the neuroprotective agent memantine induces brain-derived neurotrophic factor and trkb receptor...

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Molecular and Cellular Neuroscience 18, 247–258 (2001)

doi:10.1006/mcne.2001.1027, available online at http://www.idealibrary.com on MCN

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The Neuroprotective Agent Memantine InducesBrain-Derived Neurotrophic Factor andtrkB Receptor Expression in Rat Brain

Marketa Marvanova,* Merja Lakso,* Jarmo Pirhonen,†

Hiroyoki Nawa,‡ Garry Wong,* and Eero Castren* ,§,1

*A. I. Virtanen Institute and §Department of Psychiatry, University of Kuopio, 70211uopio, Finland; †Center for Scientific Computing, P.O. Box 405, Espoo, 02101, Finland;

and ‡Department of Molecular Biology and Neurobiology, Brain Research Institute,Niigata University, Niigata 951-8585, Japan

Memantine is a medium-affinity uncompetitive N-methyl-D-aspartate receptor antagonist and has been clinicallyused as a neuroprotective agent to treat Alzheimer’s andParkinson’s diseases. We have examined the effect ofmemantine (ip 5–50 mg/kg; 4 h) on the expression ofbrain-derived neurotrophic factor (BDNF) and trkB recep-tor mRNAs in rat brain by in situ hybridization. Memantineat a clinically relevant dose markedly increased BDNFmRNA levels in the limbic cortex, and this effect was morewidespread and pronounced at higher doses. Effects ofmemantine on BDNF mRNA were also reflected inchanges in BDNF protein levels. Moreover, memantineinduced isoforms of the BDNF receptor trkB. Taken to-gether, these data suggest that the neuroprotective prop-erties of memantine could be mediated by the increasedendogenous production of BDNF in the brain. These find-ings may open up new possibilities of pharmacologicallyregulating the expression of neurotrophic factors in thebrain.

INTRODUCTION

Memantine (1-amino-3,5-dimethyladamantane hy-drochloride) is a medium-affinity uncompetitive antag-onist of the N-methyl-d-aspartate (NMDA) subtype ofthe glutamate receptor. It has therapeutic potential innumerous conditions that include central nervous sys-

1 To whom correspondence and reprint requests should be ad-ressed at A.I. Virtanen Institute, University of Kuopio, P.O. Box
627, 70211 Kuopio, Finland. Fax: 1358-17-163030. E-mail:[email protected].
1044-7431/01 $35.00Copyright © 2001 by Academic Press

ll rights of reproduction in any form reserved.

tem (CNS) disorders (Ditzler, 1991; Gortelmeyer andErbler, 1992; Weller and Kornhuber, 1992; Kornhuber etal., 1994; Parsons et al., 1999; Skolnick, 1999). Memantineis considered to be a neuroprotective agent for thetreatment of several dementias, particularly Alzhei-mer’s disease (AD) (Gortelmeyer and Erbler, 1992;Danysz et al., 1995; Muller et al., 1995; Parsons et al.,1998). Besides dementias in the elderly, during the past10 years this drug has been used for the treatment ofParkinson’s disease (PD) (Kornhuber and Weller, 1997;Danysz et al., 1997), a disease that is caused by the lossof dopaminergic neurons located in substantia nigrapars compacta (SNC) (Lang and Lozano, 1998a and b).Since neurodegeneration in Parkinson’s disease is aprogressive process, and a significant proportion of thenigral dopaminergic neurons persist even in more se-vere states of the disease (McGeer et al., 1988; Fearnleyand Lees, 1991), it has been suggested that agents thatpromote survival and functional recovery of affecteddopaminergic neurons would have a significant thera-peutic value.

Members of the neurotrophin gene family (Lewinand Barde, 1996), and particularly brain-derived neuro-trophic factor (BDNF), exert trophic and protective ef-fects on dopaminergic neurons (Beck, 1994; Hefti, 1994,1997; Connor and Dragunow, 1998) as well as otherneuronal systems (Knusel et al., 1991; Ghosh et al., 1994;Saaralainen et al., 2000). Moreover, markedly decreasedlevels of neurotrophins, such as BDNF and nerve
growth factor (NGF), have been observed in the nigro-striatal dopamine regions (Howells et al., 2000; Nagatsu
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et al., 2000) of PD patients and in the hippocampus ofD patients (Phillips et al., 1991; Siegel et al., 2000).hus, BDNF has been suggested as a possible therapeu-

ic candidate for treatment of several neurodegenera-ive diseases, such as AD and PD.

