Nimodipine Inhibits TMB-8 Potentiation of AMPA-Induced Hippocampal Neurodegeneration Fabia ´n Bernal, Valerie Petegnief, Manuel Jose ´ Rodrı ´guez, Gloria Ursu, Marco Pugliese, and Nicole Mahy * Unitat de Bioquı ´mica, IDIBAPS, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain Human cerebral calcification has been related to dereg- ulation of intracellular calcium homeostasis. In rat basal ganglia, nimodipine and TMB-8, two commonly used calcium antagonists, worsen the chronic AMPA-induced lesion, whereas only nimodipine potentiates calcifica- tion. To investigate whether similar effects are present in the hippocampus, AMPA dose–response and calcium movement blockade were performed. A dose-related increase of both hippocampal lesion and calcification was evident in a saturable mode, mostly different from the continuous globus pallidus response previously observed. The value of 2.7 nmol AMPA, selected as yielding 60% of maximum calcification, was coinjected with nimodipine or/and TMB-8 to determine their influ- ence on tissue damage. TMB-8 increased the AMPA lesion in terms of calcified area, and nimodipine reversed this increase, with no effect alone. These results, divergent from those for the globus pallidus, reveal differences in extra- and intracellular calcium movement between the two neurodegenerative proc- esses. Future work focused on other brain areas is required to understand how control of calcium stores may influence neurodegenerative disease evolution. V V C 2008 Wiley-Liss, Inc. Key words: calcification; excitotoxicity; hippocampus; neurodegeneration; nimodipine Calcium dyshomeostasis has been strongly linked to neuronal loss in a diversity of human pathological condi- tions such as Alzheimer’s disease, Parkinson’s disease, postraumatic epilepsy, and brain ischemia (Verkhratsky, 2005; Chinopoulos and Adam-Vizi, 2006; Lindholm et al., 2006; Zatti et al., 2006). Calcium overloading in the cytosol and mitochondria plays a critical role in neu- ronal injury, but superfluous Ca 21 also induces the release of several intrinsic and extrinsic factors that acti- vate processes to rescue neurons from death (Rose and Konnerth, 2001; Pe ´rez-Garcı ´a et al., 2004; Mironov et al., 2005; Mattson, 2007). To support their low re- generative capacity and long life span, neurons can with- stand very large calcium insults through several adaptive mechanisms. For example, the formation of insoluble calcium phosphate complexes, similar to biological hydroxyapatites, reduces free cytoplasmic calcium in neurons at no energy expense (Rodrı ´guez et al., 2000; Chalmers and Nicholls, 2003; Nicholls et al., 2003; Ramonet et al., 2006). The extension of this calcifica- tion process in a brain area depends on the intensity of the acute phase and on the characteristics of each area of pathology (Rodrı ´guez et al., 2001; Ramonet et al., 2006). Thus, the differences in extension of calcification observed between the globus pallidus, the hippocampus, and the cerebral cortex in human perinatal hypoxia-is- chemia strongly correlate with hypoxia-ischemia area vulnerability (Rodrı ´guez et al., 2001). In rodent brain areas, glutamate receptor activation induces cytoplasmic calcification with the same deposit morphology and composition in neurons and astrocytes. The same area vulnerability is also observed in humans (Mahy et al., 1999; Bernal et al., 2000b; Ramonet et al., 2006), and canine brain calcification also presents similar characteristics (Ramonet et al., 2002). A plausible explana- tion for the homogenous neuronal and astroglial calcification response observed in three mammal species lies in activation of common synaptic processes to decrease neuronal suffering. Despite glutamate-related calcium dyshomeostasis being considered a main pathophysiological mechanism, few in vivo studies have investigated the combined neu- ronal and glial response to cytosolic calcium overload Present address of Fabia ´n Bernal: Group of Neuroimmunology, Depart- ment of Neurology, Heirich Heine University, Duesseldorf, Germany. Present address of Valerie Petegnief: Department of Pharmacology and Toxicology, IIBB-CSIC, IDIBAPS, Barcelona, Spain. Contract grant sponsor: Spanish Ministerio de Sanidad y Consumo (pro- ject CIBERNED); Contract grant sponsor: Spanish Ministerio de Educa- cio ´n y Ciencia; Contract grant number: SAF2005-04314; Contract grant sponsor: Generalitat de Catalunya; Contract grant number: 2005SGR00609. *Correspondence to: Dr. Nicole Mahy, Unitat de Bioquı ´mica, IDIBAPS, Facultat de Medicina, Casanova 143, E-08036 Barcelona, Spain. E-mail: [email protected]Received 13 February 2008; Revised 3 September 2008; Accepted 3 September 2008 Published online 11 November 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21930 Journal of Neuroscience Research 87:1240–1249 (2009) ' 2008 Wiley-Liss, Inc.
