Soluble amyloid beta1-42 reduces dopamine levels in rat prefrontal cortex: Relationship to nitric oxide

Neuroscience 147 (2007) 652– 663

SOLUBLE AMYLOID BETA1-42 REDUCES DOPAMINE LEVELS IN RAT PREFRONTAL CORTEX: RELATIONSHIP TO NITRIC OXIDE amyloid beta (A␤) peptide, which plays an important role in the development of AD. However, the mechanism by which A␤ causes neuronal injury and cognitive impairment is not yet clearly understood. Different lines of evidence now suggest that A␤ plays a central role in the pathogenesis of neuronal dysfunction in AD (for reviews, see Hardy and Allsop, 1991; Selkoe, 1991; Hardy and Selkoe, 2002). However, despite a substantial number of reports, the amyloid cascade hypothesis remains controversial. This may be explained by recent evidence suggesting that A␤ neurotoxicity can be mediated by multiple different assembly forms of the peptide and that cognitive deficits may be attributable, at least in part, to soluble oligomers that can initiate downstream changes (Walsh and Selkoe, 2004; Heinitz et al., 2006; Tamagno et al., 2006). While intriguing, an important unanswered question about the potential correlation of amyloid pathology, neurotransmission and resultant dementia is whether the deposition of aggregated A␤ into plaques is required to induce neurotoxic or functional changes. From a neurochemical point of view, although a “cholinergic deficit hypothesis” has been suggested in AD (Perry, 1986), noncholinergic neurochemical abnormalities that may contribute to the behavioral and cognitive disorders associated with the pathology have been identified (Zubenko et al., 1990; Camacho et al., 1996). Among these, dopaminergic transmission seems to be of particular interest. In this regard, interactions between cholinergic and dopaminergic systems seem to play a role in the modulation of memory processes. In fact, a specific cholinergic control of the dopaminergic transmission within brain areas involved in cognitive functions (hippocampus and cerebral cortex) has been shown (Memo et al., 1988). On the other hand, DA modulates cholinergic activity via two classes of DA receptors, the D1-like receptors (D1 and D5 receptor subtypes) and the D2-class (D2, D3 and D4 receptor subtypes). Cholinergic neurons projecting to the cortex are regulated in an excitatory manner by D1 receptors (Day and Fibiger, 1993). Moreover, it has been shown that the majority of cholinergic cells in the cerebral cortex express the D5 receptors (Berlanga et al., 2005) and it could be hypothesized that D5 receptor subtype may serve as an important neuroanatomical substrate involved in mediating dopaminergic influences on cholinergic neurotransmission throughout the brain. In contrast, D2-class receptors attenuate acetylcholine (ACh) release (Bertorelli and Consolo, 1990). It is worth noting that non-cognitive behavioral and neuropsychiatric symptoms often accompany AD and other forms of dementia (Assal and Cummings, 2002). It

L. TRABACE,a* K. M. KENDRICK,b S. CASTRIGNANÒ,a M. COLAIANNA,a A. DE GIORGI,a S. SCHIAVONE,a C. LANNI,c V. CUOMOd AND S. GOVONIc a

Department of Biomedical Sciences, Faculty of Medicine c/o OO.RR., University of Foggia, Viale L. Pinto, 71100 Foggia, Italy

b

Department of Neurobiology, The Babraham Institute, Babraham, Cambridge, UK CB22 3AT

c

Department of Experimental and Applied Pharmacology, University of Pavia, Viale Taramelli 14, 27100 Pavia, Italy

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Department of Human Physiology and Pharmacology, Vittorio Erspamer, University of Rome La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy

Abstract—Several studies suggest a pivotal role of amyloid beta (A␤)1-42 and nitric oxide (NO) in the pathogenesis of Alzheimer’s disease. NO also possess central neuromodulatory properties. To study the soluble A␤1-42 effects on dopamine concentrations in rat prefrontal cortex, microdialysis technique was used. We showed that i.c.v. injection or retrodialysis A␤1-42 administration reduced basal and Kⴙ-stimulated dopamine levels, measured 2 and 48 h after peptide administration. Immunofluorescent experiments revealed that after 48 h from i.c.v. injection A␤1-42 was no longer detectable in the ventricular space. We then evaluated the role of NO on A␤1-42-induced reduction in dopamine concentrations. Subchronic L-arginine administration decreased basal dopamine levels, measured either 2 h after i.c.v. A␤1-42 or on day 2 post-injection, whereas subchronic 7-nitroindazole administration increased basal dopamine concentrations, measured 2 h after i.c.v. A␤1-42 injection, and decreased them when measured on day 2 post-A␤1-42-injection. No dopaminergic response activity was observed after Kⴙ stimulation in all groups. These results suggest that the dopaminergic system seems to be acutely vulnerable to soluble A␤1-42 effects. Finally, the opposite role of NO occurring at different phases might be regarded as a possible link between A␤1-42-induced effects and dopaminergic dysfunction. © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: A␤ 1-42 toxicity, nitric oxide, dopamine, prefrontal cortex, rat, microdialysis.

