Plastic Changes in Neuropeptide Y Receptor Subtypes in Experimental Models of Limbic Seizures

E/>i/?/JAiia,41(sUppl. 6):SI 15-s121, 2000 Lippincott Williams & Wilkins, Inc., Baltimore 0 Intcrnational Lcaguc Against Epilcpsy

Plastic Changes in Neuropeptide Y Receptor Subtypes in Experimental Models of Limbic Seizures A. Vezzani, D. Moneta, F. Mul6, T. Ravizza, *M. Gobbi, and TJ. French-Mullen Departments of Neuroscience and "Biochemistry, Mario Negri Institute for Pharmacological Research, Milano, Italy; and fCV&GI Research Department, AstraZeneca Pharmaceuticals, Macclesfeld, United Kingdom

Summary: Purpose: Neuropetide Y (NPY)-mediated neurotransmission in the hippocampus is altered by limbic seizures. The functional consequences of this change are still unresolved and clearly depend on the type of NPY receptors involved. NPY Y, and Y, receptors are increased on mossy fiber terminals and decreased on granule cell dendrites after seizures, respectively. We investigated (a) whether seizures modify the NPY Y, receptors in the hippocampus, and (b) the effect of an agonist at YJY, receptors and antagonists at Y , receptors on acute and chronic seizure susceptibility. Methods: Limbic seizures were induced in rats by electrical stimulation of the dorsal hippocampus, leading to stage S kindled seizures, or by intrahippocampal or systemic injections of kainic acid. Pentylentetrazol was administered to epileptic rats to assess their enhanced susceptibility to seizures. NPY Y, receptor protein was measured in hippocampal homogenates using a specific polyclonal antibody and quantitative Western blotting.

Results: YS receptors (57-kD band) were transiently decreased (23 to 35%) in all hippocampal subregions 2 and 7 days, but not 2.5 hours, after seizures induced by systemic kainic acid. A minor band of 51 kD was reduced significantly in CA3 and dentate gyrus, although it was increased in CAJ , 3 0 days after seizures, suggesting long-term posttranslational changes in this protein. NPY Y, receptors were increased by 200% in total homogenate from the stimulated hippocampus 2 days but not 30 days after fully kindled seizures. Intracerebral injections of NPY 13-36 (Y$Y, receptor agonist) or BIBP 3225 and BIB0 3304 (selective Y , receptor antagonists) decreased seizure susceptibility in rats. Conclusions: These results indicate that NPY Y receptors change after limbic seizures and suggest that NPY receptors may provide novel target(s) for the treatment of epilepsy. Key Words: Anticonvulsant-Epilepsy-Hippocampus-Kainic acid-Kindling-Rat.

Recent evidence indicates that neuropeptide Y (NPY)mediated neurotransmission undergoes plastic changes during epileptogenesis (1). Thus, the release of the peptide in the hippocampus and entorhinal cortex was enhanced after limbic seizures (2-4). This effect was associated with changes in NPY immunoreactivity in interneurons and fiber tracts. Interestingly, the granule neurons of the dentate gyrus and their constitutive and sprouted mossy fibers expressed high levels of the peptide after seizures, whereas NPY was not measurable there under physiological conditions (1,5). Electrophysiological and biochemical studies have demonstrated that NPY inhibits glutamatergic neurotransmission acting on the presynaptic Y, receptor subtypes

(6,7). On the other hand, excitatory effects of NPY acting on postsynaptic Y, receptors have been reported (8,9). It is important, therefore, to establish whether seizures induce plastic changes in NPY receptor subtypes and the functional consequences on synaptic transmission and neuronal excitability. In this respect, we recently found that NPY Y, and Y, receptors undergo opposite changes in the rat hippocampus after generalized limbic seizures. Specifically, Y, receptor density decreases on granule cell dendrites and Y, receptor density increases on mossy fibers terminals (for review, see 1). Recently, Y, receptors were detected in the rodent hippocampus (10-12), and they appear to mediate the inhibitory effects of NPY on seizures (13,14). We have now examined the changes in Y, receptors in the rat hippocampus after limbic seizures induced by kainic acid or kindling using a specific antibody raised against this receptor protein. We also assessed the effects of a preferential agonist at Y,/Y, receptors and selective

Address correspondence and reprint requests to Dr. A. Vezzani at Laboratory of Experimental Neurology, Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Via Eritrea 62, 20 157 Milano, Italy. E-mail [email protected]

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antagonists at Y, receptors on acute and chronic seizure susceptibility in rats.

