Status of somatostatin receptor messenger RNAs and binding sites in rat brain during kindling epileptogenesis

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Neuroscience Vol. 75, No.3, pp. 857-868, 1996 Copyright © 1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain S0306-4522(96)00304-l 0306-4522/96 $15.00+0.00

STATUS OF SOMATOSTATIN RECEPTOR MESSENGER RNAs AND BINDING SITES IN RAT BRAIN DURING KINDLING EPILEPTOGENESIS C. PIWKO,* V. S. THOSS,* R. SAMANIN,t D. HOYER* and A. VEZZANIH *Preclinical Research, 360/604, SANDOZ Pharma Ltd, CH-4002, Basel, Switzerland tLaboratory of Neuropharmacology, Mario Negri Institute of Pharmacological Research, Via Eritrea 62, 20157 Milano, Italy Abstract~In

situ hybridization histochemistry with somatostatin sst 1-sst 5 receptor messenger RNAselective oligoprobes and quantitative receptor autoradiographic binding studies using [125I]Tyr 3 _ octreotide, [Leu8,o-Trp22,125I-Tyr25]somatostatin-28 and [125 I]CGP 23996 (rt 25 I]c[Asn-Lys-Asn-Phe-PheTrp-Lys-Thr-Tyr-Thr-Ser]) were performed to determine the level of expression of somatostatin receptor messenger RNA and receptor binding sites in the hippocampal formation, limbic system and cerebral cortex of adult rats electrically kindled in the dorsal hippocampus. In control rats (implanted with electrodes but not electrically stimulated), the somatostatin-l receptorselective rt 25 I]Tyr 3-octreotide and the non-subtype-selective [Leu 8 ,o-Trp22, 1251_Tyr 25 ]somatostatin-28 preferentially labelled the strata oriens and radiatum of the CAl subfield of the hippocampus, the molecular layer of the dentate gyrus, the subiculum and presubiculum of the hippocampal formation, the inner layer of the frontal cortex, and the lateral and basolateral nuclei of the amygdala. The non-subtype-selective radioligand rt 25 I]CGP 23996 (in 5 mM Mg2+ buffer) preferentially labelled the strata oriens and radiatum of the CAl subfield of the hippocampus, the subiculum and the basolateral nucleus of the amygdala. Under conditions where primarily somatostatin-2 receptors were labelled, rt 25 I]CGP 23996 (in 120 mM Na+ buffer) showed strong binding in the strata oriens and radiatum of the CAl subfield of the hippocampus and the frontal cortex, whereas the dentate gyrus, subiculum and amygdala showed only weak signals. During and after kindling, no significant differences were observed between the ipsi- and contralateral sides of the hippocampus. A significant decrease (about 40%) of somatostatin receptor binding sites was observed in the molecular layer of the dentate gyrus with all radioligands (except [125 I]CGP 23996 in Na+ buffer, which did not label this area) at stage 2 (pre-convulsive stage) and one week, but not one month, after stage 5 (generalized motor seizures). In contrast to somatostatin receptor binding, no alterations of the messenger RNA levels for sst l -sst 5 receptors were found either at stage 2 or at stage 5. Similarly, no changes in receptor binding or messenger RNA levels were observed in the brain of rats which experienced a single afterdischarge. The present study shows a significant and selective decrease of somatostatin-l receptor binding sites in the dentate gyrus of kindled rats. This is part of the plastic changes induced by kindling and may contribute to the increased sensitivity for the induction of generalized seizures during kindling. Copyright © 1996 !BRO. Published by Elsevier Science Ltd. Key words: somatostatin (SRIF, somatotropin release-inhibiting factor), sst/SRIF receptors, autoradiography, in situ histochemistry, epilepsy, kindled seizure.

Somatostatin (SRIF, somatotropin release-inhibiting factor) is a widely distributed neuropeptide with a range of actions; SRIF modulates many physiological and pathological functions, including hormone secretion and cell proliferation. 8,37 The molecular mechanisms of action of this peptide suggest modulation of potassium and calcium channels and cyclic nucleotides, mainly cyclic AMP. 16,18,19 SRIF-14 and its N-terminally extended form SRIF-28 occur in tTo whom correspondence should be addressed. Abbreviations: CGP 23996, c[Asn-Lys-Asn-Phe-Phe-Trp-