Alzheimer’s disease (AD) is the most common cause of progressive cognitive impairment in the elderly. The characteristic neuropathology of AD is an accumulation of senile plaques and neurofibrillary tangles in vulnerable brain regions. The senile plaques are primarily composed of *Corresponding author. Tel: ⫹39-0881-588056; fax: ⫹39-0881-712366. E-mail address: [email protected] (L. Trabace). Abbreviations: aCSF, artificial cerebrospinal fluid; AD, Alzheimer’s disease; ANOVA, analysis of variance; A␤, amyloid beta; BSA, bovine serum albumin; DA, dopamine; eNOS, endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; PFC, prefrontal cortex; 7-NI, 7-nitroindazole.

0306-4522/07$30.00⫹0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.04.056

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has been reported that 60% of patients with AD experience neuropsychiatric disturbances ranging from depression and anxiety to hallucinations and delusions. The non-cognitive aspects of AD usually arise, among a number of other factors, from alterations in dopaminergic function (Assal and Cummings, 2002). There is now also suggestive evidence that neurodegenerative conditions such as AD involve nitric oxide (NO) in their pathogenesis (Duncan and Heales, 2005; Guix et al., 2005). In this regard, it has been shown that positive neurons for nitric oxide synthase (NOS) are distributed all over the AD brain (Hyman et al., 1992). Furthermore, increased expression of neuronal nitric oxide synthase (nNOS) in those neurons with neurofibrillary tangles has been found in the entorhinal cortex and hippocampus of AD patients (Thorns et al., 1998). It has been reported that there are increased numbers of NADPH-diaphorase- (a histochemical marker for NOS-containing cells) positive neurons within the substantia innominata in the AD brain (Benzing and Mufson, 1995). Moreover, the density of nNOS mRNA-labeled neurons was significantly reduced in the hippocampus and in the frontal cortex of AD brains (Norris et al., 1996). The free radical gas NO is a multifunctional messenger molecule which exerts a wide spectrum of central and peripheral effects. When produced in excessive amounts, NO switches from a physiological neuromodulator to a neurotoxic effector. Indeed, NO has been reported to play a double-edged role in either neurotoxicity or neuroprotection (Gross and Wolin, 1995; Wink and Mitchell, 1998; Chiueh, 1999; Chiueh and Rauhala, 1999). Although NO is considered to have a role in both neurotoxicity and neurodegeneration, increasing recent evidence suggests that it may possess neuroprotective properties for protection against oxidative stress, apoptosis, and related neurodegenerative disorders (Rauhala et al., 2005). In particular, endogenous NO may have a normal physiological function, which may lead to prevention of caspase-dependent apoptosis and to mediation of preconditioning-induced adaptive neuroprotection in the brain (Andoh et al., 2000, 2002). It has been shown that endogenous NO is important in increasing neurogenesis (Zhu et al., 2003). Interestingly, several data are consistent with a role for NO as a neuroprotective agent in the brain acting to desensitize NMDA receptors (Kendrick et al., 1996). Taken together these results point to the hypothesis that homeostasis of NO seems to be important in protecting neurons and other brain cells against neurodegeneration. NO also possess potent neuromodulatory actions in several brain regions (Guevara-Guzman et al., 1994; Buchholzer and Klein, 2002; Feldman and Weidenfeld, 2004; Trabace et al., 2004). In particular, we have reported that when NO donors were retrodialyzed in the rat striatum, extracellular dopamine (DA) concentrations were significantly reduced (Kendrick et al., 1997; Trabace and Kendrick, 2000). This background prompted us to explore the in vivo neuromodulatory role of NO on dopaminergic transmission in an animal model of A␤ exposure. As AD is characterized

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by a highly regional-specific neuronal loss, particularly prominent in the basal forebrain and its target areas, cortex and hippocampus, we have focused our attention on the prefrontal cortex (PFC). In particular, we have investigated the acute effect of soluble A␤1-42 on basal and high K⫹stimulated DA release after retrodialysis administration or i.c.v. injection. Extracellular concentrations of DA were measured 2 h or 2 days after treatment. Then, A␤ immunoreactivity was evaluated to study the efficiency of administration and diffusion of soluble A␤1-42 peptide to brain parenchyma following retrodialysis perfusion or i.c.v. injection. Finally, we investigated whether a subchronic systemic administration of 7-nitroindazole (7-NI), a selective inhibitor of nNOS, or L-Arg, the exclusive physiological substrates of nNOS, would induce changes in basal or stimulated extracellular DA concentrations in rat PFC, measured 2 h or 2 days after i.c.v. A␤1-42 injection.

EXPERIMENTAL PROCEDURES Animals Microdialysis experiments were conducted in conscious, freely moving young, male Wistar rats (Harlan, S. Pietro al Natisone, Udine, Italy) weighing 250 –300 g. They were housed at constant room temperature (22⫾1 °C) and relative humidity (55⫾5%) under a 12-h light/dark cycle (lights on from 7:00 AM to 7:00 PM). Food and water were freely available. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (D. L. No. 116, G. U., Suppl. 40, 18 Febbraio 1992, Circolare No. 8, G. U., 14 Luglio 1994) and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, December 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996). All efforts were made to minimize the number of animals used and their suffering.

Surgery Rats were anesthetized with 3.6 ml/kg Equithesin intraperitoneally (composition: 1.2 g sodium pentobarbital; 5.3 g chloral hydrate; 2.7 g MgSO4; 49.5 ml propylene glycol; 12.5 ml ethanol and 58 ml distilled water) and secured in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). The skin was shaved, disinfected and cut with a sterile scalpel to expose the skull.