MATERIALS AND METHODS Experimental animals Male Sprague-Dawley rats (250 to 280 g) were purchased from Charles River (Calco, Italy) and were housed at a constant temperature (23°C) and relative humidity (60%) with free access to food and water and a fixed 12-hour lighddark cycle. Procedures involving animals and their care were conducted in conformity with institutional guidelines, which are in compliance with national and international laws and policies. Placement of cannulae and electrodes for electroencephalographicrecordings Rats were surgically implanted with cannulae and electrodes under stereotactic guidance as described in detail elsewhere (15). Bipolar nichrome wire-insulated electrodes (60 pm) were implanted bilaterally into the dentate gyrus of the dorsal hippocampus (septa1 pole), and a guide cannula (22 gauge) was unilaterally positioned on top of the dura for the intracerebral infusion of drugs. The electrodes were connected to a multipin socket (March Electronics, New York, NY, U.S.A.) and, together with the injection cannula, were secured to the skull with acrylic dental cement. The experiments were carried out 4 to 7 days after surgery, when the animals did not show any sign of pain or discomfort. Electroencephalographic recordings The procedures for electroencephalographic (EEG) recordings have been described (15). Before treatments, a 15- to 30-minute baseline recording was made to establish an adequate control period. In rats receiving intrahippocampal kainic acid, EEG recordings (four-channel EEG poygraph, model BP8, Battaglia Rangoni, Bologna, Italy) were made continuously during drug injection and up to 180 minutes after kainic acid infusion. Analysis of the EEG recording The EEG recording for each rat was analyzed visually to detect any activity different from baseline. Seizures consisted of the simultaneous occurrence of at least two of the following alterations in all four leads: highfrequency or multispike complexes and high-voltage synchronized spike or wave activity (9). We determined the latency to the first seizure (onset), the total number of seizures, and the total time spent in seizures in the 3 hours of recording. Seizure susceptibility in rats treated with kainic acid Rats were made spontaneously epileptic by a subcutaneous injection of kainic acid, which caused generalEpilepsia, Vol. 41, Suppl. 6, 2000

ized limbic seizures for at least 3 hours. Thirty days later, the rats were tested for enhanced susceptibility to seizures using a normally subconvulsive dose of pentylenetetrazol (PTZ) (3). Behavior was observed for 30 minutes. Myoclonic seizures (all-body twich) were counted separately from tonic-clonic seizures (tonic-clonic forelimb or hindliinb extension with loss of posture).

Kindling The electrodes were implanted in the dorsal hippocampus as described previously in detail (16), and EEG recordings were made in freely moving animals as reported above (15). Constant-current stimuli were delivered unilaterally to the dorsal hippocampus twice daily at intervals of at least 6 hours (50 Hz, 2-millisecond monophasic rectangular wave pulses for 1 second, with current intensity ranging between 60 and 200 PA). Behavior was observed, and the duration of afterdischarge was measured in the stimulated hippocampus after each stimulation for every animal. Rats kindled at three consecutive stage 5 seizures (17) and the corresponding sham animals (rats implanted with electrodes but not electrically stimulated) were killed 2 and 30 days after the last electrical stimulation. These intervals were chosen based on previous studies showing changes in Y, and Y, receptor subtypes related to kindling-induced plasticity (for review, see I). Intracerebral injection of drugs All of the injections were made in unanesthetized rats using a needle (28 gauge) inserted into the guide cannula and aimed at the dorsal hippocampus or the lateral ventricle. Convulsant drugs Kainic acid (Sigma-Aldrich, Milano, Italy) was dissolved in buffered saline (pH 7.4) and injected unilaterally into the dorsal hippocampus (0.2 nmol/0.5 pL) or subcutaneously (10 mgkg). PTZ was dissolved in buffered saline and injected intraperitoneally at dosages ranging between 30 and 40 mgkg. Yl antagonists BIBO 3304 or BIBO 3457 (inactive enantiomer) was dissolved in 25% polyethylene glycol and injected intrahippocampally at the same site as kainic acid 15 minutes before the convulsant. BIBP 3226 or BIBP 3435 (inactive enantiomer) was dissolved as a BIBO compound and injected intracerebroventricularly (15 nmol/lO pL) 10 minutes before PTZ.