Lys-Thr-Tyr-Thr-Ser]; EDTA, ethylenediaminetetraacetate; EEG, electroencephalographic; EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; [125 I]LTT_ SRIF-28, [Leu 8 ,o-Trp22, 125I-Tyr25]somatostatin-28; SRIF, somatostatin; sst, somatostatin receptor subtype. 857

several peripheral tissues,37 as well as in the brain. 8 Five different somatostatin receptor subtypes (SSTRl-5) have been identified and cloned, which belong to the family of the seven transmembrane domain receptors coupled to guanyl-nucleotide binding proteins (G_proteins).15,38 According to IUPHAR recommendations, these receptors are now named sst 1-sst 5. 15 Based on operational and structural considerations, two classes of SRIF receptors can be distinguished: 15 ,16,17 the SRIF/SS-l/SOM A family (sst 2 , sst 3 and sst 5) shows intermediate to high affinity for octreotide, seglitide and somatuline, whereas the SRIF 2/SS-2/S0M B family displays virtually no affinity for these short cyclic analogues of SRIF. In addition, Reubi and Maurer 39 showed that ions exert an effect on the binding of radioligands to

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SRIF receptors: the presence of Mg2+ ions significantly increased the SRIF1 binding, whereas that of Na+ increased the SRIF 2 binding. Increasing evidence suggests that various diseases of the CNS are associated with an imbalance of brain SRIF expression. Indeed, in post mortem brains or in cerebrospinal fluid from patients with CNS disorders such as Alzheimer's disease,5 Huntington's chorea,! ,2,25 multiple sclerosis,51 schizophrenia, 11,25 depression,43 Parkinson's disease 9 and epilepsy,2,42 the levels of SRIF have been reported to be altered. Various biochemical, immunocytochemical and pharmacological evidence in humans and/or experimental models indicates that brain SRIF is implicated in seizure phenomena?,8, 13,14,20,22,40,42,47,49,50,52,56,58 In particular, recent immunocytochemical findings have shown that SRIF-containing neurons in the hilus of the dentate gyrus degenerate following status epilepticus induced either electrically or chemically,47,52 while they show a pronounced and lasting increase in their mRNA expression and immunoreactivity in the cell bodies and/or terminal projection area after kindling. 3,46,59 Similar changes were observed in humans affected by temporal lobe epilepsy, with graded degrees of neurodegeneration in the hippocampus. 6,42 The changes observed in SRIFcontaining neurons in chronic models of epilepsy result in modifications in the release of the peptide,29,57 indicating a functional alteration in the related neurons. Pharmacological and electrophysiological findings suggest that SRIF released in the various hippocampal subfields under physiological or epileptic conditions exerts an important inhibitory control on granule and/or pyramidal cell excitability.21,30,44 Indeed, we found that the stimulation of sst 2 receptors in the hippocampus with octreotide or octastatin protected the rats from acute and chronic seizure susceptibility resulting from kainate treatment. 29 ,58 In addition, by studying the development of hippocampal kindling in rats, we have recently found that intrahippocampal infusion of an SRIF antibody enhances the rate of kindling development,24 suggesting an anti-epileptogenic role of the endogenous peptide in these models of limbic epileptogenesis. Perez et al. 29 have recently shown that SRIF receptors in the hippocampus undergo selective subtype- and area-specific changes after generalized limbic seizures induced by systemic injection of kainate. Thus, the CAl subfield displays a decrease in mRNA for the sst 3 and sst4 receptors and in the density of binding sites for [Leu 8,D_Trp22, 125 I_ 25 I]LTT-SRIF-28) and Tyr25]somatostatin-28 125 [ I]c[Asn-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-ThrSer] ([ 125 I]CGP 23996) (Mg2 + buffer), while sst 2 receptor mRNA and binding sites labelled with [125 I]Tyr 3-octreotide were spared. No changes in receptor mRNA or binding were observed in the molecular and granule cell layers.

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No data are yet available on the status of SRIF receptors in kindling, a gradual process of epileptogenesis considered to be a model of certain aspects of human epilepsy of focal onset. 12,34 This knowledge is important to add fresh information on the biochemical and molecular mechanisms which underlie kindling epileptogenesis, to elucidate the mechanism of the anti-convulsant action of SRIF and to target new pharmacological approaches that may counteract the establishment of a chronic epileptic focus. In the present study, we examined the expression of SRIF sst 1-sst 5 receptor mRNA and SRIF receptor binding sites in various subfields of the hippocampal formation, the limbic system and the cerebral cortex of adult rats at different stages of kindling and after kindling acquisition. We performed autoradiographic binding studies using [125 I]Tyr 3-octreotide, which has been reported to label primarily sst 2 receptors,31,32,45 [125 I]LTT-SRIF-28, which is believed to bind to all SRIF receptors,27 although we have recently observed a preferential binding to the SRIF1 receptors,32,53 and [125 I]CGP 23996 in two different buffers. Thus, in Mg 2+ buffer [125 I]CGP 23996 binds with high affinity to most cloned receptors,35,36 while radioligand binding to SRIF2 sites seem to be favoured by Na+. 39 EXPERIMENTAL PROCEDURES