Cannula implantation A hole (about 0.6 mm diameter) was drilled to allow the implantation of the cannula into the brain parenchyma. Unilateral 23gauge stainless steel guide cannula (Cooper’s Needles, Birmingham, UK) were implanted during the same surgery as the microdialysis probe only for the 2 h experiments and secured to the skull with two stainless steel screws and dental cement. The injections were made using the following coordinates relative to Bregma: AP⫽⫺0.5, L⫽⫹1.2, H⫽⫺3.2 with the incisor bar set at ⫺3.3 mm, according to a stereotaxic atlas (Paxinos and Watson, 1982). Thirty-gauge stainless steel stylets flush with the end of the guide cannula were inserted into the guide to prevent clogging.

Dialysis procedure A dialysis fiber was positioned in the PFC. Stereotaxic coordinates were as follows: AP⫽⫹10.7, DV⫽⫹8.0 from the interaural line according to a stereotaxic atlas (Paxinos and Watson, 1982). A

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Fig. 1. Experimental schedule. M, microdialysis.

short piece of dialysis fiber made of copolymer of acrylonitrile sodium methallyl sulfonate (AN69 Hospal S.p.A; 20,000 Da cutoff) was covered with epoxy glue to confine dialysis to the area of interest (8-mm glue-free zone). The skull of the rat was exposed, and two holes were made on the lateral surface at the level of the PFC. A dialysis fiber, held straight by a tungsten wire inside, was inserted transversely into the brain so that the glue-free zone was exactly located in the target area. The tungsten wire was withdrawn, and stainless steel cannulas (22-gauge diameter, 15 mm long) were glued to the ends of the fibers. These ends were bent up and fixed vertically to the skull using dental cement and modified Eppendorf tips. Finally, the skin was sutured, and the rats were allowed to recover from anesthesia for at least 15 h before the neurotransmitter release study. On the day of the experiment, the fibers were perfused with an artificial cerebrospinal fluid (aCSF) containing: 145 mM NaCl, 3 mM KCl, 1.26 mM CaCl2, 1 mM MgCl2 and 1.4 mM Na2HPO4 in distilled water (1 l); the solution was buffered at pH 7.3 with a 0.6 mM NaH2PO4 and filtered (0.45 ␮m). The fibers were perfused at a constant flow rate of 2 ␮l/min using a CMA/100 microinjection pump (CMA Microdialysis, Stockholm, Sweden). In all experiments, the microdialysis membrane was allowed to stabilize for 2 h at the flow rate of 2 ␮l/min without collecting samples. At the end of the stabilization period, samples were collected at 20-min intervals at a flow rate of 2 ␮l/min. Three baseline samples were collected to evaluate baseline release of DA. After this time, a challenge with high K⫹ aCSF (100 mM for 20 min) was delivered, and further samples were collected. The position of the microdialysis probe was verified by histological procedures at the end of each experiment. Only rats in which probe tracks were exactly located in the target area were considered in the Results.

A␤1-42 administration The A␤1-42 peptide was obtained from Tocris (Bristol, UK). All solutions were freshly prepared. Peptide was dissolved in sterile double-distilled water (vehicle) at a concentration of 100 ␮g/ml. On the testing day, the rat was held (duration ⬍1 min) to remove the stylet from the guide cannula and insert the injection needle (30-gauge stainless steel tubing). The rat was gently returned to the home cage. The control rats received 5 ␮l of vehicle and the treatment groups received A␤1-42 (4 ␮M) with a 25 ␮l Hamilton microsyringe. Control rats were injected with vehicle only, because reverse A␤42-1 for control showed no neurochemical changes in our preliminary experiments (our unpublished observations). The injections were delivered at an infusion rate of 2 ␮l/min for a duration of 2.5 min. The injection needle was left in place for 5

min before withdrawal to allow diffusion from the tip and prevent reflux of the solution. The injection placement of needle track was visible and was verified at the time of dissection. In a second series of experiments, animals received a retrodialysis administration of 10 ␮M A␤1-42 in the PFC.

Drugs and experimental design L-Arg was provided by Alexis Biochemicals (Vinci-Biochem, VinciFi, Italy), dissolved in saline and administered i.p. (300 mg/kg) to A␤1-42-treated rats for 7 (2 h group) or 9 (2 days group) consecutive days. 7-NI was provided by Sigma Aldrich s.r.l. (Milano, Italy), dissolved in peanut oil and administered i.p. (50 mg/kg) to A␤1-42-treated rats for 7 (2 h group) or 9 (2 days group) consecutive days. The experimental schedule is shown in Fig. 1. L-Arg or 7-NI administration began 7 days before A␤1-42 i.c.v. injection, and continued throughout the experimental period. The microdialysis study began 2 h or 2 days after A␤1-42 i.c.v. injection.

Assay of DA in the dialysate DA concentrations (20 ␮l) were determined by high performance liquid chromatography (HPLC) using LC-18 DB column (150 mm⫻ 4.6 mm; 5 ␮m SUPELCOSIL) with ESA Coulometric detection (ESA Inc., Chelmsford, MA, USA) in oxidation/reduction mode (E1: ⫹300 mV; E2: ⫺300 mV). The mobile phase used consisted of 85 mM NaH2PO4, 1 mM SDS, 0.02 mM EDTA, 0.7 mM triethylamine, acetonitrile 15%, methanol 13%, solved in distilled water (1 l) and buffered at pH 5.8 with NaOH 1 N. The flow rate was 1 ml/min. Detection limit was about 0.5 DA fmol on column (signalto-noise ratio 2).