BDNF is widely expressed in the CNS with a rela-ively abundant expression in several brain regions,ncluding hippocampus and cerebral cortex (Ernfors etl., 1990; Hofer et al., 1990; Phillips, 1991; Castren et al.,

1995), and its expression is dynamically regulated byphysiologically relevant neuronal activity (Castren etl., 1992; Lindholm et al., 1993; Thoenen, 1995). BDNFas been shown to promote the survival and differen-

iation of mesencephalic dopaminergic neurons in vitroHyman et al., 1991; Knusel et al., 1991) and in vivo (Shen

et al., 1994; Shults et al., 1994; Hagg, 1998) as well as inther neuronal systems, such as cortical (Ghosh et al.,994; Saaralainen et al., 2000) and cholinergic neuronsKnusel et al., 1991). Furthermore, application of exog-nous BDNF supports the survival of dopaminergiceurons in models of Parkinson’s disease (6-OH dopa-ine and MPTP toxicity models) both in vitro (Spina et

l., 1992a,b) and in vivo (Altar et al., 1992; Shults et al.,995). From these data it has been suggested that thencrease of production of endogenous BDNF might pro-ect vulnerable neurons and promote their survival dur-ng neurodegenerative processes.

BDNF mediates its functions through trkB tyrosineinase receptors (Barbacid et al., 1994; Lewin and Barde,996). trkB is alternatively spliced into several differentsoforms, including full-length tyrosine kinase-contain-ng isoform (trkB.TK1) and two truncated formstrkB.T1 and T2) which lack the tyrosine kinase domainnd contain a short, unique, intracellular tail (Klein etl., 1990; Middlemas et al., 1991). Since tyrosine kinaseomain dimerization and transphosphorylation areecessary to mediate BDNF signaling, truncated recep-

ors expressed in the same cells act as dominant nega-ive inhibitors of trkB signaling (Eide et al., 1996;

inkina et al., 1996; Li et al., 1998; Haapasalo et al., 2001).Here we describe our studies of the effects of an acute

dministration of memantine on the expression ofDNF mRNA and protein, as well as trkB mRNA levels

n adult rat brain, to test a hypothesis that the neuro-rotective effect of clinically relevant low doses of me-antine might be mediated by promotion of endoge-

ous synthesis of BDNF in the brain. Our dataemonstrating an increased production of BDNF elic-

ted by memantine in rat brains may provide a partial
xplanation for the neuroprotective effect of this agentn AD and PD treatments. On the other hand, toxico-
ogically relevant high doses ($50 mg/kg) cause signif-cant increases in BDNF mRNA and protein levels inhe cingulate and retrosplenial cortices, which may beelated to neurotoxic effects of the substance at the sameigh sublethal doses (Olney et al., 1989, 1991).

RESULTS

Effect of Acute Memantine Treatment on BDNFmRNA Expression

To reveal possible effects of an acute treatment withmemantine on the expression of BDNF mRNA levels, insitu hybridization was performed on brain coronal sec-tions of rats treated with saline (control) and meman-tine (ip 5–50 mg/kg, 4 h) using a rat BDNF oligonucle-otide probe. Representative in situ hybridizationautoradiograms are shown (Fig. 1). For better visualidentification of regions, 3-D transparent images werereconstructed from the 2-D autoradiograms of saline-treated (Figs. 2A–2C) and memantine-treated (50 mg/kg, 4 h) (Figs. 2D–2F) rat brains.

High doses of memantine were found to increaseBDNF mRNA levels in all cortical areas measured, withthe highest BDNF expression in the limbic cortex, espe-cially in the retrosplenial, entorhinal, and cingulate cor-tices. The increase in BDNF mRNA was also found inhippocampal areas [dentate gyrus (DG), CA1, CA2,CA3], thalamic and hypothalamic nuclei, and the dopa-minergic regions including substantia nigra and ventraltegmental area. A decrease of BDNF mRNA levels wasfound in both molecular and granular layers of theolfactory bulb (autoradiograms are not shown). Striatalexpression of BDNF mRNAs was not detectable in ei-ther saline- or memantine-treated brains.