Fabian Bernal, Valerie Petegnief, Manuel Jose Rodrıguez, Gloria Ursu,Marco Pugliese, and Nicole Mahy*
Unitat de Bioquımica, IDIBAPS, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain
Human cerebral calcification has been related to dereg-ulation of intracellular calcium homeostasis. In rat basalganglia, nimodipine and TMB-8, two commonly usedcalcium antagonists, worsen the chronic AMPA-inducedlesion, whereas only nimodipine potentiates calcifica-tion. To investigate whether similar effects are presentin the hippocampus, AMPA dose–response and calciummovement blockade were performed. A dose-relatedincrease of both hippocampal lesion and calcificationwas evident in a saturable mode, mostly different fromthe continuous globus pallidus response previouslyobserved. The value of 2.7 nmol AMPA, selected asyielding 60% of maximum calcification, was coinjectedwith nimodipine or/and TMB-8 to determine their influ-ence on tissue damage. TMB-8 increased the AMPAlesion in terms of calcified area, and nimodipinereversed this increase, with no effect alone. Theseresults, divergent from those for the globus pallidus,reveal differences in extra- and intracellular calciummovement between the two neurodegenerative proc-esses. Future work focused on other brain areas isrequired to understand how control of calcium storesmay influence neurodegenerative disease evolution.VVC 2008 Wiley-Liss, Inc.
Calcium dyshomeostasis has been strongly linked toneuronal loss in a diversity of human pathological condi-tions such as Alzheimer’s disease, Parkinson’s disease,postraumatic epilepsy, and brain ischemia (Verkhratsky,2005; Chinopoulos and Adam-Vizi, 2006; Lindholmet al., 2006; Zatti et al., 2006). Calcium overloading inthe cytosol and mitochondria plays a critical role in neu-ronal injury, but superfluous Ca21 also induces therelease of several intrinsic and extrinsic factors that acti-vate processes to rescue neurons from death (Rose andKonnerth, 2001; Perez-Garcıa et al., 2004; Mironovet al., 2005; Mattson, 2007). To support their low re-generative capacity and long life span, neurons can with-stand very large calcium insults through several adaptivemechanisms. For example, the formation of insolublecalcium phosphate complexes, similar to biological
hydroxyapatites, reduces free cytoplasmic calcium inneurons at no energy expense (Rodrıguez et al., 2000;Chalmers and Nicholls, 2003; Nicholls et al., 2003;Ramonet et al., 2006). The extension of this calcifica-tion process in a brain area depends on the intensity ofthe acute phase and on the characteristics of each area ofpathology (Rodrıguez et al., 2001; Ramonet et al.,2006). Thus, the differences in extension of calcificationobserved between the globus pallidus, the hippocampus,and the cerebral cortex in human perinatal hypoxia-is-chemia strongly correlate with hypoxia-ischemia areavulnerability (Rodrıguez et al., 2001).
In rodent brain areas, glutamate receptor activationinduces cytoplasmic calcification with the same depositmorphology and composition in neurons and astrocytes.The same area vulnerability is also observed in humans(Mahy et al., 1999; Bernal et al., 2000b; Ramonet et al.,2006), and canine brain calcification also presents similarcharacteristics (Ramonet et al., 2002). A plausible explana-tion for the homogenous neuronal and astroglial calcificationresponse observed in three mammal species lies in activationof common synaptic processes to decrease neuronal suffering.
Despite glutamate-related calcium dyshomeostasisbeing considered a main pathophysiological mechanism,few in vivo studies have investigated the combined neu-ronal and glial response to cytosolic calcium overload
Present address of Fabian Bernal: Group of Neuroimmunology, Depart-
ment of Neurology, Heirich Heine University, Duesseldorf, Germany.
Present address of Valerie Petegnief: Department of Pharmacology and
Toxicology, IIBB-CSIC, IDIBAPS, Barcelona, Spain.
Contract grant sponsor: Spanish Ministerio de Sanidad y Consumo (pro-
ject CIBERNED); Contract grant sponsor: Spanish Ministerio de Educa-
cion y Ciencia; Contract grant number: SAF2005-04314; Contract
grant sponsor: Generalitat de Catalunya; Contract grant number:
2005SGR00609.
*Correspondence to: Dr. Nicole Mahy, Unitat de Bioquımica, IDIBAPS,
Facultat de Medicina, Casanova 143, E-08036 Barcelona, Spain.
Received 13 February 2008; Revised 3 September 2008; Accepted 3
September 2008
Published online 11 November 2008 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/jnr.21930
Journal of Neuroscience Research 87:1240–1249 (2009)
' 2008 Wiley-Liss, Inc.
