Chronic hyperammonemia alters motor and neurochemical responses to activation of group i metabotropic glutamate receptors in the nucleus accumbens in rats in vivo

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Neurobiology of Disease 14 (2003) 380 –390

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Chronic hyperammonemia alters motor and neurochemical responses to activation of group I metabotropic glutamate receptors in the nucleus accumbens in rats in vivo Juan-Jose´ Canales,1 Amina Elayadi, Mohammed Errami, Marta Llansola, Omar Cauli, and Vicente Felipo* Laboratory of Neurobiology, Instituto de Investigaciones Citolo´gicas, Fundacio´n Valenciana de Investigaciones Biome´dicas, Valencia, Spain Received 16 July 2002; revised 14 May 2003; accepted 29 August 2003

Abstract Hyperammonemia leads to altered cerebral function and neurological alterations in patients with hepatic encephalopathy. We studied the effects of hyperammonemia in rats on the modulation by group I metabotropic glutamate receptors (mGluR) of motor and neurochemical functions in vivo. Locomotion induced by injection of the mGluR agonist DHPG into nucleus accumbens was increased in hyperammonemic rats. In control rats DHPG increased extracellular dopamine (ca. 400%) but not glutamate. In contrast, in hyperammonemic rats DHPG increased extracellular glutamate (ca. 600%), while DHPG-induced dopamine increase was reduced. Blocking mGluR1 receptor with CPCCOEt prevented all DHPG effects, indicating that this receptor mediates its locomotor and neurochemical effects. Hyperammonemic rats showed increased (32%) mGluR1␣, but not mGluR5 content in nucleus accumbens. These results show that modulation of locomotor and neurochemical functions by mGluRs in nucleus accumbens is strongly altered in hyperammonemia. These alterations may contribute to the neurological alterations in hyperammonemia and liver failure. © 2003 Elsevier Inc. All rights reserved. Keywords: Ammonia; Hepatic Encephalopathy; Motor behavior; Dopamine; Glutamate

Introduction Ammonia is a product of the degradation of proteins and other compounds that, at high concentrations, leads to functional disturbances of the central nervous system. To avoid these toxic effects, ammonia is usually detoxified in the liver by incorporation into urea. Liver disease (e.g., cirrhosis, hepatitis) impairs ammonia detoxification leading to hyperammonemia, which is considered the main contributing factor to the neurological alterations found in hepatic encephalopathy. Although the molecular mechanisms re-

sponsible for the neurological alterations found in hyperammonemia and liver disease remain elusive, alterations in glutamatergic neurotransmission have been suggested to play a major role in the pathogenesis of such abnormalities (Butterworth, 1992; Min˜ana et al., 1997; Corbalan et al., 2002; Monfort et al., 2002). Glutamate has two main types of receptors: metabotropic receptors (mGluRs)2 associated with G proteins which modulate different enzymes and ion channels (phospholipases C and D, adenylate cyclase, etc.), and ionotropic receptors. There are at least 8 different types of mGluRs, classified in

* Corresponding author. Laboratory of Neurobiology, Instituto de Investigaciones Citolo´gicas, Fundacio´n Valenciana de Investigaciones Biome´dicas, Amadeo de Saboya, 4, 46010 Valencia, Spain. Fax: ⫹34-96-3601453. E-mail address: [email protected] (V. Felipo). 1 Current address: Area of Psychobiology, University Jaume I, Campus C. de Borriol s/n, E-12080 Castello´n, Spain.

2 Abbreviations used: tACPD, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid; AIDA, 1-aminoindan-1,5-dicarboxylic acid; AMPA, ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; CPCCOEt, 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethylester, (S)-DHPG: (S)-3,5-dihydroxyphenylglycine; mGluRs, metabotropic glutamate receptors; MPEP, 2-methyl-6-(phenyle-thynyl)-pyridine, NMDA, N-methyl-Daspartate.

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groups I, II, and III according to their pharmacological profile. Although the effects of hyperammonemia and liver failure on ionotropic glutamate receptors have been studied by different groups, only very few studies have addressed the effects on mGluRs. Lombardi et al. (1994) reported that ammonium acetate (2– 4 mM) reduced the formation of inositol phosphates induced by tACPD, an agonist of mGluRs, in slices from cerebral cortex. However, ammonia potentiated the effects of mGluRs coupled to G proteins that inhibit adenylate cyclase, indicating that high ammonia concentrations (2– 4 mM) differentially affect the effects induced by activation of different types of mGluRs in cerebral cortex. Saez et al. (1999) showed that, in primary cultures of cerebellar neurons, activation of mGluRs with tACPD led to a significant increase in the phosphorylation of the microtubule-associated protein MAP-2 in control neurons. However, in neurons exposed to ammonia, tACPD induced a dephosphorylation of MAP-2, indicating that hyperammonemia alters the function of signal transduction pathways associated to mGluRs that modulate phosphorylation of MAP-2. These in vitro studies suggest that the processes modulated by mGluRs may be altered in brains of hyperammonemic rats in vivo. The aim of the present work was to assess, using a rat model of chronic moderate hyperammonemia (similar to that present in patients with liver cirrhosis), whether hyperammonemia affects the motor and neurochemical responses induced by activation of mGluRs in nucleus accumbens of rats in vivo. Neuroanatomical mapping studies indicate that the expression of group I mGluRs is enriched in striatum (Albin et al., 1992; Kerner et al., 1997). Activation of mGluRs in nucleus accumbens stimulates both neurotransmitter release (Bruton et al., 1999; Cartmell and Schoepp, 2000) and motor activity (Feeley Kearney et al., 1997; Swanson and Kalivas, 2000; Wang and Mao, 2000). We therefore assessed whether hyperammonemia affects responses to activation of mGluRs in brain in vivo by measuring the motor activity and the extracellular concentrations of dopamine and glutamate induced in control and hyperammonemic rats by injection of DHPG, an agonist of group I mGluRs, into the nucleus accumbens.