Y/Y5 agonist NPY 13-36 was dissolved in saline and injected intracerebroventricularly (17 nmol/lO pL) 10 minutes before PTZ.

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0 Ab + Peptide (200 pg/ml)

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low). Membranes were processed for immunoreactivity as described (18) using enhanced chemiluminescence. Densitometry was used to quantify the changes in Y, receptor levels in the immunoblots. Exposures with maximal signals below the photographic saturation point were used for densitometric analysis. Immunoreactivity of neurofilament M (145 kD; 1:lOOO; Chemicon, Temecula, CA, U.S.A.) was determined in each lane, and the value was used as an internal standard to correct for possible differences in the protein content of the samples.

Statistical analysis Data presented are means % SE (n = number of animals). The effects of treatments were analyzed by oneway analysis of variance followed by the Tukey test for unconfounded means or by the Fisher test.

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FIG. 1. Characterization of the Y, receptor polyclonal antibody. The bars depict the optical density of the 57-kD band divided by that of neurofilament M (NF), which was used as an internal standard in each sample. A Competition studies with the Y, fragment peptide. Ten micrograms of protein from whole homogenates of the va6ious forebrain areas was run in duplicate. B: Ab represents immunoblots incubated with the Y, receptor antibody (1:2500); Ab+peptide represents immunoblots incubated with the Y, receptor antibody preabsorbed with the Y, fragment peptide (EVKPEESSDAHEMR). Preabsorbtion was carried out at 4°C overnight during gentle shaking. FC, frontal cortex; HYP,hypothalamus; HIPP, hippocampus.

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0 Western blot The epileptic rats and their controls were killed by decapitation, and forebrain areas were dissected at 4°C and homogenized (200 pg/mL) in 20 mmol/L Tris-HC1 buffer, pH 7.4, containing 1 mmolL EDTA, 5 mmolL EGTA, 1 mmolL Na-vanadate, and a mixture of protease inhibitors. Samples of total proteins (10 yg) were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 10% acrylamide, and each sample was run in duplicate. Proteins were transferred to Hybond nitrocellulose membranes by electroblotting. For immunoblotting of the Y, receptor protein, we used a rabbit affinity-purified antibody (1 :2500;Genosys Biotechnologies, The Woodlands, TX, U.S.A.) directed against a synthetic peptide corresponding to amino acid residues 348 to 361 (EVKPEESSDAHEMR). This antibody recognizes a major protein band of an apparent molecular mass of approximately 57 kD.A minor band of 51 kD was also detected in some circumstances (see be-

CA 1

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NF NPY Y5 FIG. 2. Time course of changes in Y, receptors (57-kD band) after kainic acid-induced seizures. A: Micropunches of the various hippocampal subfields were obtained from three rats per experimental group. In each experimental group, tissue samples were pooled and 10 pg of protein per sample was run in duplicate in three different gels. The optical density of the 57-kD band was divided by that of neurofilament M, which was used as an internal standard in each sample. Data are means SE (n = 3). *p < 0.05, **p < 0.01 versus controls (rats injected with saline) by the Tukey test. B: Representative Western blots showing the decrease in Y, receptors in duplicate samples from CAI 1 week after kainic acid administration. NF, neurofilament M.

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RESULTS Antibody characterization The specificity of the anti-Y, receptor antibody was determined using immunoblots of brain tissue incubated with the antibody preadsorbed with the corresponding peptide. Competition studies using homogenates from rat frontal cortex, hypothalamus, and hippocampus showed that preadsorbtion with 200 1J.gof the peptide reduced by 62, 86, and 71%, respectively, the optical density corresponding to the 57-kD band (Fig. 1A). Five days of intracerebroventricular infusion of an antisense sequence specific to Y, messenger RNA (5'-AAGAGGACGTCCATTAGC-3' relative to the initiating ATG; molecular weight, 5519; 10 Kg/d per 24 p,L) using osmotic minipumps decreased by 40 f 8% (n = 4; p < 0.01 by the Tukey test) the optical density of the 57-kD band in the hippocampus compared with sense infusion (not shown).