(Rodrıguez et al., 2000). Whether the Ca21 origin isextracellular or intracellular may determine the course ofthe lesion (Frandsen and Schousboe, 1992, 1993; Svicharet al., 1997; Simpson and Russell, 1998) and may alsoinfluence the calcification process. A previous study per-formed in the rat globus pallidus provided evidence ofunexpected potentiation of excitotoxicity and calcifica-tion by coinjection of calcium channel blockers, includ-ing the widely used nimodipine (Petegnief et al., 2004).However, the variability of brain area response to exci-totoxic insults and hypoxia-ischemia (Saura et al., 1995;Nakajima et al., 2000) renders this effect difficult toextrapolate to other regions such as the hippocampal for-mation. Calcium antagonist treatments in hypoxia-ische-mia models that have an effectiveness of only 50%(Horn et al., 2001) also require a better understanding oftheir mechanism of action. Because of that, the sameglobus pallidus a-amino-3-hydroxy-5-methyl-isoxazolepropionate (AMPA)–induced lesion was characterized inthe hippocampus, including the use of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) as a spe-cific receptor antagonist (Petegnief et al., 1999).
To compare how the calcium origin influencesboth globus pallidus and hippocampal lesions, the sametwo potent calcium antagonists were coinjected withAMPA: nimodipine, a 1,4-dihydropyridine (DHP)–basedantagonist of voltage-operated Ca21 channels and the 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hy-drochloride (TMB-8), a blocker of calcium release fromintracellular stores (Malcolm et al., 1996). Our resultsprovide evidence that the globus pallidus and the hippo-campus vary in their sensitivity to AMPA receptor acti-vation and that nimodipine may only afford hippocampalneuroprotection. Because the conditions that triggerneuroprotection differ between brain areas, the potentialtherapeutic utility of calcium channel blockers requirescharacterization according to the locations of the lesionto be treated.
MATERIALS AND METHODS
Animals
For this experiment, 106 adult male Sprague-Dawleyrats (average body weight 310 g) were used. Animals werehoused individually and kept in standard light/darkness cycleconditions in a temperature-controlled room (228C), with freeaccess to food and water. All manipulations were performedin accordance with European legislation (86/609/EEC). Allefforts were made to minimize the number of animals andtheir suffering during the experiments, and procedures wereapproved by the Ethic Committee of the University of Barce-lona under the supervision of the Generalitat of Catalunya(Spain).
Surgery
Under equithesin anesthesia (a mixture of chloralhydrate and sodium pentobarbitone; 0.3 mL/100 g bodyweight, i.p.), animals were placed in a stereotaxic frame(David Kopf, Sweden) with the incisor bar set at 23.3 mm.
Coordinates were selected in accordance with the Paxinos andWatson atlas (1986) as follows: 23.3 mm anteroposterior and2.2 mm mediolateral referred to the bregma, and 22.9 mmdorsoventral referred to the exposed dura, targeting the mo-lecular layer of the hippocampus and ensuring a widespreadlesion of the whole formation. A delay of 5 min after theintroduction of the cannula allowed the tissue to recover.Each animal received a single 0.5-lL microinjection in theleft hippocampus with a CMA/100 microinjection pump(Carnegie Medicine, Sweden) at a rate of 0.125 lL/min. Theneedle was left for an additional 5 min to prevent spread ofthe excitotoxin up the cannula track on removal and was thenslowly withdrawn.
Before the AMPA experiments, a volume diffusion testwas performed in these conditions to ensure the appropriatelocalization of the treatments. Two rats received an intracere-bral microinjection of filtered 0.05% methylene blue in50 mM phosphate-buffered saline (PBS; pH 7.4). Two hoursafter the injection, the animals were killed by decapitation,and their brains were rapidly removed and frozen with pow-dered dry ice. Coronal 20-lm cryostat sections were mountedon untreated slides and stored at 2308C until use. Sectionswere postfixed for 30 min in 1.3% (w/v) picric acid and 4%(w/v) paraformaldehyde in 50 mM PBS (pH 7.4) and dried atroom temperature. When standard Cresyl violet staining wasperformed, the volume of diffusion was observed as anunstained spherical region limited to the hippocampus.
The dose–response experiment consisted of sevengroups, each with n 5 4. The doses of AMPA (Sigma, St.Louis, MO) were 0.27, 2.7, 5.4, 8.1, and 10.8 nmol in50 mM PBS (pH 7.4). The remaining rats were included inthe control and sham groups. The doses of AMPA wereselected according to previous studies (Petegnief et al., 1999;Andres et al., 2003), in which they showed their ability toinduce calcification and astroglial and microglial reactions.