Materials and methods Animals and surgery Male Wistar (150 –180 g) rats were made hyperammonemic by feeding them an ammonium-containing diet as previously described (Azorin et al., 1989). Rats were fed the ammonium-containing diet for at least 3 weeks before the commencement of the experiments. Animals were anesthetized with 4% chroral hydrate (1 ml/100 g b/w ip) and placed in a Kopft stereotaxic apparatus. Two stainless-steel

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cannula guides (26 gauge) were bilaterally lowered into the brain and placed 2 mm above the nucleus accumbens at the following coordinates (AP 1.6, L 1.4, DV 4.6 from brain surface) (Paxinos and Watson, 1996). The cannulation assembly was fixed to the skull with miniature stainless-steel screws and dental resin. Wire stylets were used to seal the indwelling cannula guides and to ensure guides remained patent throughout the experiments. For the experiments involving acute microinjections of DHPG (60 nmol/␮l) into the nucleus accumbens coupled with microdialysis, cannulae were placed bilaterally at an angle of 18° from the vertical plane (AP 1.6, ML 3.7, DV 4.6 from brain surface). An adjacent microdialysis guide (CMA/12) was placed 1.5 mm above the nucleus accumbens (AP 1.5, ML 1.4, DV 5.1). Animals were allowed to recover from surgery for at least 48 h, with free access to water and to the standard or the ammonium-containing diet. Adequate measures were taken to minimize pain and discomfort of animals. The experiments have been approved by the Institute and meet the guideliness of the European communities Council Directive 86/609/EEC. Drugs (S)-Dihydroxyphenylglycine (DHPG) (Tocris Cokson, Bristol, UK) was dissolved in artificial cerebrospinal fluid (aCSF) (composition in mM: NaCl 145, KCl 3.0, CaCl2 2.26, buffered at pH 7.4, filtered through sterile 0.45-␮mpore Millipore filters), and injected at graded concentrations of 0, 5, 10, 30, and 60 nmol/␮l. For the microdialysis studies, the dose of 60 nmol/␮l was used. d-Amphetamine sulfate was dissolved in aCSF and injected at a concentration of 106.8 nmol/␮l. CPCCOEt (Tocris Cokson) was dissolved in 50% dimethyl sulfoxide (DMSO) and artificial cerebrospinal fluid. The control injection for the CPCCOEt experiment consisted of 50% dimethyl sulfoxide and artificial cerebrospinal fluid. This injection did not affect any of the parameters measured. Dosage for all drugs was selected based on previous studies (Wang and Mao, 2000; Canales and Iversen, 1998; Swanson and Kalivas 2000). Intracerebral microinjections For the intracerebral microinjections, stylets were removed from the guide cannulae and 33 gauge injectors with a 2-mm protrusion were lowered into the nucleus accumbens. Injectors were connected with polyethylene tubing to a precision micropump. Bilateral infusions were made at a rate of 0.5 ␮l/min and the volume injected was 0.5 ␮l per side in all cases. Injectors remained in place for 1 min to allow for diffusion of the drugs into the brain and to reduce backflow through the cannula track. Rats were then returned to the chambers for behavioral analysis. Vehicle injections were carried out in a series of animals and no behavioral effects were observed in either group of rats. Vehicle injections were not performed therefore in subsequent studies.