Dose Response and Time Course of BDNF mRNAInduction by Memantine

Quantitative analysis of in situ autoradiograms wasperformed on rats treated for various periods of time(1–48 h) and with different doses (5–50 mg/kg) ofmemantine (Fig. 3). All quantified cortical layersshowed similar dose responses except layers III, V, andVI of parietal cortex, which revealed a decrease ofBDNF mRNA expression levels at a dose of 10–50mg/kg compared with increased levels at 5 mg/kg(Fig. 1).

The lowest dose of memantine (ip 5 mg/kg, 4 h) wasable to significantly increase BDNF mRNA levels in

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249Memantine Induces BDNF and trkB mRNA in Rat Brain

parietal, cingulate, and retrosplenial cortices (Figs. 1and 3). With higher doses of memantine (ip 10–50 mg/kg, 4 h), BDNF mRNA was also induced in other brainregions as shown (Figs. 1 and 3). A significant decreaseof BDNF mRNA level was observed in the hippocampaldentate gyrus region after memantine (10 mg/kg) treat-ment.

FIG. 1. The effect of memantine administration on the expressionhybridization are shown. The rats were ip injected with saline or memsections were hybridized by in situ hybridization (ISH) with a BDNF9 days. Abbreviations used: cingulate (Cg), parietal (Par), retrospleubstantia nigra (SN), ventral tegmental area (VTA), and striatum (C

Peak induction of BDNF mRNA was observed incingulate and parietal cortices at 4 h and in frontal

cortex at 4 and 8 h after administration of memantine.mRNA levels of all examined areas returned to basallevel by 24 h (Fig. 4).

Effect of Memantine on BDNF Protein Expressionin the Retrosplenial Cortex

DNF mRNA in rat brain. Representative autoradiograms of in situe (5–50 mg/kg) and sacrificed 4 h later. Fourteen-micrometer coronalbeled oligonucleotide probe. Exposure time for autoradiograms wasRS) cortices, hippocampal [dentate gyrus (DG), CA1, CA3] regions,

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An enzyme-linked immunosorbent assay (ELISA)was used to determine whether BDNF protein levels

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were influenced by memantine treatment (ip 25 and 50mg/kg, 6 h). BDNF protein levels were significantlyincreased at the highest dose (Fig. 5).

Effect of Memantine Treatment on trkB mRNAExpression

We studied the effects of memantine (ip 5–50 mg/kg,4 h) administration on the mRNA expression patternsof two trkB receptor isoforms: trkB.TK1 and trkB.T1(Fig. 6A). Representative autoradiograms for treated (ip25–50 mg/kg, 4 h) and saline (control) sections areshown (Fig. 6B). Quantitative analysis revealed that thehighest concentrations of memantine significantly in-creased trkB.TK1 mRNA levels in the DG and CA1regions and in the striatum (CPu) (Fig. 7A), whereas

FIG. 2. Three-dimensional brain image reconstruction of BDNF mRNTransparent 3-D brain reconstructions from saline (A–C) and memangraphic images of coronal sections representing BDNF mRNA expres(RS), entorhinal (Ent) cortices, hippocampus (Hipp), and olfactory bu

trkB.T1 was also significantly elevated in the frontaland retrosplenial cortices (Fig. 7B).

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DISCUSSION

In this study, we have investigated the possibilitythat memantine regulates the expression of neurotro-phins. We have focused on BDNF and its receptor trkBbecause it has been shown that BDNF through its re-ceptor (trkB.TK1) elicits neuroprotective effects on do-

aminergic neurons in vitro (Hyman et al., 1991; Knuselt al., 1991) and in vivo (Shen et al., 1994; Shults et al.,994; Hagg, 1998) as well as other neuronal systemsKnusel et al., 1991; Ghosh et al., 1994; Saaralainen et al.,000).In the present study, we observed that BDNF mRNA

evels were increased in response to the neuroprotectiverug memantine in many brain regions. The lowest

pression from saline- and memantine- (ip 50 mg/kg, 4 h) treated rats.(50 mg/kg) treated (D–F) brain were computed from 2-D autoradio-The perspective views are as indicated: cingulate (Cg), retrospleniallf).