The pharmacological study comprised 11 groups:2.7 nmol AMPA (n 5 7), 50 pmol TMB-8 (n 5 4; Sigma,St. Louis, MO), 10 pmol nimodipine (n 5 4; Quımica Farm-aceutica Bayer S.A., Barcelona, Spain), 7.5 nmol NBQX (n 54; Tocris Cookson Ltd., Bristol, UK), 2.7 nmol AMPA plus50 pmol TMB-8 (n 5 8), 2.7 nmol AMPA plus 10 pmolnimodipine (n 5 7), 2.7 nmol AMPA plus 50 pmol TMB-8plus 10 pmol nimodipine (n 5 8), 2.7 nmol AMPA plus7.5 nmol NBQX (n 5 7), 50 pmol TMB-8 plus 10 pmolnimodipine (n 5 3), 50 mM PBS, pH 7.4 (n 5 4), and a con-trol group (n 5 4). After the initial results, two more groupswere added to the pharmacological study: 8.1 nmol AMPA(n 5 8) and 8.1 nmol AMPA plus 5 pmol nimodipine (n 58). Nimodipine had been dissolved in 15% polyethylene gly-col and 15% ethanol (Bielenberg et al., 1990) to a workingconcentration of 50 lM and NBQX in NaOH 0.1M (pH9.5) to a working solution of 20 mM. Final doses werereached with 50 mM PBS (pH 7.4). Previous studies haveshown this vehicle has no effects (Petegnief et al., 2004). Inaccordance with Petegnief et al. (1999), we selected the doseof NBQX that blocked the formation of calcium deposits.The doses of TMB-8 and nimodipine were selected to havethe highest effectiveness and no significant cytotoxic effect incultures (Koh et al., 1995; Malcolm et al., 1996).
Fifteen days after the injections (Bernal et al., 2000a),the animals were killed by decapitation and their brains rapidlyremoved and frozen with powdered dry ice. One of two adja-cent coronal 12-lm cryostat sections was mounted onuntreated slides and stored at 2308C until use for histologicalor autoradiographical procedures.
Histological Staining
Sections were used for standard Nissl staining to ensurethe proper localization of the microinjection and to evaluatethe general tissue damage. Alizarin red S (Merck, Darmstadt,Germany) counterstained with Fast Green (Sigma, St. Louis,MO) was used to visualize calcium precipitates, according toMahy et al. (1999). These two procedures were applied to allthe animals. The lesion was quantified in a blinded fashion interms of affected area, atrophy of the whole hippocampus, andcalcified area by computer-assisted image analysis with a digitalcamera Kodak DCS 200 and OPTIMAS morphometric soft-ware (BioScan, Inc., Edmonds, WA) using 3–5 consecutivesections around the injection scar and extracting the meanvalue. Other specific histological techniques were applied, forexample, Perls method for iron, the solochrome azurinemethod for aluminum, and Congo red for amyloidosis, but nopositive staining was observed in any case.
In Vitro Autoradiography
The in vitro autoradiographic experiments were per-formed only in the pharmacological study, centered at theAMPA dose of 2.7 nmol. Astroglial reaction was studied byMonoamine oxidase B (MAO-B) autoradiography. MAO-Bwas labeled with [3H]lazabemide (a generous gift from Dr. G.Richards, Hoffmann-La Roche, Basel, Switzerland), asdescribed by Saura et al. (1995). A radioligand concentrationof 20 nM with a specific activity of 20.2 Ci/mmol and abuffer solution containing 50 mM Tris (pH 7.4), 120 mMNaCl, 5 mM KCl, 1 mM MgCl2, and 0.5 mM EGTA wereused. Incubation was performed at room temperature for90 min, followed by a 30 1 30 1 60 sec wash in 48C buffer.Nonspecific binding was determined in the presence of 1 lML-deprenyl (RBI, Natick, MA).
Microglial reaction was assessed by peripheral benzodia-zepine receptor (PBR) autoradiography (Saura et al., 1995).PBRs were labeled with [3H]PK-11195 (specific activity 85Ci/mmol; Du Pont-NEN, Boston, MA). Tissue sections wereincubated for 2 hr at room temperature in a 50 mM Trisbuffer (pH 7.7) containing 0.8 nM of the radiolabeled ligand(Rodrıguez et al., 2004). Thereafter, sections were rinsedtwice for 5 min each in buffer at 48C. For nonspecific bindingdetermination, the unlabeled compound (RBI, Natick, MA)was used at a concentration of 1 lM.
After drying under a stream of cold air overnight, theslides were apposed, along with plastic standards (3H-micro-scales, Amersham, Bucks, UK), to 3H-sensitive film (Hyper-film, Amersham) for 15 days. Film was developed and ana-lyzed densitometrically after calibration using a Kodak DCS200 digital camera and OPTIMAS software. Four sections perbrain for total binding quantification and two sections per
brain for nonspecific binding quantification were measured,selecting the anatomical areas of the hippocampus and the lat-erodorsal thalamic nucleus (LDT). The values obtained foreach rat after nonspecific binding subtraction were averagedand used for statistical comparisons.
Statistical Analysis
For each parameter, Kurtosis and Skewness momentswere calculated to test the normal distribution of the data.Then one-way ANOVA, followed by the least-significance-difference post hoc test, was used to compare differencesbetween groups. When normality was not reached, the valuesof all groups were compared using the nonparametric Krus-kal–Wallis (KW) test, followed by the Mann–Whitney U test.All results are expressed as means 6 SEM. Correlationbetween parameters was also studied. All analyses were per-formed with the Statgraphics computer program (STSC Inc.,Rockville, MD).
RESULTS
When a standard Nissl staining was performed inthe volume diffusion study, microinjection of 0.5 lLwas observed as an unstained spherical region within thehippocampus, suggesting a restricted affected area whendrugs were applied (data not shown).