Experimental animals

Male Sprague-Dawley rats (250-280 g; Charles River, Calco, Italy) were used. The animals were housed at constant temperature (23°C) and relative humidity (60%), with a fixed l2-h light-dark cycle and free access to food and water. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (4D.L. N.l16, G.U., supp!. 40, 18 February 1992) and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, 12 December 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985). Kindling

The electrodes were implanted in the dorsal hippocampus under Equithesin anaesthesia (1 % pentobarbital/4% chloral hydrate; 3.5 mllkg, i.p.), according to the following coordinates (mm) from bregma: nose bar - 2.5 below the interaural line, AP - 3.5, L ± 2.3, H 2.9 below dura. Electroencephalographic (EEG) recordings were made using bilateral cortical and hippocampal electrodes in unanaesthetized, freely moving animals, as described previously.57 Kindling was started after a postoperative period of seven days, when the animals showed no behavioural signs of pain or discomfort. The rats were allowed to acclimatize in a Plexiglas cage and an EEG recording was made for at least 10 min to assess the spontaneous EEG pattern. Constant-current stimuli were delivered unilaterally to the dorsal hippocampus through a bipolar electrode (recording electrode) twice daily for five days then once daily for two days (weekend), at intervals of at least 6 h. The stimulation parameters were 50-Hz, 2-ms monophasic rectangular wave pulses for 1 s, the current intensity ranging between 60 and 200 llA. Behaviour was observed and the duration of afterdischarge was measured in the stimulated hippocampus after each stimulation for every anima!.

Somatostatin receptor subtypes in kindling Before electrical stimulation, the rats were randomly assigned to two groups and received 12 ± I and 27 ± 2.5 stimuli (mean ± S.E.) to reach respectively stages 2 (stereotypies, occasional retraction of a forelimb; n = 5) and 5 (tonic--elonic seizures with rearing and falling; n = 8) of kindling according to Racine's c1assification. 34 Animals were considered fully kindled when they experienced at least three consecutive stage 5 seizures. Controls were implanted with electrodes, but were not electrically stimulated (referred to here as sham stimulation; n = 8). Rats kindled at stages 2 and 5 and the corresponding shams were killed respectively two days or one week and one month after the last electrical stimulation. These intervals were chosen on the basis of previous studies showing mRNA, 3 immunocytochemical 46 and release41 ,57 changes for SRIF and neuropeptide Y related to kindling-induced plasticity, but not to the recent experience of seizure activity. A different group of animals received a single stimulation inducing an afterdischarge and was killed two (n = 3) or seven (n = 3) days later. Tissue treatment

Rats after one afterdischarge or kindled to stages 2 and 5 and their respective controls were decapitated and their brains rapidly removed from the skull. The brains were then immediately immersed in -70'C isopentane for 3 min and stored in tightly sealed vials at - 70'C. Sections from rat brains were cut in either lO-~m-thick slices for receptor autoradiography analysis or 20-~m-thick slices for in situ hybridization analysis using a microtome-cryostat; they were thaw-mounted onto "Super Frost Plus" microscope slides (Menzel Glaeser) and stored at - 20'C. Receptor autoradiography and in situ hybridization analysis were carried out in the cerebral cortex, the limbic system and the hippocampus, identified according to the atlas of Paxinos and Watson?8 Receptor autoradiography

Receptor autoradiography was performed in lO-~m brain sections of control rats, stage 2 and 5 kindled rats and rats which experienced a single afterdischarge, according to the following procedure. After 20 min of preincubation in buffer containing 50 mM Tris-HCI (pH 7.4), 0.2% bovine serum albumin, 10 ~g/ml bacitracin, 2 mM EGTA and 5 mM MgCI 2 (for 2s l]Tyr 3-octreotide, 251]LTT-SRIF-28 and C2s l]CGP 23996) or 120 mM NaCI (for C251]CGP 23996) at room temperature, the slides were incubated for 2 h at room temperature in the same medium supplemented with approximately 50 pM C251]ligand. Non-specific binding was determined in a set of adjacent slices by incubation in the presence of I ~M SRIF-14. The washing steps of labelled sections were carried out as follows. A brief dipping in ice-cold distilled water was followed by two 10-min washes in the former buffer and a brief dipping in ice-cold distilled water to remove the salts. Finally, the sections were quickly dried under a stream of cold air. Autoradiograms were generated by apposing the labelled tissues and lO-~m-thick autoradiographic 1251 micro-scales (Amersham, Buckinghamshire, U.K.) to 3H-Hyperfilms (Amersham) at 4'C for two to three days (for C25 1]LTT-SRIF-28 and C2s l]Tyr3 -octreotide) or for one week (for C25 1]CGP 23996). Radioligands were custom synthesized by Anawa (Wangen, Switzerland).