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In vivo microdialysis Rats were placed in plexiglass cylinders (BAS System, West Lafayette, IN). Following a brief habituation period of 15 min, a 3-mm-long microdialysis probe (CMA/12, Solna, Sweden, 500-␮m outer diameter) was carefully lowered into the nucleus accumbens and perfused with aCSF at a constant rate of 3 ␮l/min. Following a stabilization period of at least 2 h, 18 dialysates (20 min each) were collected in 0.01 M perchloric acid, quickly frozen on liquid nitrogen, and kept at ⫺80° until further processing. With the microdialysis probe in position, animals were gently held, bilateral microinjections of DHPG were made through the indwelling cannulae into the nucleus accumbens at the beginning of fractions 4 and 11, and animals were immediately returned to the cylinders. A further injection of d-amphetamine was made at the start of fraction 16. Determination of glutamate and dopamine The glutamate contents in the microdialysis samples were analyzed using a Waters reverse-phase HPLC system with fluorescence detection and precolumn o-phtalaldehyde derivatization (Waters Corp., Milford, MA). The HPLC system consisted of a solvent delivery system (Waters 515 pump) coupled to a fluorescence detector (Waters 474, excitation filter set at 340 nm and emission filter at 460 nm). Sample injections were performed using a Waters 717plus refrigerated Autosampler. Ten microliters of the collected dialysate samples was automatically derivatized by mixing with the working OPA solution and injected into the HPLC system after 2 min. The column used was a reverse-phase C18, 5-␮m particle size, 250 ⫻ 4.6 mm (ODS 2, Waters Spherisorb) and was maintained at constant temperature. The chromatogram was performed with a gradient program of two mobile phases at a constant flow rate of 1 ml/min. Solution A was 95/5 (vol/vol) mixture of 50 mM sodium acetate buffer (pH 5.67) and methanol, to which 12.5 ml of isopropyl alcohol per liter were added; solution B was a 70/30 (vol/vol) methanol/water mixture. These conditions allowed amino acids to be detected within 20 min. Amino acids were quantified using the Millennium 32 software (Waters). Standard solutions were prepared fresh weekly and stored in the refrigerator at 4°C. The content of dopamine in the microdialysis samples was analyzed by reverse-phase HPLC with electrochemical detection. Samples were injected in a Rheodyne injector (20-␮l loop) running first in a C18 precolumn (Waters Resolve) and then in a 3.9 ⫻ 150-mm C18, 5-␮m particle size precolumn (Waters). The mobile phase consisted of 0.05 mM acetate/citrate buffer, 0.15 mM sodium EDTA, 0.4 mM sodium octyl sulfonate, 15% methanol, and 1.1 mM n-dibutylamine. Twenty microliter samples were manually injected with a Rheodyne 7125 injector (20-␮l loop, Rheodyne Inc., Rohnert Park, CA). The flow rate was kept at 1 ml/min. Theses conditions allowed monoamines to be de-

tected within 10 min. Monoamines detection was performed by a coulometric detector (Coulochem II Model 5200A; ESA). A conditioning cell (ESA 5021) was set at ⫹50 mV and an analytical cell (ESA 5011) at ⫹340 mV (Cell 1) and at ⫺250 mV (Cell 2). Chromatograms were processed using the Millennium 32 Waters software. Internal standard DHBA was used for quantification and identification of the peaks. The detection limit in 20 ␮l samples was 80 fmol/20 ␮l for dopamine. Determination of ammonia Ammonia concentration was determined both in blood and in microdialysis samples in a parallel group of animals using an adaptation of the procedure described by Kun and Kearney (1974) as described previously (Hermenegildo et al., 2000). Blood (150 ␮l) was taken from the tail vein and added to a tube containing 150 ␮l of ice-cold trichloroacetic acid. Proteins were precipitated by centrifugation (14,000g, 15 min, 4°C), and the supernatant was neutralized and used to measure ammonia. To increase the sensibility and to measure large number of samples, we set up a procedure to determine ammonia simultaneously in multiple samples by using 96-well plates and measuring fluorescence (excitation, 360 nm; emission, 460 nm) in a fluorometer (Fluoroskan Ascent, Labsystems Oy, Helsinki, Finland). Samples contained, in a final volume of 100 ␮l, 25 or 50 ␮l of sample, 30 mM ␣-ketoglutarate and 0.5 mM NADH in potassium phosphate buffer, pH 8.0. After recording initial fluorescence, reactions were started by addition of 5 ␮g of glutamate dehydrogenase (Boehringer Mannheim, Germany) and followed in the Fluoroskan for at least 60 min. Standards containing up to 20 nmol of ammonia were included in each assay. Assays were carried out using Costar 96-well UV plates Cat. No. 3635 (Corning Costar Corporation, Cambridge, MA). Blood ammonia concentration was 106 ⫾ 25 ␮M in control rats and was significantly (P ⫽ 0.023) increased to 163 ⫾ 42 ␮M in hyperammonemic rats. The concentration of ammonia in the cerebellum extracellular fluid taken by microdialysis was 127 ⫾ 18 ␮M in control rats and was significantly (P ⫽ 0.001) increased to 210 ⫾ 38 ␮M in hyperammonemic rats. Statistical analysis of these data was carried out using the Student t test. Histology and immunohistochemistry On completion of the experiments, animals were perfused transcardially with 0.1 M NaKPO4 and the brains were removed for general histology and immunohistochemistry. Transverse 30-␮m sections through the nucleus accumbens were cut on a sliding microtome and stored in protecting solution (30% ethylene glycol/30% glycerol in 0.1 M NaKPO4) at ⫺20° until processing. Some sections were stained with cresyl violet for verification of injection sites in the nucleus accumbens (Fig. 1). Other sections were

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Fig. 1. Verification of the location of microdialysis probes and injection sites in the nucleus accumbens. Photomicrograph showing the placement of a microdialysis probe in the nucleus accumbens of a representative animal. Coronal sections were cut through the nucleus accumbens and routinely stained with cresyl violet for verification of injection sites and probe locations. IsC, Islands of Calleja; ac, anterior commissure.