Acute injection of AMPA in the dorsal hippocam-pus induced cell death, gliosis, and calcification in adose-dependent manner. Calcification appeared not onlyin the hippocampus but also in the LDT. The curves oftotal calcification and lesion area were saturable andshowed a plateau at the 5.4 nmol dose (Fig. 1a,b), incontrast with the data obtained in the globus pallidus insimilar conditions by Petegnief et al. (1999), which fol-lowed a nonsaturated curve (Fig. 1c). In the hippocam-pus, as in the sham-operated animals, the lowest dose ofAMPA, 0.27 nmol, produced no calcification and onlygliosis near the injection scar (Fig. 2A).
Injection of 2.7 nmol AMPA induced discrete neu-ronal loss and gliosis in CA1, whereas the dentate gyruswas well preserved. Calcium deposits were observed inthe CA1 region, mainly in the radiatum and lacunosummoleculare layers (mean area 0.46 6 0.12 mm2;Fig. 2B). Two animals presented calcification in themedial part of CA3 and the polymorph layer of the den-tate gyrus, and one of them also showed calcification inthe LDT (2.59 3 1023 mm2). Calcium deposits appearedas small, red concretions, isolated or in tightly packed clus-ters, with an apparent mean diameter of 46 0.5 lm.
When 5.4 nmol AMPA was injected, the lesionshowed strong gliosis that affected most of the hippo-campus and the dentate gyrus and part of the LDT.Only the molecular layer of the dentate gyrus main-tained an apparently normal cellular density. The loss ofNissl-stained neurons was also evident in the pyramidaland granular layers. Necrosis was observed in theinjected area. Calcification was more extended than withthe previous doses, affecting the same regions as thelesion, with 0.48 6 0.11 mm2 of calcium deposits in the
1242 Bernal et al.
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hippocampus and dentate gyrus and 0.18 6 0.02 mm2
in the LDT (Fig. 2C). Unidentified translucent bodies,visible without staining under an optical microscope,were observed in the CA1 and CA3 pyramidal layers
and in the granular layer of the dentate gyrus. Theywere strongly stained with Fast Green but not with Aliz-arin red, Nissl, or other specific stains like the Perlsmethod for iron, Congo red for amyloidosis, or solo-chrome azurine for aluminum, indicating the absence ofmetals other than calcium.
AMPA at a dose of 8.1 nmol induced the maximaleffects in terms of total area of calcification. The wholehippocampus, the dentate gyrus, and the LDT showedintense gliosis. There were also signs of necrosis and at-rophy of the injected area. In all animals, calcificationaffected the whole hippocampus and part of the dentategyrus (0.42 6 0.1 mm2), and densely covered the LDT(0.33 6 0.04 mm2; Fig. 2D).
A wider necrosis was associated with the highestdose, 10.8 nmol AMPA, which also led to atrophy ofthe hippocampal structure. The area of calcification wasreduced in the hippocampus (0.32 6 0.08 mm2) butunchanged in the LDT (0.29 6 0.04 mm2), whichshowed no necrosis. Several large deposits (10–20 lm)and Fast Green–positive bodies were present in thepyramidal layer of CA1 (Fig. 2E).
In one animal, 8.1 nmol AMPA also inducedwidespread calcification from the frontal nuclei to thecaudal piriform cortex (Fig. 2F). Calcium precipitateswere present bilaterally in the anterior olfactory nucleus,as well as in the tecta tenia, the accumbens nucleus nearthe anterior commissure, the bed nucleus of the stria ter-minalis, the lateral septal nucleus, and in several specificmediodorsal thalamic nuclei. Calcification in the wholeipsilateral hippocampus was observed along with intensenecrosis and atrophy. Contralateral calcification was alsodetected, mainly in the CA1 and CA3 oriens and radia-tum layers of the hippocampus, but also in the piriformcortex, the entorhinal cortex, and several basolateralamygdaloid and amygdalohippocampal nuclei. When thehippocampi extended ventrally, the calcium depositsremained. No calcification was observed in other areassuch as the globus pallidus or the pineal gland, whichare frequently calcified in rodent and human brains.Intense Fast Green–stained bodies were also detected,mostly in neuron-rich areas, for example, the pyramidallayers of both hippocampi and the contralateral piriformcortex. Nissl staining revealed general gliosis and neuro-nal loss that matched the calcified areas. As the treat-ments included in this study were expected to enhanceor to reduce the calcification process, 2.7 nmol AMPAwas selected as the dose yielding 60% of the maximumresponse.
Fig. 1. Comparison of hippocampal and globus pallidus results fromAMPA dose–response study. Hippocampal lesioned area (a) and hip-pocampal calcified area curves (b), in mm2, showing the plateau ofthese parameters at higher doses. c: Logarithmic transformation ofcalcified area values in the hippocampus (l) compared with the sameparameter in the globus pallidus (*), after Petegnief et al. (1999).Data are represented as mean 6 SEM *P < 0.05 when comparedwith the AMPA maximal effect.