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ose used (ip 5 mg/kg, 4 h) was able to significantlyncrease the levels of mRNA in limbic cortical regions. It

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is important to note that the peak concentrationsachieved after intraperitoneal injection of 5.0 mg/kg ofmemantine in rats are comparable to the clinically rel-evant steady-state plasma concentrations in patientstreated with memantine (Danysz et al., 1997; Parsons etal., 1999) and are essentially devoid of many of the sideeffects associated with high-affinity NMDA channelblockers, such as PCP and MK-801 (Kornhuber et al.,1997; Parsons et al., 1999). This suggests that BDNFmRNAs may be increased by clinically relevant doses ofmemantine.

Higher doses of memantine (ip 10–50 mg/kg, 4 h)revealed more widespread increases in BDNF mRNAexpression in rat brain. The highest mRNA density wasfound in the cingulate, retrosplenial, and entorhinalcortices, which belong to the limbic cortex. Further-more, increases in BDNF mRNA levels were found indopaminergic brain areas, the ventral tegmental area(VTA), the substantia nigra (SN), and the thalamus. Inagreement with previous studies (Castren et al., 1995;

FIG. 3. Region-specific induction of BDNF mRNA expression by merain regions are shown. The rats were injected with saline or meman

treated with saline and different doses of memantine were hybridizedsignal of the BDNF mRNA was quantified from olfactory bulb (Olf),hippocampal [dentate gyrus (DG), CA1, CA3] regions, the substantiaData expressed as percentages of saline (100% represents the mean

btained from four to nine brains in each treatment group. Significa*P , 0.01; *P , 0.05; ANOVA followed by Dunnett’s post-hoc test. V

(layer III).

Conner et al., 1997) we did not find any alterations inexpression of BDNF mRNA in the striatum before or

after memantine administration. However, cell bodiesof neurons from the cortical, nigral, and thalamic neu-ron groups that project to the striatum contain highlevels of BDNF mRNAs (Phillips et al., 1990; Seroogyand Gall, 1993; Lindsay, 1994). Additionally, it has beensuggested that BDNF protein undergoes anterogradetransport by axons to the terminals of BDNF-expressingneurons (Altar et al., 1997; Conner et al., 1997; Altar andDiStefano, 1998), and BDNF protein is widely distrib-uted in nerve terminals, even in the brain areas such asthe striatum that lack BDNF mRNA (Altar et al., 1997).It is therefore possible that increases in BDNF mRNAproduction in the cortex, substantia nigra, and thalamuscaused by memantine are also able to influence striatalneurons.

Additionally, a large increase in BDNF mRNA ex-pression was found in the retrosplenial and entorhinalcortices as well as in dentate gyrus after ip injection ofthe highest dose of memantine (50 mg/kg) used in thisstudy. It is important to emphasize that this dose of

tine in the rat brain. The histograms from the quantitative analysis of5–50 mg/kg) and sacrificed 4 h later. Coronal brain sections from rats

a BDNF 33P-labeled oligonucleotide probe. The in situ hybridizational [frontal (Fr), parietal (Par), cingulate (Cg), retrosplenial (RS)], and

a (SN), and striatum (CPu) by the MCID/M4 image analysis system.hybridization signal of rats treated with saline) are means 6 SEM

fferences between saline- and memantine-treated rats are indicated:s shown are from medial layers (layer IV) except retrosplenial cortex

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memantine induces about 20-fold higher peak plasmaconcentrations than the dose used in clinical settings to

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treat AD as well as PD (i.e., 20 mg/day final doseachieved by weekly increments of 5 mg/day). Sincetolerance of memantine is dose dependent in humans aswell as in rats, the massive increase of BDNF message in

FIG. 4. The time course of memantine effect on BDNF mRNA ex-ression. The rats were ip injected either with saline and sacrificedfter 4 h or with memantine (25 mg/kg) and sacrificed 1, 4, 8, 24, or8 h later. Optical densities of BDNF mRNA expression were quan-ified by a MCID/M4 analysis system from frontal cortex (A), cingu-ate cortex (B), and parietal cortex (C). Values expressed as percent-ges of saline are means 6 SEM from three rats in each treatment

group. Significant differences between saline- and memantine-treatedrats are indicated: **P , 0.01; *P , 0.05; ANOVA, Dunnett’s post-hoctest.

several brain regions induced by a 50 mg/kg dose mayreflect a neurotoxic response. This response is reminis-

cent of those seen following administration of high-affinity uncompetitive NMDA receptor antagonists.