processed for single-antigen immunohistochemistry with antisera against mGluR1␣ and mGluR5. The mGluR1␣ antibody was from Chemicon Int. (Temecula, CA) and the mGluR5 antibody from Calbiochem (San Diego, CA). Sections were washed in PBS with 0.1% Triton X-100, treated with 0.3% H2O2, incubated with 5% blocking serum, and subsequently exposed overnight to the primary antisera. Sections were then washed, treated with biotinylated secondary antisera and incubated with HRP-conjugated avidinbiotin complex (Vector Labs, Burlingame, CA). Reaction products were visualized by treating the sections with 3,3diaminobenzidine-H2O2. In control sections, the primary antibody was omitted in the overnight incubation. The immunostained sections were studied by light microscopy and sampled images through the nucleus accumbens were analyzed with image analysis software (Spot Advanced, Leica Qwin). Levels of specific stain were determined densitometrically on a 256-point black-to-white scale and expressed in arbitrary units. Images were captured using a digitizing software (Spot Advanced) and processed with an image analysis system (Leica Qwin). Conditions of light exposure and sensitivity were kept constant for capturing the images, and therefore densitometric differences were

likely to specifically reflect changes in protein expression. The boundaries of the Acb were first delineated against the background of surrounding tissue, images were turned to gray scale, and the density of stain was determined on a 256-point scale. To correct for background stain, from the absolute values calculated we subtracted a constant blank value computed as the optical density of stain in the surrounding corpus callosum, where the intensity of stain was virtually null. The values obtained were subjected to statistical analysis by ANOVA. Analysis of the content of mGluR1␣ and mGluR5 proteins in nucleus accumbens by immunoblotting Control or hyperammonemic rats were killed by decapitation and the nucleus accumbens was rapidly removed and homogenized in 5 vol of buffer containing Tris-HCl 50 mM (pH 7.5), NaCl 50 mM, EGTA 10 mM, EDTA 5 mM, sodium pyrophosphate 2 mM, p-nitrophenyl phosphate 4 mM, sodium orthovanadate 1 mM, phenylmethylsulfonyl fluoride 1 mM, leupeptin 20 ␮g/ml, and aprotinin 4 ␮g/ml. Samples were subjected to SDS-polyacrylamide gel electrophoresis (using 8% gels) and immunoblotting was carried

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out as previously described (Felipo et al., 1988) using antibodies against mGluR5 (Upstate Biotechnology-1 ␮g/ml, polyclonal) and mGluR1-alpha (Chemicon Int.; 0.5 ␮g/ml, polyclonal). This antibody does not recognize the other forms of mGluR1. After development using anti-rabbit IgG conjugated with alkaline phosphatase (Sigma) and alkaline phosphatase color development (Sigma, St. Louis, MO), the image was captured with a Hewlett Packard precision scan Scanjet 5300C, and the intensities of the bands were measured using the 1D Main program from AAB Software (Advanced American Biotechnology, 1166E). Similar experiments were carried out with five rats per group. Locomotor activity For the motor activity studies, animals received injections of DHPG or vehicle into the nucleus accumbens according to a fully randomized, repeated-measures design. At least 48 h elapsed between injections. Vehicle injections were carried out in a series of animals and no behavioral effects were observed in either group of rats. Vehicle injections were not performed therefore in subsequent studies. To measure locomotor activity, rats were placed in transparent plastic boxes containing a bed of sawdust. Animals were habituated to the experimental chambers for 10 min before the injections. Locomotor activity was measured by counting crossovers through a line dividing the boxes into two compartments. Following intracerebral injections, locomotor activity was monitored for 1 h and subtotal counts were accumulated at 10-min intervals. Statistical analysis. Data were analyzed by analysis of variance (ANOVA) followed by mean comparisons where appropriate. Post hoc mean comparisons were carried out according to the method of Newman-Keuls using the sample error from the ANOVA as denominator. Significance levels were set at ␣ ⫽ 0.05. Statistics were generated by the Statview 5.0 or Graph Pad Prism software. The statistic analysis used in each experiment is given under Results and in the corresponding figure legend.

Results Chronic hyperammonemia potentiates locomotion induced by activation of group I mGluRs in the nucleus accumbens Control and hyperammonemic rats did not differ in their baseline levels of locomotor activity measured following vehicle injections into the nucleus accumbens. DHPG injections into the nucleus accumbens produced dose-dependent locomotor stimulation in both hyperamonemic and control rats. However, the response was significantly in-

Fig. 2. Chronic hyperammonemia increases locomotor response induced by injection of DHPG into the nucleus accumbens. To measure locomotor activity, rats were placed in transparent plastic boxes containing a bed of sawdust. Animals were habituated to the experimental chambers for 10 min before the injections. Control or hyperammonemic rats received bilateral injections of different doses of DHPG (5, 15, 30, or 60 nmol/␮l; 0.5 ␮l per injection) or vehicle into the nucleus accumbens. Locomotor activity was measured by counting crossovers through a line dividing the boxes into two compartments. Following intracerebral injections, locomotor activity was monitored for 1 h (values given in a) and subtotal counts were accumulated at 10-min intervals (values given in b). Basal values have been subtracted in a. These were 4.7 ⫾ 0.8 for control and 5.3 ⫾ 0.8 for hyperammonemic rats. Values are the mean ⫾ standard error of 5 assays of 9 rats per group. Statistical analysis was performed using two-way ANOVA.