Immunohistochemical analysis After neurochemical analysis, each brain was frozen and stored at ⫺80 °C. Serial sections of brain were cut at 20 ␮M on a cryostat and stored at ⫺20 °C. For immunodetection of infused A␤ peptide, sections were fixed in 4% formaldehyde for 15 min, washed with PBS and then were treated with 70% formic acid for 30 min at room temperature to re-expose epitope; nonspecific binding with A␤ was blocked by incubation for 30 min with PBS containing 1% bovine serum albumin (BSA). Sections were incubated for 1 h with a primary monoclonal antibody recognizing A␤ protein (clone 4G8, Chemicon International, Temecula, CA, USA), diluted 1:100 in PBS/1% BSA solution. Sections were washed with PBS and then incubated for 30 min at room temperature with a mouse anti-IgG antibody RPE conjugated (Dako, Carpenteria, CA, USA) diluted 1:40 in PBS/1% BSA. After the fluorescent labeling procedures, sections were finally counterstained for DNA for 5 min with a

L. Trabace et al. / Neuroscience 147 (2007) 652– 663 0.1 ␮g/ml HOECST 33342 solution in PBS, and mounted in a drop of Mowiol (Calbiochem, Inalco S.p.A., Milan, Italy).

Statistical analysis Neurochemical data were expressed in fmol/20 ␮l. Actual data were analyzed by one-way or two-way analysis of variance (ANOVA) for repeated measures with treatment (tr) as the between-subject factor and time (t) as the within-subject factor. Conservative F tests using the Greenhouse-Geisser correction were performed to account for possible violations of the sphericity assumption. Post hoc comparisons were made by Dunnett’s and Tukey’s tests where appropriate. Values missing because of occasional problems in sample collection or analysis were replaced by the mean of the samples immediately before and after.

RESULTS Presence of A␤1-42 in rat brain parenchyma and ventricular space after i.c.v. injection In order to test the effect of A␤ infusion on neurotransmitter release we first studied the efficiency of administration and diffusion of A␤1-42 (4 ␮M) to the brain parenchyma following i.c.v. injection. After 2 h from the injection of the peptide, A␤ immunoreactivity was detected within the ventricle system and on its walls (Fig. 2A). The peptide injected in the right ventricle was indeed found at the site of injection as well as at other levels of the ventricular system (III

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ventricle, Fig. 2C; and contralateral ventricle, data not shown) and in the choroid plexus (Fig. 2B). In particular, by using a stereotaxic atlas (Paxinos and Watson, 1998) to recognize the brain areas, we detected the A␤ peptide starting from the sections where only the lateral ventricles were visible (stereotaxic coordinates: Bregma 1.20 mm) until to slices where III ventricle is evident as well as at the level of the first portion of the hippocampus (stereotaxic coordinates: Bregma ⫺1.60 mm). No gross signs of neurodegeneration were observed within the area of A␤ diffusion in the periventricular parenchyma as shown by Hoechst immunocytochemistry, and as expected using a low micromolar freshly prepared A␤1-42 solution. By 48 h after the i.c.v. injection of A␤1-42, the peptide was not detectable in the ventricular space (Fig. 2D) at any of the levels examined, but it was still evident in some of the tissues adjacent to the ventricle system (Fig. 2D–F) as well as at the level of the choroid plexus (Fig. 2E). The decreased content of A␤1-42, observed after 48 h from the injection of this peptide suggests a putative clearance of the protein from the ventricular space by the cerebral tissue. Hoechst immunocytochemistry did not show differences between control and A␤1-42-treated animals at this time, in our experimental and staining conditions. Furthermore, at the level of PFC we did not detect the presence of A␤1-42 peptide, probably because this area is too far from the range of diffusion of the peptide (data not shown).

Fig. 2. Fluorescence micrographs showing the presence of human beta-amyloid protein in the ventricular system. Micrographs A, B, C show the presence of beta-amyloid peptide immunoreactivity (red staining, white arrow) at 2 h after i.c.v. injection, whereas micrographs D, E, F highlight beta amyloid peptide at 48 h after the killing of the rat. Nuclear DNA was counterstained with Hoechst 33342 (blue staining) (magnification: ⫻20).

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Effects of i.c.v. A␤1-42 administration on basal and stimulated extracellular DA concentrations in the PFC of rats Microdialysis samples were collected from rats at 2 h and 2 days after i.c.v. administration of A␤1-42 (4 ␮M), for measurements of extracellular concentrations of DA in the PFC. A two-way ANOVA for repeated measures of mean basal DA concentrations (three consecutive samples collected 2 h, 2 days after A␤1-42 or vehicle injection) showed the following differences:Ftr(2,13)⫽8.69,P⬍0.01;Ft(2,4)⫽0.01n.s.;Ftrxt(2,26)⫽ 0.01 n.s. These results indicate that basal DA levels were significantly decreased when measured either 2 h or 2 days after i.c.v. A␤1-42 injection in the PFC of rats (Fig. 3 inset). As far as the effects of local 100 mM K⫹ aCSF perfusion (20 min) on extracellular DA concentrations in the PFC of i.c.v. A␤1-42-treated rats were concerned, a twoway ANOVA for repeated measures of changes in DA levels (actual values, last basal value and seven consecutive samples after high K⫹ retrodialysis administration) showed the following differences: Ftr(2,13)⫽14.12, P⬍0.001; Ft(6,78)⫽ 1.69 n.s.; Ftrxt(12,78)⫽1.71 n.s. In order to exclude the possibility that the altered responsiveness to a high K⫹ challenge exhibited by