Like in sham-operated rats, intrahippocampal injec-tion of NBQX, TMB-8, nimodipine, or a combinationof the last two did not induce calcium deposits or visu-ally detectable histological alteration of the tissue. Allfive 2.7 nmol AMPA-injected groups combined or notwith drugs presented gliosis, neuronal death, and calcifi-cation to different extents. The hippocampal area in allthese AMPA-lesioned groups was significantly differentfrom that of sham-operated animals (25% mean atrophy,P 5 0.03, data not shown) but was similar among theAMPA-lesion groups.
Compared with 2.7 nmol AMPA, 2.7 nmolAMPA plus NBQX treatment significantly reduced thelesioned area (40% reduction, Fig. 3A), as well as thecalcified area, which reached the sham value (84%reduction; Figs. 3B and 4a–c). Combined injection of2.7 nmol AMPA plus nimodipine did not modify the
lesioned or calcified areas when compared with theAMPA-injected group (Fig. 3B) and affected the samehippocampal areas, namely, most of CA1 and part of thepolymorph layer of the dentate gyrus (Fig. 4d,f).
The dose of 2.7 nmol AMPA plus TMB-8 did notsignificantly increase the 2.7 nmol AMPA-lesioned area(Fig. 3A). The lesion was characterized by a slight ne-crosis near the injection site, neuronal loss, and stronggliosis within most of the hippocampal formation. Incontrast, the calcification area was higher than that pres-ent in any of the other four groups lesioned withAMPA (148% versus the AMPA group, KW 5 37,055,P < 0.00001; Fig. 3B) and reached an extension similarto that of the 8.1 nmol dose-response AMPA-injectedgroup. Calcification was observed in the whole hippo-campus, including the CA3, the polymorph layer of thedentate gyrus, and the LDT, with an area of 0.73 6
Fig. 2. Alizarin red staining of AMPA dose–response study showingdistribution of calcium deposits in hippocampus. Images correspondto (A) 0.27 nmol, (B) 2.7 nmol, (C) 5.4 nmol, (D) 8.1 nmol, and(E) 10.8 nmol AMPA-operated animals. Laterodorsal thalamic nu-cleus calcification is already visible after microinjection of 5.4 nmolAMPA. F: Section near the injection level of the 8.1 nmol AMPA-lesioned animal showing widespread calcification. Calcification, ne-crosis, and atrophy of ipsilateral hippocampus (white arrowhead), as
well as calcification of contralateral hippocampus (black arrowhead)can be observed. Calcifications were present in other structures, suchas the ipsilateral laterodorsal thalamic nucleus (LDT), the bilateralmediodorsal thalamic nuclei (MDT) and dorsal hypothalamic areas(DHA), and the contralateral basolateral amygdaloid nucleus (BLA)and piriform cortex (Pir). Scale bar 5 1.25–2 mm (A–E). [Color fig-ure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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Journal of Neuroscience Research
0.07 mm2 (Fig. 4e). As also observed in the 8.1 nmolAMPA-injected group, one animal showed widespreadcalcification that included frontally the tenia tecta, theanterior olfactory, and the lateral septal nuclei and bilat-erally the reticular thalamic area, the ventral reuniensnucleus, both hippocampi, and the LTD. The piriformcortex and amygdaloid nuclei were calcified only con-tralaterally. Fast Green–positive bodies were present inthe lesioned area, even in absence of calcification. Whennimodipine was added to the 2.7 nmol AMPA plusTMB-8 coinjection, the TMB-8 potentiation of theAMPA lesion was reversed, and no difference fromthe AMPA-injected group was observed for either thelesioned or the calcified area. Finally, when a correlationstudy of lesioned and calcified areas was performed inthe treated groups, a highly significant positive correla-tion was found (r 5 0.83, P < 0.0001).
To investigate whether nimodipine’s effects dependon the presence of TMB-8, two additional groups ofrats were lesioned, with 8.1 nmol AMPA and with 8.1nmol AMPA plus 5 pmol nimodipine. No differencesbetween these two groups in the calcified and lesionedareas were observed (data not shown).
To study the astroglial reaction, [3H]lazabemide au-toradiography was performed. In agreement with previ-ous data (Bernal et al., 2000b), the excitotoxic lesion ofthe hippocampus significantly increased [3H]lazabemidebinding by almost 26% when compared with that insham-injected rats, even in the AMPA plus NBQXgroup (Fig. 5A). The distribution was spatially heteroge-neous, with the molecular layer of the dentate gyrusalmost unaffected in all groups. Within the five AMPA-injected groups, only the ones coinjected with eithernimodipine or TMB-8 presented a significantly highernumber of binding sites. LTD [3H]lazabemide bindingremained unchanged.