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In situ hybridization In situ hybridization was performed in sections consecutive to those used for receptor autoradiography for control rats and stage 2 and 5 kindled rats as follows. The oligoprobes specific for sstl-ssts receptor mRNAs, as used previously,54 were labelled at their 3' end with 3 P]a-dATP (Amersham) and terminal deoxynucleotidyltransferase

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(Boehringer Mannheim). Cryostat sections (20 ~m) were fixed for 20 min at room temperature in a freshly prepared solution containing 4% (w/v) paraformaldehyde and incubated in a freshly prepared solution of predigested pronase of 24 U/ml. The 33P-Iabelled DNA probes were diluted to a final concentration of 1-2 x 107 c.p.m./ml labelled probe (i.e. 0.03-0.06 pmol/slide), 50% forrnamide (for sst l-SSt 3) or 40% forrnamide (for sst4 and sst s), 600 mM NaC!, 10 mM Tris-HCI (pH 7.5), I mM EDTA, I x Denhardt's, 500 ~g/ml yeast tRNA and 10% dextran sulphate. After incubation in a humid chamber overnight at 3TC, sections were washed at 60'C in a buffer containing 600 mM NaCI, 20 mM Tris-HCI (pH 7.5) and I mM EDTA with four changes of I h each. The tissues were then dehydrated by immersion in 70% and 95% ethanol containing 300 mM ammonium acetate (pH 7.0). Autoradiograms were obtained by exposure to ~-max films (Amersham) for one (sst 3), three (sst Io sst2 and sst4) or four weeks (sst s). Analysis of non-specific signals was performed in consecutive sections simultaneously hybridized with radioactive and an excess (20-fold) of corresponding non-labelled probes. Following autoradiography and in situ hybridization, sections were stained with 0.5% Cresyl Violet and regions were localized according to the atlas of Paxinos and Watson?8 Data analysis

Autoradiograms from in situ hybridization and binding experiments were quantified densitometrically with a computerized image analysis system (MCID, Imaging Research, St Catherines, Ontario, Canada). Optical density was measured in several subfields of the hippocampal formation, the limbic system and in different regions of the cerebral cortex, and non-specific values were subtracted to obtain the specific optical density in the respective area investigated. The values listed in Table I represent the mean fmol/mg protein ± S.E.M. of five animals per group. To calculate the amount of protein in fmol/mg we used lO-~m-thick 1251 micro-scales (Amersham) and followed the manufacturer's instructions.

RESULTS

Receptor autoradiography Figure 1 illustrates the total and non-specific binding obtained with the four radioligands tested in the present study, and depicts the differences between the distribution pattern of binding sites labelled with the various SRIF receptor radioligands in adjacent sections of control rats. In these rats, [125 I]Tyr 3 _ octreotide (Fig. 1.1) and [125 I]LTT-SRIF-28 (Fig. 1.2) preferentially labelled the strata oriens and radiatum of the CAl subfield of the hippocampus, the molecular layer of the dentate gyrus, the presubiculum and subiculum of the hippocampal formation, the inner layer of the frontal cortex, and the lateral and basolateral nuclei of the amygdala. 125 [ I]CGP 23996 (Mg 2 + buffer) (Fig. 1.3) preferentially labelled the strata oriens and radiatum of the CAl subfield of the hippocampus, the subiculum, the basolateral nucleus of the amygdala and the inner layer of the frontal cortex. The strongest signals for 125 [ I]CGP 23996 (Na+ buffer) (Fig. 1.4) were found in the strata oriens and radiatum of the CA1 subfield of the hippocampus, the subiculum and the frontal cortex.

2NS·

3NS

Fig. 1. Autoradiographic distribution sites in sections of control rat brains using P25 I]Tyr 3-octreotide (IC, INS), preferentially labelling receptors of the SRIF j family, P25 I]LTT-SRIF-28 (2C, 2NS) and P25 I]CGP 23996 (Mg2 + buffer) (3C, 3NS), which bind to most cloned receptors, and [125 I]CGP 23996 (Na+ buffer) (4C, 4NS), preferentially labelling receptors of the SRIF 2 family in the absence (lC-4C) and in the presence of I IlM SRIF-14 (lNS-4NS). Scale bar = 2 mm. Abbreviations in Figs 1-3: CAl, CAl subfield of the hippocampus; CA3, CA3 subfield of the hippocampus; DG, dentate gyrus (Fig. 2: molecular layer; Fig. 3: granule cell layer); MHb, medial habenular nucleus.