␤1-42-treated rats with respect to control animals could reflect differences in basal extracellular DA concentrations, a two-way ANOVA for repeated measures of absolute extracellular DA increases induced by high K⫹ challenge with respect to the last basal sample was performed. This analysis showed the following differences: Ftr(2,13)⫽9.40, P⬍0.01; Ft(5,65)⫽2.03 n.s.; Ftrxt(10,65)⫽2.06 P⬍0.05. Within-group comparisons (Tukey’s test) indicated that the high K⫹ challenge induced a significant increase in extracellular DA concentrations compared with the last basal sample only in vehicle-treated rats (846%, Fig. 3). Furthermore, between-groups comparisons (Dunnett’s test) indicated that the high K⫹-induced increase in extracellular DA levels (absolute increase) was significantly attenuated in A␤1-42-treated animals compared with vehicle-treated rats. In particular, the increase in DA release stimulated by 100 mM Kⴙ was greatly reduced in either 2 h or 2 days groups after A␤1-42 injection. These data indicate that the dopaminergic hypofunction induced by i.c.v. A␤1-42 injection is revealed both when the system is under basal unstimulated conditions and when it is activated by Kⴙ depolarization.

Fig. 3. Effects of i.c.v. vehicle (⽧) or A␤1-42 administration ( 2 h) ( 2 days) on basal (inset) and stimulated (local 100 mM K⫹ aCSF perfusion, horizontal black bar) extracellular DA concentrations in microdialysis samples from PFC of conscious, freely moving rats treated with subchronic administration of saline (7 or 9 days, i.p.). Data are mean⫾S.E. (n⫽5– 6 rats). * P⬍0.01 versus basal values (Tukey’s test); # P⬍0.01 versus vehicle (Dunnett’s test). Inset: * P⬍0.01 versus vehicle (Dunnett’s test).

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time (min) Fig. 4. Effects of local A␤1-42 perfusion (10 ␮M, ⽧), A␤1-42/100 mM K⫹ aCSF coperfusion () (horizontal black bars) on extracellular DA concentrations in microdialysis samples from PFC in conscious, freely moving rats. Data are mean⫾S.E. (n⫽7– 8 rats). * P⬍0.01 versus A␤1-42 (Dunnett’s test); # P⬍0.05 versus basal values (Tukey’s test). Inset: Effects of 100 mM K⫹ aCSF on DA levels of local A␤1-42-treated or i.c.v. A␤1-42-treated rats. * P⬍0.01 versus basal values (Tukey’s test).

Effects of local A␤1-42 perfusion on basal and stimulated DA levels in the PFC of rats As illustrated in Fig. 4, in vivo microdialysis sampling showed that A␤1-42 (10 ␮M for 40 min), administered via the dialysis probe in the PFC of freely moving rats, did not alter extracellular DA concentrations. In order to investigate whether the dopaminergic system was responsive to a high K⫹ challenge in the presence of A␤1-42, the PFC was perfused locally with A␤1-42 for 20 min before and during a high K⫹ challenge (100 mM for 20 min). A twoway ANOVA for repeated measures of changes in DA levels (actual values) showed the following differences: Ftr(1,15)⫽7.25, P⬍0.02; Ft(10,150)⫽0.05 n.s.; Ftrxt(10, 150)⫽ 0.05 n.s. Since significant differences between treatments were time-independent, individual comparisons between marginal means (pooled data of 11 samples for each treatment) were made. Results showed that the high K⫹ challenge, in the presence of A␤1-42, significantly increased extracellular DA concentrations in the PFC (126% P⬍0.05; Fig. 4 inset). However, the presence of A␤1-42 significantly attenuated high K⫹-induced increase in DA levels, when compared with controls (846% P⬍0.05; Fig. 4 inset). Moreover, when high K⫹ aCSF (100 mM for 20 min) was perfused 2 h after A␤1-42, DA levels were not altered. Nevertheless, the effects of locally applied A␤1-42 on the extracellular concentrations of DA were determined for 4 h after administration of the peptide. DA levels were signifi-

cantly reduced at both 3 and 4 h time points after A␤1-42 perfusion (Fig. 4). Effects of L-Arg and 7-NI treatment on basal extracellular concentrations of DA in the PFC of A␤1-42-injected rats As far as the effects of L-Arg administration were concerned, a two-way ANOVA for repeated measures of basal DA concentrations (three consecutive samples collected 2 h and 2 days after i.c.v. A␤1-42 injection) showed the following differences: [2 h: Ftr(2,14)⫽6.78, P⬍0.05; Ft(2,4)⫽0.01 n.s.; Ftrxt(2,28)⫽0.03 n.s.; 2 days: Ftr(2,14)⫽4.23, P⬍0.05; Ft(2,4)⫽0.02 n.s.; Ftrxt(2,28)⫽0.05 n.s.]. These results indicate that daily administration of L-Arg (300 mg/kg, i.p.) for 7 (2 h group) or 9 days (2 days group) significantly reduced basal extracellular concentrations of DA in the PFC when measured either 2 h after A␤1-42 injection (Fig. 5A) or on day 2 post-injection (Fig. 5B). As regards the effects of 7-NI administration, a twoway ANOVA for repeated measures of basal DA concentrations (three consecutive samples collected 2 h and 2 days after A␤1-42 injection) showed the following differences: [2 h: Ftr(2,14)⫽6.78, P⬍0.05; Ft(2,4)⫽0.01 n.s.; Ftrxt(2,28)⫽0.03 n.s.; 2 days: Ftr(2,14)⫽4.23, P⬍0.05; Ft(2,4)⫽0.02 n.s.; Ftrxt(2,28)⫽0.05 n.s.]. These results indicate that daily administration of 7-NI (50 mg/kg, i.p.) significantly increased basal DA concentrations when mea-