creased (F(20.320) ⫽ 96.855, P ⬍ 0.002) in hyperammonemic animals for all the concentrations of DHPG tested (Fig. 2a). In both sets of animals, and for all doses considered, DHPG stimulated locomotor activity immediately after the injections. The ambulatory response peaked during the first 10 min and showed a progressive decay within the 1-h session (Fig. 2b). Chronic hyperammonemia enhances the increase in extracellular glutamate induced by activation of group I mGluRs in the nucleus accumbens We measured extracellular levels of glutamate and dopamine, before and after acute injections of DHPG into the nucleus accumbens. We chose to make the acute injections

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⫽ 2.034, P ⬍ 0.016) than those observed in control rats, which reached 258 and 119% of baseline, respectively (Fig. 3). These data clearly indicate that chronic hyperammonemia alters modulation of extracellular glutamate concentration by group I mGluRs in the nucleus accumbens. Chronic hyperammonemia reduces the increase in extracellular dopamine induced by activation of group I mGluRs

Fig. 3. Chronic hyperammonemia potentiates the increase in extracellular glutamate induced by injection of DHPG into the nucleus accumbens. Control and hyperammonemic rats were operated as described in the methods section to insert a microdialysis guide and a cannula in the nucleus accumbens. After recovery from the operation (at least 48 h) a microdialysis probe was carefully lowered into the nucleus accumbens and perfused with aCSF at a constant rate of 3 ␮l/min. Following a stabilization period of at least 2 h, 18 dialysates (20 min each) were collected in 0.01 M perchloric acid, quickly frozen on liquid nitrogen, and kept at ⫺80° until further processing. With the microdialysis probe in position, animals were gently held, bilateral microinjections of DHPG (60 nmol/␮l; 0.5 ␮l per injection) were made through the indwelling cannulae into the nucleus accumbens at the beginning of fractions 4 and 11, and animals were immediately returned to the cylinders. A further injection of d-amphetamine was made at the start of fraction 16. The glutamate contents in the microdialysis samples were analyzed by HPLC as indicated in the methods section. Values are the mean ⫾ standard error of duplicate samples from 11 rats per group. Values that are significantly different from controls are indicated by asterisks (*P ⬍ 0.05, Newman-Keuls test).

through indwelling cannulae adjacent to a microdialysis probe, rather than perfusing the drugs through the probe itself, to match the conditions used for the behavioral experiments described above, avoiding the interference by the membrane of the dialysis probe with the transport of DHPG. Before performing the injections of DHPG, we collected three consecutive 20-min samples for determining the baseline concentrations of glutamate and dopamine. These and subsequent data are not corrected for recovery values. There were no significant differences (F(1,20) ⫽ 0.385, P ⬍ 0.542) between the basal extracellular concentrations of glutamate in nucleus accumbens from hyperammonemic and control rats (n ⫽ 11 per group). Mean baseline glutamate concentrations were 0.74 ⫾ 0.10 ␮M for control rats and 0.87 ⫾ 0.18 ␮M for hyperammonemic rats. Allowing for recovery factor, these baseline values are consistent with previous observations (Xi et al., 2002). There were, however, striking differences between control and hyperammonemic rats in extracellular glutamate following DHPG injection. In hyperammonemic rats DHPG induced peaks of increased glutamate concentration reaching 654 and 514% of baseline in fractions 5 and 12, respectively (DHPG was injected at the beginning of fractions 4 and 11). Such peaks of enhanced extracellular glutamate were significantly higher (F(14,280)

We measured extracellular dopamine levels in the nucleus accumbens in the same rats (n ⫽ 11 per group) we used for determining glutamate concentrations after group I mGluR activation with DHPG. As was the case for glutamate, chronic hyperammonemia did not affect basal concentrations of dopamine (F(1,18) ⫽ 0.001, P ⬍ 0.978). Mean baseline dopamine levels were 2.09 ⫾ 0.78 nM in control rats and 2.13 ⫾ 1.14 nM in hyperammonemic rats. These values are well within the range of previous studies (Parsons and Justice, 1992). In control rats, DHPG induced major increases in extracellular dopamine in the nucleus accumbens, which peaked to ca. 400% of baseline following each of the two injections of DHPG (Fig. 4). Dopamine levels raised steadily and remained elevated for approximately 1 h after DHPG injection. Chronic hyperammonemia significantly (F(14,280) ⫽ 1.769, P ⬍ 0.043) attenuated DHPGevoked increase in extracellular dopamine. In hyperammonemic rats, the first injection of DHPG enhanced extracellular dopamine to ca. 350% of baseline, but the increase was significantly less prolonged than that in controls. Contrary to what occurred in control rats, the second injection of DHPG completely failed to increase extracellular dopamine in hyperammonemic rats. These results sug-

Fig. 4. Chronic hyperammonemia reduces the increase in extracellular dopamine induced by injection of DHPG into the nucleus accumbens. The experiments were exactly as described in Fig. 3. Dopamine was measured (by HPLC as described in the methods section) in the same samples used to measure glutamate. Values are the mean ⫾ standard error of duplicate samples from 11 rats per group. Values that are significantly different from controls are indicated by asterisks. (*P ⬍ 0.05, Newman-Keuls test).