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"3AD Fig. 2. Cresyl Violet-stained (I) and autoradiographic distribution sites in sections showing the hippocampus of control rats (lC-4C), stage 5 kindled rats (IK-4K) and rats subjected to a single afterdischarge (IAD-4AD), using 25 I]Tyr3-octreotide (2), [125 I]LTT-SRIF-28 (3) and [125 I]CGP 23996 (Mg2+ buffer) (4). The rats were killed one week after the electrical stimulation (AD) or after the last seizure (K). See legend to Fig. I for details. Scale bar =2 mm.

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Figure 2.1 shows the morphological features of Cresyl Violet-stained sections of the hippocampus of

controls (Fig. 2.1 C), kindled rats one week after stage 5 (Fig. 2.lK) and rats which experienced a single

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Table 1. Receptor autoradiographic analysis of somatostatin receptors in the hippocampus of control rats and rats kindled at stages 2 and 5 Stage 5

Stage 2

eFrontal I]Tyr -octreotide cortex layers V and VI 25

Kindling

Control

Kindling Control (fmol/mg protein)

160 116 39 88 85 31 33 23 23 182 86 10 21 46 38 39 11 98 164 49 74

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

12 7 5 4 6 2 2 2 3 13 3 2 4 5 2 I 2 3 2 4 2

153 ± 68 ± 34 ± 86 ± 89 ± 31 ± 35 ± 25 ± 24 ± 180 ± 82 ± 12 ± 20 ± 47 ± 39 ± 38 ± 10 ± 96 ± 162 ± 51 ± 73 ±

5 4** 5 4 4 2 2 2 3 II 4 2 3 6 3 2 I 4 5 5 4

154 113 35 87 86 29 32 22 24 169 88 12 22 44 36 40 10 97 169 47 72

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6 8 4 3 5 3 2 2 2 15 5 2 3 4 4 3 2 3 5 4 2

150 ± 69 ± 36 ± 85 ± 90 ± 29 ± 33 ± 23 ± 23 ± 172 ± 85 ± II ± 22 ± 45 ± 36 ± 41 ± 9 ± 99 ± 167 ± 51 ± 75 ±

115 72 27 85 83 25 24 19 19 115 58 10 21 44 28 31 64 90 40 45

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6 5 2 6 3 2 2 3 2 4 5 2 3 5 3 2 5 6 4 3

117 45 24 86 85 24 23 21 21 112 56 10 20 45 26 33 63 95 44 48

3 3** 2 5 3 3 2 2 3 6 4 I 4 6 2 3 4 5 5 4

124 70 29 82 81 26 23 20 21 120 54 9 21 47 27 32 55 89 41 47

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6 5 3 6 4 3 2 2 4 9 3 I 3 4 3 3 5 6 4 3

109 ± 39 ± 26 ± 87 ± 84 ± 25 ± 24 ± 22 ± 20 ± 109 ± 53 ± 8 ± 21 ± 44 ± 30 ± 33 ± 58 ± 93 ± 43 ± 50 ±

Area 3

Molecular layer of the DG Granular layer of the DG Stratum oriens of CAl Stratum radiatum of CAl Stratum oriens of CA2 Stratum radiatum of CA2 Stratum oriens of CA3 Stratum radiatum of CA3 Subiculum Presubiculum Outer layers of the entorhinal cortex Intermediate layers of the entorhinal cortex Deep layers of the entorhinal cortex Piriform cortex Posterolateral cortical amygdala nucleus Central amygdala nucleus Lateral amygdala nucleus Basolateral amygdala nucleus Medial amygdala nucleus Posteromedial cortical amygdala nucleus

eFrontal I]LTT-SRIF-28 cortex layers V and VI

4

3** 3 2 4 2 3 I 2 10 4 3 2 5 3 3 I 5 6 3 4

25

Molecular layer of the DG Granular layer of the DG Stratum oriens of CAl Stratum radiatum of CAl Stratum oriens of CA2 Stratum radiatum of CA2 Stratum oriens of CA3 Stratum radiatum of CA3 Subiculum Presubiculum Outer layers of the entorhinal cortex Intermediate layers of the entorhinal cortex Deep layers of the entorhinal cortex Piriform cortex Posterolateral cortical amygdala nucleus Lateral amygdala nucleus Basolateral amygdala nucleus Medial amygdala nucleus Posteromedial cortical amygdala nucleus