Memantine increased the production of BDNF pro-tein as well as mRNA levels in the retrosplenial cortex,although the induction of protein was less pronounced.BDNF mRNA levels are typically induced to a higherextent than protein levels. The levels of induction ob-served here are reminiscent of the induction of BDNFmRNA and protein levels in the hippocampus by MK-801 treatment, another noncompetitive NMDA receptorantagonist. Although this treatment increases BDNFmRNA levels up to 20-fold (Castren et al., 1993; Lindenet al., 2000), only a 1.4-fold induction in BDNF proteinhas been observed with MK-801 (Linden et al., 2000). Asignificant increase of BDNF protein levels was foundonly at the highest dose of memantine. This suggeststhat altered BDNF protein levels may be related to toxicside effects, which become apparent at the dose 50mg/kg and above. Alternatively, BDNF is a stable pro-tein even when taken up by neurons and glial cellsthrough receptor-mediated internalization and retro-grade transport (Rubio, 1997; Watson et al., 1999), andhe protein levels measured represent the total pool ofDNF in all cellular compartments. Therefore, even ifDNF protein levels in this total pool do not change, it

s still possible that BDNF protein is being locally re-eased and interacting with the receptors.

trkB mRNA levels are often increased concomitantlyith increased production of BDNF mRNA in the brain

Merlio et al., 1993; Ferrer et al., 1998). A similar regu-ation of full-length and truncated isoforms in the adult

FIG. 5. Expression of BDNF protein after memantine treatment.Rats were injected with memantine (ip 25 or 50 mg/kg) for a 6-h timeperiod, and protein from the dissected retrosplenial cortex was ob-tained and analyzed with an ELISA. Values are expressed as percent-ages of saline. Data shown are means 6 SEM from three rats per-
formed in triplicate. *P , 0.05, significantly different compared tosaline-treated rats (ANOVA, Dunnett’s post-hoc test).

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rat brain after memantine treatment was observed. Thelarge increase of trkB.TK1 and trkB.T1 isoform levels inhe DG might reflect morphological changes in hip-ocampal neurons after administration of the highestose of memantine. The differential induction of trkB

FIG. 6. Expression of mRNA of TrkB receptor isoforms after me-mantine treatment. (A) Schematic pictures of receptor isoformstrkB.TK1 and trkB.T1. (B) Brain coronal sections from rats treated

ith saline and memantine (ip 5–50 mg/kg) were hybridized with aDNF 33P-labeled oligonucleotide probe by in situ hybridization. Rep-esentative in situ hybridization autoradiograms are shown (bregma,.8 mm). Symbols used are identical to those in Fig. 1.

soforms in the different brain areas may be due to theact that expression of trkB.TK1 mRNA is restricted to

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neurons whereas trkB.T1 is expressed in neurons aswell as glial cells (Frisen et al., 1993; Armamini et al.,1995; Biffo et al., 1995).

The mechanism through which the levels of endoge-nous BDNF are influenced by memantine in rat brain isunclear. Since memantine acts as an antagonist of theNMDA receptor, it can be hypothesized that the effectmight be elicited via blockade of NMDA receptors.However, other receptor systems, such as sigma orserotonin, might also be involved in these effects.

The induction of BDNF mRNA expression inducedby higher doses of memantine was more widespreadthan that induced by MK-801. Additionally, whereasBDNF mRNA and protein levels were decreased byMK-801 in hippocampus (Castren et al., 1993; Linden etl., 2000), memantine predominantly increased BDNFRNA levels in this brain area. Differences in BDNFRNA regulation in hippocampal regions could be due

o different kinetic and voltage-dependent properties ofemantine binding to the PCP binding site of theMDA receptor ion channel. The binding of meman-

ine to the PCP site of the NMDA receptor ion channelas a strong voltage dependency, rapid blocking/un-locking kinetics to the PCP binding site, and modestffinity to the receptor-associated ion channel (Kornhu-er et al., 1991; Parsons et al., 1993). Thus, memantine at

a clinically relevant dose is generally well tolerated andlargely devoid of psychotomimetic-like side effectscharacteristic of higher affinity noncompetitive NMDAreceptor antagonists, such as PCP and MK-801 (Korn-huber et al., 1997; Parsons et al., 1999).