To study the microglial reaction, [3H]PK-11195autoradiography was performed. When compared withthat in the sham-operated animals, hippocampal AMPAmicroinjection produced an increase in [3H]PK-11195binding sites that colocalized with calcification of almost500% (Fig. 5B). Injection of NBQX, nimodipine,TMB-8, or a mixture of the last two did not produceany differences with the control or sham values. Com-pared with that in the AMPA group, coinjection ofNBQX reduced the number of [3H]PK-11195 bindingsites by 36% but was still 320% of the sham value.AMPA coinjection with nimodipine and/or TMB-8gave [3H]PK-11195 binding values similar to thoseobtained after AMPA injection alone. Compared withthat in the control groups, when [3H]PK-11195 bindingwas measured in the LDT, a significant mean increase of190% was observed in all groups.
When a correlation study was performed, only[3H]PK-11195 autoradiography had an excellent correla-tion with the lesioned area (r 5 0.91, P < 0.0001) and agood correlation with the calcified area (r 5 0.69, P <0.0001). No good correlation was found with hippo-campal atrophy data.
Fig. 3. A: Hippocampal lesioned area representation (mm2) of thepharmacological study showing the effect of nimodipine on 2.7 nmolAMPA plus TMB-8 treatment. B: Total calcified area representation(mm2), including hippocampus and laterodorsal thalamic nucleus, ofthe pharmacological study [Nim, nimodipine; *significant difference(P < 0.05 with sham-operated animals; �significant difference (P <0.05) with respect to 2.7 nmol AMPA-lesioned rats; #significant dif-ference between 2.7 nmol AMPA plus TMB-8 and 2.7 nmol AMPAplus TMB-8 plus nimodipine; data are the mean 6 SEM].
Our study demonstrates that fostering glutamate re-ceptor activation by increasing doses of AMPA producesdose-dependent neuronal loss and calcification in thehippocampus. Our data also provide evidence that theextent of the lesioned area and calcification reached pla-teaus at 5.4 nmol, in opposition to the results of a similarstudy done in the globus pallidus characterized by a non-saturable process and a focal necrotic zone at the highestdoses (Petegnief et al., 1999).
A direct, unequivocal association between AMPAreceptor activation and calcification was found in thehippocampus but not in the globus pallidus, in whichadditional specific temporal or spatial factors would alsodetermine calcification. The hippocampal calcificationpattern showed isolated clusters of small deposits, differ-ent from the dense small and large deposits of the globuspallidus organized in a concentric mode extending pro-gressively to neighboring areas (Saura et al., 1995; Peteg-nief et al., 1999; Bernal et al., 2000b). This contrastswith the similar diameters of deposits formed in brainareas of rodents and humans suffering from several path-ophysiological processes (Saura et al., 1995; Rodrıguezet al., 2001; Ramonet et al., 2002, 2006). This hippo-campal specificity may underlie differences in the tightmechanisms that control astroglial and neuronal destabili-zation of Ca21 homeostasis, with the capacity of cyto-
plasmic Ca21 sequestration to inversely correlate in eachbrain area with the extension of calcification. For exam-ple, together with a higher number of glutamate recep-tors (Young et al., 1991; Hollmann and Heinemann,1994), the rat hippocampus shows higher neuronal andastrocytic density than the globus pallidus. Thus, anAMPA hippocampal microinjection and, by extension,an ischemic episode will acutely activate AMPA recep-tors in a larger number of cells, leading to a degenerativeprocess that calcifies the entire area. Crucial factors mayalso be the heterogeneity of the astroglial compartmentevidenced in the hippocampus by its high susceptibilityto a-aminoadipic acid–induced astroglial ablation(Rodrıguez et al., 2004) and by the differences betweenneuronal and glial capacity to buffer Ca21 (Saura et al.,1995; Bernal et al., 2000a; Sapolsky, 2001).
We also demonstrate the hippocampal similarity of8.1 nmol AMPA and 2.7 nmol AMPA plus 5 pmolTMB-8 widespread calcification in the same brain areasthat degenerate after systemic or intrahippocampal kainic(Sperk, 1994) and quisqualic acid injections (Jarrard andMeldrum, 1993). As most of the distal calcified areasreceive direct efferents of the hippocampal formation,this process may rely on a transsynaptic excitatory effect.In a similar way, LDT calcification observed at AMPAdoses higher than 2.7 nmol may result from the far-rang-ing loop of projections extending from the hippocampus
Fig. 4. Alizarin red staining in the pharmacological study showing intensity of calcification whenthe various drugs were coinjected with AMPA. The images correspond to (a) sham-operated ani-mals, (b) 2.7 nmol AMPA-operated rats, (c) AMPA plus NBQX, (d) AMPA plus nimodipine, (e)AMPA plus TMB-8, and (f) AMPA plus TMB-8 plus nimodipine. Scale bar 5 1.25 mm. [Colorfigure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
1246 Bernal et al.
Journal of Neuroscience Research
to the thalamus. As the volume of injection is restrictedto the hippocampal area, we can discard the toxin diffu-sion in this distal process, except at the highest dose, inwhich slow AMPA diffusion might progressively reachthe thalamus. In these two rats with widespread calcifica-tion, the absence of calcification in the globus pallidus,close to the calcified thalamic nuclei and without directhippocampal connection, also favors a distal transsynapticeffect.