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Fig. 5. (A) Effects of saline, L-Arg or 7-NI subchronic administration (7 days, i.p.) on basal extracellular DA concentrations in microdialysis samples from PFC of conscious, freely moving rats, measured 2 h after i.c.v. A␤1-42 administration. Data are mean⫾S.E. (n⫽6 –7 rats). * P⬍0.01 versus saline (Dunnett’s test). (B) Effects of saline, L-Arg or 7-NI subchronic administration (9 days, i.p.) on basal extracellular DA concentrations in microdialysis samples from PFC of conscious, freely moving rats, measured 2 days after i.c.v. A␤1-42 administration. Data are mean⫾S.E. (n⫽5– 6 rats). * P⬍0.01 versus saline (Dunnett’s test).

sured 2 h after A␤1-42 injection (Fig. 5A). On the contrary, basal DA levels were significantly decreased in 7-NItreated animals when measured on day 2 post-A␤1-42injection (Fig. 5B). Effects of L-Arg and 7-NI treatment on 100 mM Kⴙstimulated extracellular concentrations of DA in the PFC of A␤1-42-injected rats As far as the effects of L-Arg on DA levels were concerned, because a two-way ANOVA for repeated measures

showed that significant differences between treatments were time-independent, both when measured 2 h and 2 days after A␤1-42 injection [2 h: Ftr(2,88)⫽5.93, P⬍0.05; Ft(8,88)⫽0.03 n.s.; Ftrxt(16,88)⫽0.03 n.s.; 2 days: Ftr(2,88)⫽ 3.54 P⬍0.05; Ft(8,88)⫽0.03 n.s.; Ftrxt(16,88)⫽0.04 n.s.], individual comparisons between marginal mean values (pooled data of nine samples for each treatment) were made. Results showed that L-Arg treatment significantly decreased DA levels in the PFC at both 2 h and 2 days in A␤1-42-injected animals. Furthermore, 100 mM Kⴙ aCSF

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Fig. 6. (A) Effects of local 100 mM K⫹ aCSF perfusion (horizontal black bar) on extracellular DA concentrations of i.c.v. saline- (⽧), L-Arg- () or 7-NI( ) treated animals (7 days, i.p.), measured 2 h after i.c.v. A␤1-42 administration in microdialysis samples from PFC of conscious, freely moving rats. Data are mean⫾S.E. (n⫽6 rats). (B) Effects of local 100 mM K⫹ aCSF perfusion (horizontal black bar) on extracellular DA concentrations of i.c.v. saline- (⽧), L-Arg- () or 7-NI- ( ) treated animals (9 days, i.p.), measured 2 days after i.c.v. A␤1-42 administration in microdialysis samples from PFC of conscious, freely moving rats. Data are mean⫾S.E. (n⫽6 –7 rats).

‘

‘

perfusion (20 min) did not modify DA levels when measured 2 h or 2 days after A␤1-42 injection (Fig. 6A–B, respectively). In terms of the effects of 7-NI on extracellular DA concentrations, a two-way ANOVA for repeated measures also showed that significant differences between treatments were time-independent, both when measured 2 h and on day 2 after A␤1-42 injection [2 h: Ftr(2,88)⫽5.93, P⬍0.05; Ft(8,88)⫽0.03 n.s.; Ftrxt(16,88)⫽0.03 n.s.; 2 days: Ftr(2,88)⫽3.54 P⬍0.05; Ft(8,88)⫽0.03 n.s.; Ftrxt(16,88)⫽0.04

n.s.], and therefore individual comparisons between marginal mean values (pooled data of nine samples for each treatment) were made. Results showed that 7-NI treatment significantly increased DA levels in the PFC 2 h after A␤1-42 injection (Fig. 5A), whereas extracellular DA concentrations were significantly decreased when measured after 2 days (Fig. 5B). Moreover, 100 mM Kⴙ depolarization (20 min) did not alter extracellular concentrations of DA in 7-NI-treated rats, when measured 2 h or 2 days after injection of A␤1-42 (Fig. 6A–B, respectively).