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4 3** 2 5 5 3 3 I 2 7 4 I 2 3 4 3 6 6 3 2

Continued opposite

afterdischarge (Fig. 2.1AD). Figures 2.2, 2.3 and 2.4 depict the autoradiograms of the total binding of SRIF receptor ligands in the corresponding experimental groups. The quantitative assessment of the autoradiograms is reported in Table 1. The density of 25 I]Tyr 3 _ octreotide binding sites was significantly decreased by 41% (P < 0.01) at stage 2 of kindling and by 39% (P < 0.01) at stage 5 in the molecular layer of the dentate gyrus (Fig. 2.2). Decreases of 38% (P < 0.01) and 44% (P < 0.01) were also observed in the same region with [125 I]LTT-SRIF-28 (Fig. 2.3) at stages 2 and 5, respectively. [125 I]CGP 23996 (Mg 2 + buffer) binding sites were selectively diminished by 35% (P < 0.05) at stage 2 and by 38% (P < 0.01) at stage 5

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in the molecular layer of the dentate gyrus (Fig. 2.4). No significant difference was observed between the ipsi- and contralateral sides of the hippocampal formation; therefore, the results of both sides were pooled. In all other regions of the rat brain examined, the binding of none of the radioligands was significantly altered by kindling. Similarly, no differences were observed in the binding of [125 I]CGP 23996 in Na+ buffer, which predominantly labels the SRIF2 receptor family (Table 1). In the molecular layer of the dentate gyrus, no significant differences in autoradiographic signals were observed in rats two days (not shown) or one week after a single afterdischarge compared to controls with all four radioligands used (Figs 2.2, 2.3, 2.4AD).

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Table I (continued). Stage 2 Kindling Control (fmol/mg protein)

Kindling

45 26 9 53 72 22 23 16 16 44 21 6 12 16 12 19 17 71 13 25

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 2 2 4 6 2 2 I 2 3 1 I 2 2 2 3 2 5 2 3

49 17 8 55 68 21 26 15 18 46 24 5 14 19 II 21 17 65 14 26

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5 3' I 4 6 3 3 2 3 5 3 I 3 3 2 3 3 4 3 3

44 29 9 49 67 19 27 18 14 49 19 5 II 15 12 18 15 68 15 27

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6 3 I 5 4 3 2 2 2 5 3 I 2 3 3 2 2 3 2 3

46 18 7 54 65 22 22 19 15 45 21 4 13 14 13 17 14 66 13 23

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 2" 2 4 5 3 2 1 2 4 3 1 1 2 2 2 3 4 3 2

25 19 22 24 18 II 19 17

± ± ± ± ± ± ± ±

3 2 3 4 2 2 2 2

22 17 18 21 16 12 21 15

± ± ± ± ± ± ± ±

4 2 3 2 6 3 3 3

27 20 19 19 20 12 22 14

± ± ± ± ± ± ± ±

3 3 2 3 3 2 3 2

23 21 17 23 17 13 18 15

± ± ± ± ± ± ± ±

2 2 2 3 2 2 2 4

Area [

[

Stage 5

Control

125

I]CGP 23996 in Mg2 + buffer Frontal cortex layers V and VI Molecular layer of the DG Granular layer of the DG Stratum oriens of CAl Stratum radiatum of CA1 Stratum oriens of CA2 Stratum radiatum of CA2 Stratum oriens of CA3 Stratum radiatum of CA3 Subiculum Presubiculum Outer layers of the entorhinal cortex Intermediate layers of the entorhinal cortex Deep layers of the entorhinal cortex Piriform cortex Posterolateral cortical amygdala nucleus Lateral amygdala nucleus Basolateral amygdala nucleus Medial amygdala nucleus Posteromedial cortical amygdala nucleus

125

I]CGP 23996 in Na+ buffer Frontal cortex layers V and VI Stratum oriens of CAl Stratum radiatum of CAl Subiculum Presubiculum Piriform cortex Basolateral amygdala nucleus Posteromedial cortical amygdala nucleus

Data represent fmol/mg protein ± S.E.M. measured in five rats per experimental group. The animals were killed two days and one week after stages 2 and 5, respectively. DG, dentate gyrus. 'P<0.05; "P
When the receptor binding was measured one month after the last stage 5 seizure, an average 14% decrease was found in the binding of C2S I1Tyr 3octreotide (optical density: control, 515 ± 20; kindled, 453 ± 12), C2s I1LTT-SRIF-28 (control, 354 ± 12; kindled, 314 ± 9) and C2s I1CGP 23996 (control, 326 ± 23; kindled, 276 ± 14; n = 3) in the molecular layer of the dentate gyrus.

tor mRNA signals in all examined regions of kindled rat brains compared to controls at both stages of kindling (Fig. 3).