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Fig. 5. Chronic hyperammonemia does not affect the increase in extracellular dopamine and glutamate induced by injection of d-amphetamine into the nucleus accumbens. The experiments were exactly as described in Fig. 3. d-Amphetamine was injected at the start of fraction 16 and dopamine and glutamate were measured as above in fractions 16 –18. Values are the mean ⫾ standard errors of duplicate samples from 11 rats per group. Values that are significantly different from controls are indicated by asterisks. The levels of dopamine are very high already in fraction 16 due to the rapid effect of the d-amphetamine injections on dopamine release. Percentage of increase was calculated relative to baseline values (fractions 1–3, see Figs. 3 and 4).

gest altered control of dopamine release in the nucleus accumbens by group I mGluRs in hyperammonemia. To assess whether the altered responses of extracellular dopamine and glutamate in hyperammonemic rats are specifically linked to group I mGluRs activation, we determined extracellular levels of dopamine and glutamate after d-amphetamine injections into the nucleus accumbens. Measurements were carried out in samples 16 –18 in the same rats used for determining glutamate and dopamine concentrations following DHPG injections. The results showed that d-amphetamine induced vast increases in dopamine release in both hyperammonemic and control rats (Fig. 5), and that neither the magnitude nor the kinetics of such changes differed between the two groups of rats, (repeated measures ANOVA, F(2,40) ⫽ 0.235, P ⬍ 0.792). Moreover, d-amphetamine-induced changes in extracellular glutamate were very mild and were not different in both groups of animals (F(2,40) ⫽ 0.953, P ⬍ 0.394). These results indicate, first, that dopamine bioavailability and damphetamine-induced dopamine release are not impaired in hyperammonemia and, second, that extracellular levels of glutamate are not differentially affected by injections of d-amphetamine into the nucleus accumbens neither in control nor in hyperammonemic rats. Thus the striking differences in extracellular glutamate and dopamine concentrations in hyperammonemic rats after DHPG treatment likely represent alterations specifically linked to group I mGluRs. Chronic hyperammonemia increases the content of mGluR1␣, but not mGluR5 in the nucleus accumbens We used immunohistochemistry to visualize the content mGuR1␣ and mGluR5 in the nucleus accumbens. In normal

rats, specific immunostaining for both mGluR1␣ and mGluR5 was prominent in the limbic forebrain, especially in the nucleus accumbens, olfactory tubercle, and extended amygdala. At the level of the nucleus accumbens, immunolabeling was present mostly in neuropil, allowing densitometric analysis of overall levels of stain and estimations of receptor content. We selected matching sections through the rostral nucleus accumbens from control and hyperammonemic rats. The results showed that mGluR1␣ contents in the nucleus accumbens were significantly increased (ca. 40%, P ⬍ 0.013, one-way ANOVA) in hyperammonemic rats compared to controls (Fig. 6). By contrast, the contents of mGluR5 did not differ significantly between hyperammonemic and control rats (P ⬍ 0.849). To confirm in a more quantitative way the effects of hyperammonemia on the content of mGluR1␣ and mGluR5, we analyzed by immunoblotting the content of these receptors in the nucleus accumbens. The content of mGluR1␣ was significantly increased by 32 ⫾ 8% in hyperammonemic rats (F(1,6) ⫽ 7.564, P ⬍ 0.021), while no significant change was found in the content of mGluR5 (F(1,6) ⫽ 1.024, P ⫽ 0.351). Representative immunoblottings are shown in Fig. 7. Both the immunohistochemistry and immunoblotting experiments show that chronic hyperammonemia increases mGluR1␣ receptor content in the nucleus accumbens. This may provide a potential explanation for the altered neurochemical and behavioral responses to DHPG injections in chronic hyperammonemia. Blocking mGluR1 receptors completely prevents the increase in locomotion and in extracellular glutamate and dopamine induced by DHPG in both control and hyperammonemic rats To further confirm that the altered responses to DHPG injections in chronic hyperammonemia are due to the increase in mGluR1␣ receptor content we assessed whether these altered responses are prevented by blocking mGluR1 receptors. As shown in Fig. 8, the increase in locomotion induced by DHPG is completely prevented by previous injection of CPCCOEt, a selective antagonist of mGluR1 receptors, in both control and hyperammonemic rats. Moreover, CPCCOEt also prevents the increase in glutamate induced by DHPG in hyperammonemic rats (Fig. 9) and the increase in dopamine induced by DHPG in control rats (Fig. 10). These results confirm that both neurochemical and locomotor responses to DHPG are mediated by activation of mGluR1 both in control rats and in hyperammonemic rats.

Discussion The present study was designed to assess whether chronic hyperammonemia leads to altered responses to activation of group I mGluRs in the nucleus accumbens of rats

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Fig. 6. Chronic hyperammonemia increases the content of mGluR1␣ but not of mGluR5 in nucleus accumbens. Brains were prepared for immunohistochemistry as described in the methods section. Transverse 30 ␮m sections through the nucleus accumbens were cut and incubated with antisera against mGluR1␣ or mGluR5. Sections were washed, treated with biotinylated secondary antisera, and incubated with HRP-conjugated avidin-biotin complex. Reaction products were visualized by treating the sections with 3,3-diaminobenzidine-H2O2. The immunostained sections were studied by light microscopy and sampled images through the nucleus accumbens were analyzed with image analysis software. All images were captured and analyzed under identical conditions of light exposure. Some representative images are shown. Levels of specific stain were determined densitometrically on a 256-point black-to-white scale and expressed in arbitrary units in E. Values are the mean ⫾ standard error of 4 rats per group. Values that are significantly different from controls are indicated by asterisks. (*P ⬍ 0.05, ANOVA). AC, anterior commissure.

