Considering that some of these structures are implicated in the generation and propagation of epileptic activity (Fernandez-Guardiola et al., 1990; Tuff et al., 1983a,b; Wada and Okamoto, 1986), the LFS-induced BZD and ␮ receptor binding changes in these areas may restrain the spread of epileptic activity. Interestingly, the decreased ␮ receptor binding produced by LFS is similar to the changes detected after repeated application of electroconvulsive shocks (Nakata et al., 1985), a type of electrical stimulation that also inhibits amygdala kindling (Post et al., 1984). The decreased BDZ and ␮ receptor levels may be associated with reduction of neural activity and conse-

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quent functional deafferentation of structures receiving the specific outputs (Ackermann et al., 1984). An interesting result is that low receptor binding could result from the repetitive exposure to endogenous ligands (Lyons et al., 2001; Stecee et al., 1989). Then, future experiments should be carried out to evaluate the GABA and opioid peptide release in specific brain areas involved in the antiepileptic effects of LFS. However, preliminary results obtained in our laboratory indicate that LFS does not modify in vivo GABA release in rat amygdala (López-Meraz et al., 2002). Studies support that ␣1 -adrenergic receptor activation induces an inhibitory effect in epileptogenesis (Gundlach et al., 1995; Weinshenker et al., 2001), whereas the activation of Gs-AC-cAMP system in rat brain facilitates epileptic seizures (Ludvig et al., 1992; Williams et al., 1993; Higashima et al., 2002; Iwasa et al., 2000). Our autoradiography experiments indicate that LFS does not modify either ␣1 receptor levels or adenylyl cyclase binding. These results lead to the idea that LFS induces selective changes on the BZD and ␮ receptors. Additional studies examining the effects of LFS in specific neurotransmitter systems such as GABA and opioids will add to the understanding of the mechanisms of LFS and may give additional insight into its applications. LFS-induced BZD receptors binding alterations could be mediating, almost partially, the anti-anxiety effects produced by LFS in cats (Adamec, 1999; Adamec and Young, 2000). In addition, LFS could be effective in the treatment of other neurological disorders in which the GABA neurotransmission and opioid system are involved. However, it remains to be determined whether these changes are permanent and whether such stimulation can be effective in the treatment of neurological or psychiatric disorders in humans. In the future, it will be necessary to examine different receptor binding levels in rats receiving both kindling and LFS in order to determine changes in specific brain areas related with the LFS-induced antiepileptic effects and their relationship with behavioral changes.

Acknowledgements We are indebted to Mrs. Magdalena Briones, Mr. Héctor Vázquez, and Mr. Adrian Mart´ınez for their

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