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FIG. 4. Effect of hippocampal kindling on Y, receptors (57-kD band). Each sample (10 pg of protein from total hippocampal homogenate) represents an individual rat. Sham indicates rats implanted with electrodes but not stimulated. Rats and their controls were killed 2 and 30 days after the last of three consecutive stage 5 seizures. lpsi and contra indicate the stimulated and contralateral hippocampus, respectively. The optical density of the 57-kD band was divided by that of neurofilament M, which was used as an internal standard in each sample. Data are means SE (n = 5). **p < 0.01 versus sham animals by the Tukey test.

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FIG. 3. Changes in Y, receptors (51-kD band) 30 days after kainic acid-induced seizures. A: Micropunches of the various hippocampal subfields were obtained from three rats per experimental group. In each group, tissue samples were pooled and 10 pg of protein per sample was run in duplicate in three different gels. The optical density of the 51-kD band was divided by that of neurofilamentM (NF), which was used as an internal standard in each sample. Data are means SE (n = 3). ""p < 0.01 versus controls (rats injected with saline) by the Tukey test. B: Representative Western blots showing the changes in Y, receptors in duplicate samples from CA1 and DG 30 days after kainic acid administration.

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Effect of limbic seizures on Y, receptors After kainic acid treatment, micropunches of hippocampal CA1, CA3, and dentate gyrus (DG) subregions revealed a second band with an apparent molecular mass of 51 kD that was not apparent in the total homogenate fraction. This band probably represents a deglycosylated form of the receptor. Quantitative Western blot analysis showed that Y, receptor protein (57-kD band) did not change in any hippocampal subregion 2.5 hours after injection when seizure activity was observed in CA1 (2 f 0.7%, n = 5 animals), CA3 (-4.7 2 0.8%, n = 5), and DG (1.9 f 0.8%, n = 4) subregions compared with controls. In sharp contrast, however, the Y, receptor protein exhibited a decrease in the CA1 (-36.5 7%, n = 5, p < 0.05) and CA3 (-24 +- 8%, n = 5, p < 0.05) subregions and an

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increase in the DG (42 k lo%, n = 4, p < 0.01). Y, receptor protein (57 kD) was significantly reduced by 23 to 35% in the various hippocampal subfields 2 and 7 days after seizures, whereas no changes were observed after 30 days (Fig. 2). However, the 51-kD band was significantly reduced in CA3 and DG, whereas it was increased in CA1 30 days after seizures, suggesting longterm posttranslational changes in this protein (Fig. 3). Kindling Fig. 4 shows a transient but significant (200% on average) increase in Y, receptors (57 kD) 2 days after fully kindled seizures. This effect was restricted to the stimulated hippocampus and was not observed after 30 days. Pharmacological studies Fig. 5 shows the anticonvulsant effects of NPY 13-36, a preferential Y,/Y, agonist (lo), and BIBP 3226, a selective Y, receptor antagonist (19), on chronic seizure susceptibility in spontaneously epileptic rats. When seizures were triggered in the epileptic animals by an otherwise subconvulsive dose of PTZ, the onset to generalized tonic-clonic convulsions was delayed significantly by both compounds (p < 0.05). NPY 13-36 significantly decreased the number of rats that showed generalized convulsions (p < 0.05), whereas BIBP 3226 reduced the number of tonic-clonic seizures (p < 0.05). Intrahippocampal injection of 1.5 nmol of BIBO 3304, a nonpeptide selective antagonist at Y, receptors (20), reduced the number of EEG seizures [control, 26.0 k 4.4 (n = 15); BIBO 3304, 10.0 f 2.0 (n = 8); p < 0.051 and the total time spent in seizures (control, 29.0 1- 5.0 minutes; BIBO 3304, 10.0 k 3.0 minutes; p < 0.05) induced by intrahippocampal kainate compared with its inactive

J-3-

enantiomer (control). No changes were observed in the time to onset of EEG seizures (control, 13.0 f 3.0 minutes; BIBO 3304, 15.0 5 2.0 minutes).