DISCUSSION

Experimental evidence suggests that brain SRIF is involved in kindling epileptogenesis. Thus, SRIFcontaining neurons in the hippocampus are functionIn situ hybridization histochemistry ally activated during and after kindling acquisition, Representative sections of the dorsal hippocampus as indicated by tissue levels,14,s7,s9 immunocytofor each oligoprobe (sst l -sst S) are reported in Fig. 3. chemistry,33,46 release S7 ,20 and in situ hybridization The sst2-sst4 receptor oligoprobes labelled the studies of preprosomatostatin mRNA. 3 ,49,S6 Pharmapyramidal and granular cell layers of the hippo- cological evidence indicates that SRIF has an anticampus in control rats. The sst 2 receptor mRNA was convulsant role in kindling and other models of particularly expressed in the CAl sector and granule limbic seizures, predominantly mediated by the sst 2 cell layer; the sst3 mRNA was expressed in all hippo- ' subtype receptors. 24,29,S8 Previous evidence has shown that SRIF receptors campal layers to a similar extent, while the sst4 receptor mRNA was predominant in the pyramidal may change after seizures. Thus, generalized limbic cell layer as compared to granule neurons. The sst) convulsions induced by systemic injection of kainand sst s receptor mRNAs were virtually absent in the ate 29 decreased sst3 and sst4 receptor mRNAs and dorsal (septal) aspect of the hippocampus. The quan- SRIF binding sites in the hippocampus assessed titative assessment of the autoradiograms (not using [12s I1CGP 23996 (Mg 2 + buffer) and C2s I]LTTshown) showed no significant changes in SRIF recep- SRIF-28, and a reduction of SRIF receptors,

Fig. 3. Hybridization signals for SRIF sst, (1), sst z (2), sst 3 (3), sst4 (4) and sst s receptor probes (5) in the hippocampus of control (1 C-5C) and stage 5 kindled rats killed one week after the last seizure (1K-5K). Displacement of specific hybridization signals is shown (INS-5NS) using an excess of corresponding non-labelled probe. Scale bar = 2 mm.

Somatostatin receptor subtypes in kindling assessed using (D-Tyr8)-SRIF, was observed in the amygdala of fully kindled rats. 13 The present study shows that [125 I]Tyr 3-octreotide, 125 [ I]LTT-SRIF-28 and [125I]CGP 23996 (Mg 2+ buffer) SRIF binding sites were significantly and selectively decreased in the molecular layer of the dentate gyrus at stages 2 and 5 of kindling, although no significant changes in SRIF sst l-sst 5 receptor mRNA signals were detected in sections consecutive to those used for receptor autoradiography. The fact that SRIF receptor binding changed in the molecular layer of the dentate gyrus at stage 2 but not after a single afterdischarge indicates that this effect is linked to kindling progression and is not a simple consequence of the electrical stimulation or the experience of generalized seizure activity. The different results obtained in the receptor autoradiographic and in situ hybridization studies may be due to a different turnover of the mRNA expressing the receptor protein and the receptor protein itself, or to a non-linear correlation between the level of mRNA and the corresponding protein. Receptor autoradiography using 25 I]CGP 23996 in Na+ buffer was also performed in this study, since Reubi and Maurer 39 showed that ions exert an effect on the binding of radioligands to SRIF receptors. In fact, the presence of Na+ ions increases SRIF2 (in particular the sst l subtype) binding, whereas that of Mg2+ ions significantly increases SRIF I (in particular the sst 2 subtype) binding. Thoss et al. 55 found a highly significant correlation between monkey cortex 25 I]CGP 23996 binding in Na+ buffer and human recombinant sst l and sst4 receptors, but no correlation was observed with the binding profile of any member of the SRIF I receptor family (e.g., sst 2, sst3 and sst 5). The present findings indeed show that [125I]CGP 23996 (in the presence of high Na+ concentration) labelled sites in the rat brain which are different from those labelled in Mg 2+ buffer. In particular, we could detect almost no autoradiographic signal in the molecular layer of the dentate gyrus using 25 I]CGP 23996 in Na+ buffer, thus suggesting that the SRIF I receptor family is most probably involved in the changes induced by kindling in this layer. Our experimental conditions do not allow us to discriminate whether changes in the density and/or in the affinity of the receptors underlie the observed effects. The similar pharmacological characteristics of sst 2 and sst 5 receptors 4.10 • 16,35,36 prevent us from conclusively establishing whether the functional effects produced by the short synthetic analogues of SRIF such as octreotide are mediated by one or the other of these two receptors. Similarly, the functional effects of SRIF mediated by the sst/sst4 receptors l5 cannot yet be distinguished pharmacologically. However, the expression of mRNA for sst l and sst5 receptors is virtually absent in the dorsal hippocampus, suggesting that the related receptor proteins are not significantly present in this area.