The 2.7 nmol AMPA lesion indicates that neuronaldeath is associated with hippocampal atrophy, calciumprecipitates, and astroglial and microglial reactions. Allparameters but the astroglial one were reduced or sup-pressed by NBQX. Microglial reaction has been directlyassociated with the formation of calcium deposits in neu-rons and astrocytes, whereas the astroglial reaction islinked to neuronal degeneration (Saura et al., 1995; Tac-coni, 1998). Maintenance of astrogliosis in AMPA plusNBQX treatment suggests that calcification requires a setpoint of neuronal suffering. In similar conditions, the50% reduction in astrogliosis characterized in the globuspallidus argues for astroglial heterogeneity between thetwo areas, although differences in the affinity to NBQXcannot be discarded as an explanation (Porter andGreenamyre, 1994).
A major result of this study is the finding of re-gional differences in nimodipine effectiveness during anexcitotoxic episode, at least when the hippocampus andthe globus pallidus are compared. Nimodipine and otherDHPs like nifedipine and nitrendipine, used to reducedamage in a large number of calcium-mediated excito-toxic processes, have shown contradictory results(Frandsen and Schousboe 1993; Rami and Krieglstein,1994; Berg et al., 1995; Small et al., 1997; Fryer et al.,1999; Horn et al., 2001). The direct acute intracerebralcoinjection and the low nimodipine concentration usedin our study were chosen to ensure a specific localizedaction with a final concentration similar to that from pe-ripheral administration. The lack of 5 pmol nimodipineinhibition of AMPA-induced hippocampal neurodegen-eration that our results showed suggests that in theseconditions, Ca21 entry via L-type channels is notdirectly involved. We have demonstrated that nimodi-pine reversed the TMB-8 enhanced AMPA excitotoxic-ity in both calcified and lesioned area parameters. Block-ade of IP3 receptors with TMB-8 (Fellner andArendshorst, 2008) results in misregulation of calciumhomeostasis and unveils the participation of L-type cal-cium channels. L-type calcium channels are primarilyactivated by depolarization, but a large number of factorsregulate their activity. Thus, TMB-8 potentiation of2.7 nmol AMPA action would indirectly enhance pro-tein kinase C activation, leading to added positive mod-ulation of L-type channels and negative modulation ofN-type channels. The lack of TMB-8 effectiveness inthe globus pallidus (Petegnief et al., 2004) may be aresult of the 4 times lower level of IP3 receptors in thisarea compared with that in the hippocampal stratumradiatum corresponding to the injection site (Fotuhi
Fig. 5. Histograms of (A) monoamine oxidase B (MAO-B) concen-tration (pmol/mg protein) and (B) peripheral benzodiazepine recep-tor (PBR) concentration (fmol/mg protein) measured in hippocam-pus and laterodorsal thalamic nucleus (LDT) after the pharmacologi-cal treatments [Nim, nimodipine; *significant difference (P < 0.05)in hippocampus with respect to sham-operated animals; significantdifferences (P < 0.05) in both anatomical areas with respect to sham-operated animals; �significant differences (P < 0.05) in hippocampuswith respect to AMPA-lesioned rats; data are given as the mean 6SEM].
et al., 1993). That nimodipine did not modify the effectsof 8.1 nmol AMPA is also consistent with the involve-ment of a common pathway in TMB-8 and nimodipineactions that is not directly activated by low or high dosesof AMPA.
In addition, in the activated excitotoxic process,direct calcium influx through acutely activated AMPAreceptors or indirect NMDA receptor activation(Rothman and Olney, 1995) cannot be ruled out.Another possibility is the essential participation of othervoltage-dependent calcium channels, such as N- or P/Qtypes, as shown in hypoxia models (Small et al., 1997),whose activation would be potentiated by impairment ofglial glutamate transporters (Bogaert et al., 2001; Yehet al., 2005; De Yebra et al., 2006).
Further studies are needed to better understand themechanisms involved in the nimodipine hippocampalneuroprotective effect, such as the role of the L-type-related calmodulin-MAPK pathway (Dolmetsch et al.,2001). Nevertheless, our results provide evidences thatL-type calcium channel activity participates in the differ-ences between globus pallidus and hippocampal neuro-degeneration associated with differences in extra- and in-tracellular calcium movement. Future work focusing onother brain areas and rodent models will ultimatelyincrease our knowledge about the role of calcium storesin the human neurodegenerative diseases and explain thecontroversial effectiveness of calcium channel antagonistsin experimental and clinical assays.
ACKNOWLEDGMENTS
The authors thank Dr. J. G. Richards (Hoffmann-LaRoche, Basel, Switzerland) for the generous gift of[3H]lazabemide and Ricard Canadell for the volume dif-fusion study.
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