in vivo. The results presented include several novel findings. First, it is shown that chronic hyperammonemia induced supersensitivity to the motor stimulant effects evoked by group I mGluRs activation in the nucleus accumbens. Second, it is shown that changes in extracellular concentration of neurotransmitters induced by activation of group I mGluRs in the nucleus accumbens are strongly altered in chronic hyperammonemia, resulting in enhanced glutamate and reduced dopamine concentrations in the extracellular fluid, when compared with control animals. Third, it is shown, by using immunohistochemical and immunoblotting techniques, that the content of mGluR1␣ in the nucleus accumbens is significantly increased in hyperammonemic rats, while the content of mGluR5 is not affected. Fourth, it is shown that the effects of DHPG are mediated by activation of mGluR1 and are completely prevented, in both

control and hyperammonemic rats by blocking this receptor. These results therefore unveil major alterations induced by chronic hyperammonemia in both neurotransmission and motor function modulated by group I mGluRs in the nucleus accumbens. The results reported show a differential sensitivity of hyperammonemic rats to the locomotor stimulant effects of DHPG injections into the nucleus accumbens. The nucleus accumbens has been widely implicated in the motor stimulant effects of both dopaminergic (Jones et al., 1981; Canales and Iversen, 1998) and glutamatergic agents (Donzanti and Uretsky, 1983; Shreve and Uretsky, 1988; Wu et al., 1993). Recent evidence specifically indicates that motor activity can be modulated by group I mGluRs in the nucleus accumbens. Microinjections of DHPG into this area increased locomotor activity dose dependently (Swanson and

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Fig. 7. Effect of chronic hyperammonemia on the content of mGluR1␣ and mGluR5 in nucleus accumbens. Control rats (C) and hyperammonemic (A) rats were sacrificed and the nucleus accumbens was immediately removed and homogenized and the homogenates were subjected to immunoblotting as indicated in the methods section. Figures indicate the amount of protein applied to the gel: 25 and 50 ␮g for both mGluR1␣ or mGluR5. Similar results were obtained in experiments with 5 rats per group. The content of mGluR1a was significantly (P ⫽ 0.021) increased by 32 ⫾ 8% in hyperammonemic rats while no difference was found in the content of mGluR5. Statistical analysis of these data was carried out using the Student t test.

Kalivas, 2000; Wang and Mao, 2000). The results reported here are in agreement with these observations and, moreover, demonstrate that hyperammonemia induces an enhanced locomotor response following activation of group I mGluRs in the nucleus accumbens. The experiments reported do not allow us to distinguish the contribution of different compartments within the nucleus accumbens (core, shell, rostral pole) to the differential responses in hyperammonemic rats. Group I mGluRs have been shown to regulate transmitter release in the nucleus accumbens. As an initial approach to delineate the mechanisms that could account for the increased locomotor response of hyperammonemic animals to group I mGlu receptor activation, we assessed whether hyperammonemia alters neurotransmitter release in response to activation of these receptors.

Fig. 8. Blocking mGluR1 receptors with CPCCOEt prevents the increase in locomotion induced by injection of DHPG into the nucleus accumbens. Control and hyperammonemic rats were injected bilaterally with DHPG alone, with CPCCOEt alone, or with DHPG plus CPCCOEt. CPCCOEt alone did not induce any locomotor effect. CPCCOEt (10 nmol/␮l) was injected 10 min before DHPG (60 nmol/␮l) injection. Right after DHPG injection motor activity was measured for 1 h by counting crossovers through a line dividing the boxes into two compartments as in Fig. 2. Rats were previously habituated for 1 h to the experimental box in which locomotor activity was recorded. Values are the mean ⫾ standard error of 2 assays of 8 rats per group. Statistical analysis was performed using two-way ANOVA. Values that are significantly different from controls (P ⬍ 0.05) are indicated by asterisks.

Fig. 9. Blocking mGluR1 receptors with CPCCOEt prevents the increase in extracellular glutamate induced by injection of DHPG into the nucleus accumbens. Experiments were carried out essentially as in Fig. 3. A microdialysis probe was carefully lowered into the nucleus accumbens and perfused with aCSF at a constant rate of 3 ␮l/min. Following a stabilization period of at least 2 h, 10 dialysates (20 min each) were collected in 0.01 M perchloric acid, quickly frozen on liquid nitrogen, and kept at ⫺80° until further processing. With the microdialysis probe in position, animals were gently held, and bilaterally injected with CPCCOEt (10 nmol/␮l; 0.5 ␮l per injection) followed, 10 min later, by DHPG (60 nmol/␮l; 0.5 ␮l per injection) through the indwelling cannulae into the nucleus accumbens at the beginning of fraction 4. Values are the mean ⫾ standard error of duplicate samples from 8 hyperammonemic rats and 7 control rats.