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The distribution of binding sites in the various hippocampal subfields, together with the expression of their respective mRNAs in pyramidal and granule cells, suggest that SRIF receptors are mostly localized on the dendritic projections of CAl pyramidal cells in the strata radiatum and oriens and of granule neurons in the stratum moleculare of the dentate gyrus. This conforms to the electrophysiological evidence that SRIF, when applied to those hippocampal regions, affects neuronal activity.7.21.26,6o A recent electrophysiological study44 showed that, although SRIF may induce somatic depolarization on pyramidal neurons, long-duration pressure ejection or bath application of SRIF induce dendritic hyperpolarization. The author suggested that, during longer and widespread release, as might occur in kindling, if the peptide were released from many sites of peptidergic neurons simultaneously, hyperpolarization may override any depolarizing effect in the hippocampus. Indeed, the release of SRIF is enhanced in the hippocampus at stages 2 and 5,57 and recent immunocytochemical studies indicate that the peptide content is enhanced in fibres in the stratum lacunosum moleculare and, more significantly, in the outer molecular layer,46 the terminal areas of pyramidal and granule cell dendritic projections, respectively. This evidence may give insights into the mechanism underlying the decrease in SRIF1 receptors in kindling. The enhanced spontaneous and depolarization-evoked release of SRIF may lead to an increased extracellular concentration of the peptide, which in turn causes a decrease in receptor density and/or affinity. We found the both the release of SRIF 57 and its immunocytochemical changes 46 were increased to a lesser extent one month as compared to one week after stage 5 seizures, in accordance with a minor decrease in binding observed at the later time. Since previous evidence suggests that SRIF has a tonic inhibitory action on kindling epileptogenesis 24 and the sst 2 receptors mediate the anti-convulsant action of the peptide in the hippocampus,29,58 the decrease in SRIF 1 receptor binding may have some role in favouring hyperexcitability in the kindled tissue. This phenomenon, however, does not appear to be involved in the long-term sequelae of events underlying the maintenance of the kindling state. n is interesting to note that an enhanced GABA and benzodiazepine receptor binding has been found in the stratum moleculare of kindled rats 24 h but not 28 days after the last stage 5 seizure. 48 Since GABA is the classical neurotransmitter co-released with SRIF (see Ref. 46), these receptor changes may play a significant role in modulating granule cell excitability and the likelihood of recurrent seizures. The consequences of kindling and kainate-induced seizures on SRIF receptor subtypes are clearly different. Thus, kindling induced a decrease in receptor binding (essentially of the SRIF I sites without affecting mRNA levels for any of the receptors), while

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kainate-induced seizures were followed by a decrease in sst 3 and sst4 receptor mRNAs in CAL No modifications in sst 2 receptor sites were found, while a decrease in C25 I]LTT-SRIF-28 and 25 I]CGP 23996 (Mg2+ buffer) binding was observed. 29 These differences can be explained as follows. (1) The changes induced by kainate are very likely related to seizureinduced nerve cell loss in the corresponding neuronal populations carrying the receptors. In kindling, we previously found a 21-27% decrease ofhilar neurons, while pyramidal and granule cells were preserved. 46 (2) Kainate-treated rats showed a degeneration of a population of SRIF-containing neurons in the hilus and no increase was observed in the peptide's immunoreactivity in the outer molecular layer. 52

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CONCLUSION

The decrease in SRIF 1 receptor binding on presumed dendrites of granule cells in the outer molecular layer of the dentate gyrus may be part of

the plastic modifications in synaptic transmIssIon during kindling epileptogenesis. Although the functional consequences of this effect are still unknown, we suggest that it may impair inhibition of granule cell dendrites, thus increasing their level of excitability. It is known that granule neurons have a key position in determining hippocampal threshold to seizure activity.23.5o These results may therefore have important implications for the development of the kindling process and suggest that SRIF receptors of the SRIF 1 family represent an interesting target for pharmacological attempts to control neuronal excitability which is altered during epileptogenesis.

Acknowledgements-We thank Mr E. Schupbach and Mr D. Langenegger for expert technical assistance and Mr M. Rizzi and Dr M. Gariboldi for helping in kindling procedures. This work was partly supported by the CNR (National Research Council, Rome, Italy), Convenzione Psicofarmacologia.

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