DISCUSSION Although the role of NPY as an endogenous anticonvulsant has been substantiated by in vivo and in vitro studies, the involvement of specific receptor subtypes was not clearly defined until recently. Recent studies have demonstrated a role for the Y, (6) and Y, (13,14) receptors. In particular, Y, receptor (-/-) mice were more sensitive to kainic acid-induced seizures, did not exhibit spontaneous seizure-like activity, and showed no antiepileptic effects of exogenously applied NPY (14). Here we show significant changes in NPY Y, receptor protein in two models of limbic seizures in the rat, i.e., kindling and status epilepticus induced by kainic acid, using a selective antibody against a specific peptide sequence. The observed changes in Y, receptors were different depending on the experimental model of seizures. At 2.5 hours after a kainic acid injection, when seizure activity was observed there was no change in Y, receptor protein in the hippocampal subregions compared with Y, receptor protein, which exhibited a decrease in CAI and CA3 and an increase in DG. In accordance with recent studies measuring Y, receptor by autoradiographic binding (21), we found a significant decrease in the receptor protein up to 7 days after kainate-induced seizures. Dramatic modifications occurred in a minor protein band that likely represents a deglycosylated form of the receptor (22). In particular, this band increased in CAI but was reduced in 100

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FIG. 5. Effect of a YJY, agonist and Y, antagonists on chronic seizure susceptibility in rats. Bars in the middle and right panels represent the percentage of rats without seizures or showing myoclonic or tonic-clonic seizures after a subcutaneous injection of 30 to 40 mg/kg PTZ. These rats were pretreated 30 days before with 10 mglkg kainic acid subcutaneously to make them spontaneously epileptic (see “Materials and Methods” for details). NPY 13-36 (17 nmol) or BlBP 3226 (15 nmol) was injected in 10 pL of saline in the lateral ventricle 10 minutes before PTZ administration. In the left panel, vehicle includes the individual control rats used for NPY 13-36 (saline) or BlBP 3226 (inactive enantiomer) because they did not differ significantly. Data are means SE (n = 19 to 24). *p < 0.05 by the Tukey test (left panel) or the Fisher test (middle and right panels).

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CA3 and DG. Changes in glycosylation of the receptor may represent a long-term posttranslational process that affects its responsiveness to the endogeous ligand. In kindling, the 57-kD Y, receptor protein was selectively increased in the stimulated hippocampus. Because total hippocampal homogenates were used in these experiments, our data did not allow us to distinguish the hippocampal subregions in which these changes occurred. However, recent evidence has shown increased Y, messenger RNA levels 2 to 4 hours after a rapid kindling protocol confined to the dentate granule cell layer (23). The differences between kainic acid and kindling may be attributable to the dissimilar neuropathology associated with seizures in the two models. Thus, pyramidal and hilar cell loss that occurs after kainate-induced seizures (24) may contribute to the decrease in receptors. The late recovery to control values may represent upregulation of the spared receptors on surviving neurons. In the classic kindling protocol, only minor loss of hilar interneurons was described (16). Alternatively, the observed opposite receptor changes in the two models may be caused by the different mechanisms of seizure induction [i.e., activation of N-methyl-D-aspartate versus nonN-methyl-D-aspartate glutamate receptor subtypes (see 24,25)] or the cell populations primarily recruited in the epileptic activity (16). As for the functional consequences of these changes, mice deficient in Y, receptors appear to be more sensitive to kainic acid-induced seizures (14), suggesting an inhibitory role of Y, receptors in limbic seizures. In addition, pharmacological studies using Y,/Y, receptor agonists have shown protection from kainate-induced seizures in rats (13). This is in accordance with our findings showing that NPY 13-36 decreases chronic susceptibility to PTZ seizures in epileptic rats. Therefore, a loss of Y, receptors may contribute to hippocampal hyperexcitability after seizures. In contrast to Y, and Y, receptors, NPY Y, receptors mediate an excitatory component of NPY neurotransmission. Thus, in agreement with our previous studies (9), selective blockade of these postsynaptic receptors has anticonvulsant activity on kainate-induced EEG seizures and decreases chronic seizure susceptibility in rats. The present studies have demonstrated that Y, receptors are transiently modified by seizures compared with the long-lasting changes in Y, and Y, receptors (1,2628,29). Late posttranslational changes in Y, receptors may occur and play a role in the plastic alterations in neurotransinission in the epileptic tissue. Pharmacological evidence shows that NPY has a dual modulatory action in seizures depending on the receptor subtypes involved. The specific role of the NPY receptor subtypes in seizure susceptibility and in epileptogenenesis will be further clarified by the recent availability of Epilepsia, Vol. 41. Suppl. 6,2000

selective agonists and antagonists. The available data suggest that NPY receptors may provide a novel target(s) for the treatment of epilepsy.

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