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DISCUSSION Given the numerous difficulties involved in assessing disease progression dynamically in the human brain, significant work has been performed to create animal models that might replicate at least some aspects of AD pathogenesis. In this regard, a considerable number of transgenic mouse lines have been generated. Most of these transgenic models replicate some of the neuropathological features of the disease (including dopaminergic dysfunction, Perez et al., 2005) but nontransgenic animal models are also considered to be a useful complement to transgenic approaches to Alzheimer’s pathology. In this latter respect, rodents given brain injection/infusion of A␤ have been frequently used as an animal model for AD-type pathology. In this regard, it has been shown that i.c.v. administration of A␤ causes memory deficits and neuronal dysfunctions (Flood et al., 1991; Nitta et al., 1994; Maurice et al., 1996; Nakamura et al., 2001; Wang et al., 2006). Moreover, A␤ injections into the cortex, hippocampus, amygdala or nucleus basalis magnocellularis have been reported to produce neuronal loss and cholinergic degeneration (Chen et al., 1996; Emre et al., 1992; Kowall et al., 1992; Sigurdsson et al., 1997; Song et al., 1998; Giovannini et al., 2002). One of the major findings of the experiments reported here was that the dopaminergic system seems to be acutely vulnerable to the effects of A␤. Indeed, a single i.c.v. injection of freshly prepared A␤1-42 induced a marked reduction in extracellular concentrations of basal DA levels in the PFC, when measured 2 h and 2 days after peptide administration. Moreover, the increase in DA release stimulated by local 100 mM K⫹ perfusion was abolished in A␤1-42-injected rats. In addition, A␤1-42 given locally via dialysis probes in the PFC also produced a significant decrease in DA levels, starting 180 min after the administration and for at least a further 1 h and 40 min. A high K⫹-induced depolarization, performed 2 h after A␤1-42 perfusion, did not elicit DA increase and a reduced K⫹induced increase in DA levels (⫹126%) was observed when co-perfused with A␤1-42, compared with vehicle-infused controls (⫹834%). Thus, the present results clearly show that A␤1-42 is producing significant impairment within the dopaminergic system in the PFC which is affecting both tonic and dynamic functioning. The effects of a single infusion can occur quite rapidly (by 2–3 h) and can also be seen after 2 days. In the present study, a soluble form of A␤1-42 has been used. The effects observed after A␤ retrodialysis administration on dopaminergic transmission are likely due to soluble A␤1-42, diffusing through the membrane of the microdialysis probe. Indeed, A␤1-42 aggregates would be retained by the dialysis membrane of the probe. After i.c.v. injection, the observation that A␤ can decrease extracellular DA levels in the PFC within 2 h also supports this hypothesis, indicating a relatively rapid mechanism of action. Previous findings have demonstrated that A␤1-40 and A␤1-42 can enter brain synaptosomes within minutes of application in the external medium (Plant et al., 2006). In

addition, the hypothesis concerning the effect of A␤1-42 in its soluble form is also supported by immunofluorescence experiments, showing that after 48 h from i.c.v. injection A␤1-42 peptide was no longer detectable in the ventricular space, but was still present mainly at level of the choroid plexus, thus suggesting a putative clearance of the protein by the cerebral tissue. The presence of A␤ mainly at the level of the choroid plexus is intriguing, since Crossgrove et al. (2005) have recently showed that the choroid plexus is able to clear A␤ from the CSF rapidly through a nondiffusional uptake process and that A␤ uptake by it favors its efflux from CSF into the blood rather than its influx from blood into the CSF. It seems likely therefore from our findings that A␤induced alterations in the dopaminergic system of the PFC can precede the development of actual A␤ deposits and that further investigation of the long-term functional effects of soluble A␤ is warranted. Our results are also consistent with previous observations showing that non-fibrillar A␤ aggregates, protofibrils, oligomers and amyloid-derived diffusible ligands, are neurotoxic and may be responsible for synaptic dysfunction in AD brains (for review see Hardy and Selkoe, 2002). In this regard, there is now mounting evidence suggesting that soluble A␤ plays a critical role in the pathogenesis of AD. Indeed, analyses of human brains have demonstrated strong correlations between cortical levels of soluble A␤ and the extent of synaptic loss and severity of cognitive impairment. Specifically, soluble A␤ peptide concentrations have been suggested to be a useful predictor of synaptic change in AD (Lue et al., 1999) and a determinant of severity of neurodegeneration (McLean et al., 1999). It has been shown that pre-fibrillar assemblies of A␤ induce neurotoxicity, electrophysiological changes and disruption of cognitive functions (Hartley et al., 1999; Walsh et al., 1999, 2002; Wang et al., 2002; Kayed et al., 2003; Heinitz et al., 2006). Moreover, in AD-affected brains there is a better correlation between the pool of soluble A␤ and the severity of neurodegeneration than that observed for insoluble A␤ plaques (McLean et al., 1999). Collectively, these findings suggest that synaptic dysfunction is an early event in the pathogenesis of AD. They are also consistent with results from a different animal model of AD in which disruption of neural circuits were found to occur independently of plaque accumulation (Hsia et al., 1999). The underlying mechanism(s) associated with soluble A␤1-42-induced inhibition of basal and stimulated DA release are unknown. Given the ability of A␤ to interact with the cell membrane (Chauhan et al., 1993), it might be hypothesized that A␤1-42 can act either via an as yet undefined association to alter membrane permeability or via the activity of some associated protein. In this regard, it has been demonstrated that treatment of fibroblasts with soluble A␤ induced the same K⫹ channel dysfunction shown to occur specifically in fibroblasts from patients with AD (Etcheberrigaray et al., 1994). Cp2O, an extremely potent regulator of K⫹ channels (Nelson et al., 1990) and a GTP-binding protein, was significantly decreased in fibroblasts from AD patients. Normal control fibroblasts ex-