The results of these experiments revealed striking differences between control and hyperammonemic rats in the regulation of extracellular glutamate and dopamine, two transmitters critically involved in the control of motor behavior by the basal ganglia (Carlsson, 1993; Hauber, 1998). Extracellular glutamate in the nucleus accumbens was strongly increased in hyperammonemic rats by DHPG, but almost no effect was observed in control rats. Microinjections of d-amphetamine did not elevate glutamate levels in the nucleus accumbens in control rats, confirming prior observations (Kalivas and Duffy, 1997), nor in

Fig. 10. Blocking mGluR1 receptors with CPCCOEt prevents the increase in extracellular dopamine induced by injection of DHPG into the nucleus accumbens. Experiments were carried out as in Fig. 9. Dopamine was measured (by HPLC as described in the methods section) in the same samples used to measure glutamate. Values are the mean ⫾ standard error of duplicate samples from 8 hyperammonemic rats and 7 control rats.

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hyperammonemic rats, indicating that the effects of DHPG on extracellular glutamate were pharmacologically specific. The results reported show that in control animals acute DHPG injections produced large, sustained, and reproducible enhancement of local dopamine release, which is in agreement with a previous report in which DHPG was applied in the nucleus accumbens by means of reverse dialysis (Bruton et al., 1999; but see Hu et al., 1999). The results reported show that DHPG-induced elevations in extracellular dopamine are significantly reduced in hyperammonemic rats. Moreover, d-amphetamine-induced dopamine release was not affected by hyperammonemia. These findings suggest that hyperammonemia does not impair synthesis and vesicular release and/or transport of dopamine per se, but specifically alters the modulation of dopamine release by group I mGluRs. It should be noted that glutamate exerts biphasic effects upon dopamine release in corticostriatal preparations: low concentrations of glutamate stimulate dopamine release, while high concentrations inhibit it (Cheramy et al., 1986; Leviel et al., 1990; Whitton, 1997). Thus, the large increase in extracellular glutamate observed in hyperammonemic rats after DHPG treatment may be responsible for the reduced increase in extracellular dopamine in hyperammonemic rats in response to DHPG and especially for the absence of increase in dopamine following the second injection of DHPG. In addition to this interaction between glutamatergic and dopaminergic neurotransmission, a direct effect of hyperammonemia on the transduction mechanisms associated to group I mGluRs leading to the release and/or transport of dopamine cannot be ruled out. The results reported raise the possibility that in normal rats, but not in hyperammonemic rats, locomotor activity induced by activation of group I mGluRs in the nucleus accumbens is mediated by enhanced dopamine release. This is in agreement with recent data indicating that motor activity evoked by mGluRs activation in the nucleus accumbens with tACPD is blocked by 6-hydroxydopamine lesions of this nucleus (Meeker et al., 1998) or by dopamine receptor antagonists (Attarian and Amalric, 1997; Kim and Vezina, 1997). In contrast, the results reported here suggest that in hyperammonemic rats the enhanced motor stimulation induced by group I mGluRs activation would be mediated by increased glutamatergic transmission, an adaptation that may be linked to the elevated content of mGluR1␣ in the nucleus accumbens. To assess this possibility we tested whether blocking mGluR1 receptors with CPCCOEt prevents the altered responses to DHPH in hyperammonemic rats. It is shown that CPCCOEt prevents the increase in locomotion induced by DHPG in both control and hyperammonemic rats. Moreover, it also prevents the increase in extracellular glutamate induced by DHPG in hyperammonemic rats and the increase in extracellular dopamine in control rats (Figs. 8 –10). These results confirm that the locomotor and neurochemical effects of DHPG in nucleus

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accumbens are mediated by mGluR1 receptors in both control and hyperammonemic rats and support the idea that the altered responses in hyperammonemia are also mediated by this type of receptors. These results are in agreement with a previous report showing that, in normal rats, locomotor activity induced by DHPG injections into the nucleus accumbens is blocked by the selective mGluR1 antagonist CPCCOEt (Swanson and Kalivas, 2000). Moreover, increased levels of mGluR1 have been detected in the nucleus accumbens of animals showing increased locomotor activation after chronic exposure to amphetamine (Mao and Wang, 2001) or cocaine (Churchill et al., 1999). Taken together, these studies highlight the importance of potentiated glutamatergic neurotransmission, preferentially through actions on mGluR1, in the development of sensitized motor responses. In summary, the results reported show significant alterations in the behavioral and neurochemical responses to activation of group I mGluRs in the nucleus accumbens of hyperammonemic rats. These rats also show increased content of mGluR1␣ in the nucleus accumbens, and this receptor mediates the altered responses. These alterations may contribute significantly to some of the cognitive and motor alterations characteristic of hyperammonemia and hepatic encephalopathy.

Acknowledgments This work was supported by grants (PM99-0018, SAF2002-00851, and SAF97-0001) of the Ministerio de Educacio´ n y Ciencia and of Plan Nacional de I ⫹ D and from Ministerio de Sanidad (Red G03-155) of Spain; by a grant (2P/00) from the Agencia Espan˜ ola de Cooperacio´ n Internacional. Ministerio de Asuntos Exteriores. Programa de Cooperacio´ n entre Espan˜ a y Marruecos and by grant from Escuela Valenciana de Estudios para la Salud (EVES) Consellerı´a de Sanidad. Generalitat Valenciana (BM-028/ 2002).

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