BDNF can support survival of dopaminergic neurons(Hyman et al., 1991; Knusel et al., 1991; Shen et al., 1994;Shults et al., 1994; Hagg, 1998) and other neuronal sys-tems (Knusel et al., 1991; Ghosh et al., 1994; Saaralainent al., 2000) but it does not penetrate the blood–brainarrier and therapeutic administration of BDNF isherefore possible only by intraventricular infusionGalpern et al., 1996), by transplantation of embryonicissue, or by engineered cells secreting BDNF in therain (Cirulli et al., 2000). These techniques are invasivend can be applied only to a limited number of patients.hus, memantine, and similarly acting agents that inlinically relevant doses can influence the synthesis ofndogenous neurotrophins in the CNS, may open upew possibilities of pharmacologically regulating thexpression of neurotrophic factors in the brain. Ourata suggest that the neuroprotective properties of me-antine might be mediated by its ability to promote the

ndogenous production of BDNF in the central nervousystem. Thus these results should strengthen the ratio-

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nale for the use of memantine in the treatment of ADand PD, as well as other neurodegenerative diseases.

EXPERIMENTAL METHODS

Animals and Treatments

Male Wistar rats (initial weight 200–280 g, NationalLaboratory Animal Center, University of Kuopio, Kuo-pio, Finland) were kept under standardized tempera-

FIG. 7. Effect of an acute administration of memantine on TrkB rmemantine (ip 5–50 mg/kg) were hybridized with a BDNF 33P-labelefrom olfactory bulb (Olf), cortical [frontal (Fr), parietal (Par), cingulaCA3] regions, the substantia nigra (SN), and striatum (CPu) by a MCtrkB.T1 mRNA (B) levels are shown. Data are means 6 SEM obtainedbetween saline- and memantine-treated rats are indicated: **P , 0.01are from medial layers (layer IV) except retrosplenial cortex (layer II

ture, humidity, and lighting conditions (12-h light/darkcycles) with free access to water and food. All animal

treatments were approved by the Experimental AnimalCommittee of the University of Kuopio and have beencarried out in accordance with the guidelines issued bythe Society for Neuroscience.

Rats were injected intraperitoneally (ip) with 5, 10, 25,and 50 mg/kg of memantine (akatinol–memantine z

Cl, gift from Dr. G. Quack, Merz 1 Co., Frankfurt/Main, Germany) and sacrificed 1–48 h afterward. Twolower doses (5 and 10 mg/kg) did not produce anyobservable behavioral changes, and after the higher

or isoforms mRNA. Brain sections from rats treated with saline orgonucleotide probe by in situ hybridization. Signals were quantifiedg), retrosplenial (RS)], and hippocampal [dentate gyrus (DG), CA1,

M4 image analysis system. Histograms of trkB.TK1 mRNA (A) andfour to nine brains in each treatment group. Significant differences

, 0.05; ANOVA followed by Dunnett’s post-hoc test. Values shown

eceptd olite (CID/from

doses (25 and 50 mg/kg) rats appeared sedated. How-ever, we did not attempt to observe quantitative behav-

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ioral differences. The rats were narcotized with CO2

before decapitation. Brains were rapidly removed,rinsed in phosphate-buffered saline (PBS) (pH 7.5),placed on dry ice, and stored at 280°C. Coronal brainsections (14 mm) were cut on a Leica CM 3000 cryostatand thaw-mounted onto SuperFrost/Plus (Menzel-Glaeser, Germany) slides. Brain sections from saline-treated (control) and memantine-treated rats were co-mounted onto the same slides, fixed in 4%paraformaldehyde solution, and stored in 95% ethanolat 4°C until used.

In Situ Hybridization (ISH)

In situ hybridization with oligonucleotide probes wasperformed as described by Wisden and Morris (1994).The oligonucleotide probes for rat BDNF (59-GATT-

GGTAGTTCGGCATTGCGAGTTCCAGTGCCT-39),at trkB.TK1 (59-TGCCTTGGGAAGGGCCGTCTGG-AGAAGAGGGAGT-39), and rat/mouse trkB.T1 (59-TGTCTTTCCTTTATGTCAGCTACCCATCCAG-39)ere 39 end-labeled to a specific activity of 1–2 3 107

cpm/pmol using terminal deoxynucleotidyl transferase(MBI Fermentas, Vilnus, Lithuania) and a 30:1 molarratio of [a-33P]dATP (2000 Ci/mmol, New England Nu-lear, Boston, MA) to probe. Hybridization was per-ormed overnight (42°C) on paraformaldehyde-post-xed sections in the presence of 1–3 3 103 cpm/ml of