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posed to A␤, at the same concentration that induced ADlike K⫹ changes in control fibroblasts, showed a similar decrease in Cp2O (Kim et al., 1995). Interestingly, the observed changes not only occur in peripheral tissues, such as fibroblasts, but also in neural tissue, the primary site of AD pathology (Etcheberrigaray et al., 1996). It is worth noting that K⫹ channels play a pivotal role in the regulation of several cellular responses, including the release of neurotransmitters (Roberts, 1986; Hille, 1992; Etcheberrigaray et al., 1994). It has also been reported that K⫹ channels are important in the acquisition of memory (Roberts, 1986; Hille, 1992). On the other hand, it has been shown that A␤ produces amnestic effects and potently impairs post-training memory processing when injected into different brain structures (Flood et al., 1991, 1994). Taken together, these data and our results suggest that interference with K⫹ channels may be associated with the modulatory effects of A␤ on DA release. Previous findings have also demonstrated that A␤-related peptides inhibit K⫹-evoked acetylcholine release from rat hippocampal slices (Kar et al., 1996). In another study, the increase in acetylcholine release stimulated by 100 mM K⫹ was significantly reduced in A␤1-42-injected rats (Giovannini et al., 2002). Overall, these results suggest that soluble A␤1-42 acts as an acute potent inhibitor of DA release and may serve as a basis for the functional inter-relationship between acute A␤ dysfunction and the vulnerability of dopaminergic transmission in AD. While the modulatory effect of A␤ on DA release may be relevant to the known impairment of dopaminergic functions in AD (Nazarali and Reynolds, 1992; Storga et al., 1996; Alisky, 2006) other non-dopaminergic transmission systems may also be affected. Among these, the nitrergic pathway is thought to be of special significance. Increasing evidence suggests that NO may directly, or indirectly, be involved in neuronal death in AD and other neurodegenerative disorders (Law et al., 2001; Duncan and Heales, 2005). In this regard, the relevance of NO in both physiological and pathological scenarios is considerable. NO generated by NOS is a unique endogenous molecule modulating vital physiological functions (Moncada et al., 1991; Murad, 2003). The relationship between NO and AD may not be limited to NO-mediated neurotoxicity and the potential neuroprotective properties of NO should also be considered. The free-radical gas NO has been shown to have a number of actions as an intercellular messenger, or novel type of neurotransmitter in the brain (Garthwaite, 1991; Snyder and Bredt, 1991). Under normal physiological circumstances, NO possesses neuromodulatory actions. We have previously reported that NO donors produce effects on a number of brain neurotransmitter systems and can significantly decrease DA levels (Kendrick et al., 1997; Trabace and Kendrick, 2000). In the present study, we have found that subchronic treatment with L-Arg, a precursor of NO biosynthesis, significantly decreased basal extracellular DA concentrations in the PFC when measured either 2 h after i.c.v. A␤1-42 injection or on day 2 post-injection, whereas subchronic

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administration of a selective nNOS inhibitor, such as 7-NI, significantly increased basal DA concentrations when measured 2 h after i.c.v. A␤1-42 injection and decreased them when measured on day 2 post-A␤1-42-injection. No response of dopaminergic activity was observed after high K⫹ stimulation in all groups considered. The possible, although speculative, explanation for the consistent increased effect of L-Arg could be that it promoted greater formation of damaging NO from the outset. If we assume that L-Arg, as an NO precursor, would have acted to boost NO release, then the optimal tonic protective levels of NO were exceeded and additional damage was done. In fact, it is possible that the presence of tonic levels of NO derived from nNOS and endothelial nitric oxide synthase (eNOS) was protective in the first instance but that increased levels caused by later induction of inducible (iNOS) were damaging. Several reports have shown that 7-NI selectively inhibits nNOS (Babbedge et al., 1993; Moore et al., 1993). If we assume that 7-NI does not influence iNOS, then this treatment would have reduced the initial protection via nNOS and eNOS but not the damage caused by NO release from iNOS. Indeed, this hypothesis was supported by the observation that the 7-NI-induced effects on DA levels were different at 2 h and 2 days. In the first place the higher DA levels could represent an index of exaggerated ongoing injury to the system (consistent with the lack of K⫹ response). This would imply that tonic NO release (derived from eNOS/nNOS) could be neuroprotective, as previously data have already shown (Kendrick et al., 1996) although excess NO release could also be damaging, as demonstrated by the effects of L-Arg treatment. Thus, the altered ability of NO to modulate neurotransmission during neurodegenerative pathologies may particularly impact on the functional consequences of NO on the dopaminergic system. To our knowledge, this is the first in vivo report demonstrating an acute effect of soluble A␤ on a transmitter function, such as dopaminergic system, which may suggest that cognitive deficits, executive and emotional dysregulation may take place a long time before A␤-induced perturbing effects. Moreover, from our results it could be suggested that the effects of A␤1-42 on dopaminergic system in the PFC take place on the dopaminergic terminals directly, since drugs administered by reverse dialysis diffuse only in the near surroundings of the membrane, or indirectly, through NO involvement.

CONCLUSION In conclusion, the data presented here strongly suggest that the dopaminergic system in the PFC seems to be acutely vulnerable to soluble A␤1-42 effects. Furthermore, the opposite role of NO occurring in the different phases, early neuroprotective (2 h) and then detrimental (2 days), might be regarded as a possible link between A␤1-42induced effects and dopaminergic dysfunction. Acknowledgment—This study was supported by a grant from FIRB 2006 to V. Cuomo.

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(Accepted 5 April 2007) (Available online 7 June 2007)