labeled probe in buffer containing 50% formamide, 43standard saline citrate (SSC) (13 SSC:150 mM NaCl, 15mM sodium citrate), 10% dextran sulfate, and 10 mMdithiothreitol (DTT). After overnight hybridization at42°C, the sections were dipped into 13 SSC at roomtemperature, washed for 30 min at 55°C in 13 SSC, andwashed sequentially for 3 min each at room tempera-ture in 13 SSC, 0.13 SSC, 70% ethanol, and then 94%ethanol. Following 2–3 weeks exposure, Hyperfilm-bmax films (Amersham, Buckinghamshire, UK) weredeveloped in D-19 (Kodak, France). Nonspecific hybrid-ization was determined by adding 100-fold excess ofunlabeled oligonucleotide probe to the hybridizationbuffer. Nonspecific hybridization was not significantlyabove the background.

BDNF ELISA

BDNF ELISA was performed essentially as described(Nawa et al., 1995). Brain tissue containing retrosplenialcortex was dissected, taking care that other underlying
brain regions were not included, and rapidly homoge-nized by sonication in 10 vol of the ice-cold homogeni-
zation buffer (50 mM Tris, pH 7.5, 0.3 M NaCl, 0.1%Triton X-100, 1% bovine serum albumin (BSA), fractionV, pH 7.5, 200 kallikrein U/ml of aprotinin, 0.1 mMphenylmethylsulfonyl fluoride, 0.1 mM benzethoniumchloride, and 1 mM benzamidine). Samples were mi-crofuged 15,000 rev/min for 20 min at 4°C, diluted 1:5in the ELISA buffer (50 mM Tris, pH 7.5, 0.3 M NaCl,0.1% Triton X-100, 1% BSA, fraction V, pH 7.5, and 1%gelatin), and assayed overnight at room temperature.

Data Analysis

BDNF mRNA was quantified from in situ autoradio-grams by a video-based computer image analysis sys-tem (MCID/M4, Imaging Research, Inc., St. Catharines,Ontario, Canada). Brain regions examined were deter-mined according to Paxinos and Watson (1986) andincluded frontal (Fr), parietal (Par), cingulate (Cg), ret-rosplenial (RS), occipital (Occ), temporal (Te), andperirhinal (PRh) cortices, hippocampal [dentate gyrus(DG), CA1, CA2, CA3], thalamic, and hypothalamicregions, the ventral tegmental area (VTA), substantianigra (SN), striatum (CPu, caudate putamen), and ol-factory bulb (Olf). Superficial, medial, and deep layerswere examined from the cortex areas. Quantitative datawere obtained from four to nine brains performed induplicate. 14C microscales (ARC Inc., St. Louis, MO)were simultaneously exposed together with the hybrid-ized brain sections to convert optical densities to radio-activity (nCi/g).

3-D Reconstruction Method

Images of sections hybridized with the BDNF probewere taken by a video-based computer image system(MCID/M4, Imaging Research, Inc.). Digitized imageswere registered and rendered as an alignment of orig-inal images on a silicon graphics workstation (SGI On-yx2, Mountain View, CA) with an Irix 6.5 operatingsystem. A created 3-D grid was visualized applying avolume-rendering technique by using a volumizer tool-kit (SGI OpenGL Volumizer Toolkit), which allowed usto make transparent 3-D reconstructions of the wholerat brain.

Statistical Analysis

Values for treated rats were converted to percentagesof control values (saline treated) obtained from the
sections comounted with the treated sections in order toovercome the experimental variation. Statistical analy-

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ses for significant differences between saline- and me-mantine-treated rats were performed by one-way anal-ysis of variance (ANOVA) followed by Dunnett’s post-hoc test.

ACKNOWLEDGMENTS

The authors thank Anne Lehtela and Laila Kukkonenfor their professional technical assistance and Anna-kaisa Haapasalo for expert discussion. We are indebtedto Dr. G. Quack at Merz 1 Co. (Frankfurt/Main, Ger-many) for endowment of akatinol–memantine z HCl tomake this research possible. This study has been sup-ported in part by the Academy of Finland, Center forInternational Mobility (CIMO), and National Technol-ogy Agency of Finland (TEKES).

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Received May 15, 2001Revised July 11, 2001

Accepted July 27, 2001

the neuroprotective agent memantine induces brain-derived neurotrophic factor and trkb receptor...

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