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Research Report

Melatonin promotes distal dendritic ramifications in layer II/IIIcortical pyramidal cells of rats exposed to toluene vapors

Rodrigo Pascual⁎, Carlos BustamanteLaboratorio de Neurociencias, Escuela de Kinesiología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso,Avenida Brasil 2950, Valparaíso, Chile

A R T I C L E I N F O

⁎ Corresponding author. Fax: +56 32 2274395.E-mail address: rodrigo.pascual@ucv.cl (RAbbreviations: CNS, central nervous system

0006-8993/$ – see front matter © 2010 Elsevidoi:10.1016/j.brainres.2010.07.086

A B S T R A C T

Article history:Accepted 24 July 2010Available online 30 July 2010

We have previously shown that toluene inhalation produces significant impairments in thebasilar dendritic outgrowth of pyramidal cortical cells. This neurotoxic effect was markedlyinhibited by melatonin administration at a dose of 5 mg kg−1. The present study wasdesigned to determine whether toluene and melatonin equally affect all basilar dendriticsegments or if a differential response exists between the segments. Twenty-eightmalemicewere weaned at postnatal day 21 (P21) and randomly assigned to either the control (C; n=10,)or toluene (T; n=18) group. Between P22-P32,male rats were placed into a glass chamber andexposed to either toluene vapors (5–000–6000 ppm) or clean air for 10 min a day. Whentoluene exposure ended (P32), animals were further assigned to the following experimentalgroups: (a) control/saline (C/S; n=10), (b) toluene/saline (T/S; n=10), or (c) toluene/melatonin5 mg kg−1 (T/M; n=8). Melatonin or vehicle solutions were administered daily between P32and P38. Forty-eight hours after the final toluene exposure, the animals were sacrificed, andthe pyramidal cortical cells were stained using the Golgi–Cox–Sholl procedure. The numberof basilar dendritic branches/order was counted using the centrifugal ordering method. Theresults indicate that (i) toluene inhalation significantly impairs both proximal and distalbasilar dendritic ramifications (in the parietal and frontal/occipital cortices, respectively)and (ii) melatonin both protects neurons from toluene neurotoxicity in all cortical areasstudied and increases the complexity of the dendritic tree above control values.

© 2010 Elsevier B.V. All rights reserved.

Keywords:MelatoninToluene inhalationDendritic overgrowthNeocortical pyramidal cell

1. Introduction

In recent years, many publications have highlighted theprotective role of melatonin (N-acetyl-5-methoxytryptamine)in the central nervous system (CNS). In many tissues(including the CNS), melatonin acts either via the nuclearhormone receptor family (primarily RZRβ) or through themembrane G-protein-coupled receptors MT1/MT2 (Hekmek-

. Pascual).; P, postnatal day; C/S, c

er B.V. All rights reserved

ekcioglu, 2006). In addition, melatonin can act in a receptor-independentmanner as amajor scavenger of both oxygen andnitrogen reactive species (Hibaoui et al., 2009). Becauseoxidative/nitrosative stress is a pathophysiological pathwaythat is common in many brain disorders (Barja and Herrero,2000; Korkmaz et al., 2009), several preclinical studies haveused this hormone in animal models of brain damage toevaluate its therapeutic potential. For example, it was found

ontrol/saline; T/S, toluene/ saline; T/M, toluene/melatonin

.

Fig. 1 – Basilar dendritic order quantification.Photomicrograph of a typical layer II/III Golgi-stained corticalpyramidal cell. The basal dendritic tree was redrawn tohighlight the branches located outside the photographicplane.White numbers indicate dendritic orders (1: first-orderdendrites arising from cell body; 2: second-order dendritesarising from first branching point, and so on). Bar=50 μm.

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that melatonin can minimize behavioral and neural impair-ments induced by cerebral ischemia (Bouslama et al., 2007;Chen et al., 2009; Duan et al., 2006; García-Chávez et al., 2008;González-Burgos et al., 2007; Letechipía-Vallejo et al., 2007;Rennie et al., 2008;), hypoxia (Kaur et al., 2010), traumatic braininjury (Samantaray et al., 2009), and neurodegeneration (Lin etal., 2008; Weishaupt et al., 2006).

In addition, the chronic abuse of volatile solvents, espe-cially toluene, is a common practice among children andadolescents at social risk—primarily for its anxiolytic effectand its role in activating the brain's reward pathways(Chouanière et al., 2002; Kurtzman et al., 2001). Thesepsychotropic actions have led to the widespread recreationaluse of toluene and related volatile compounds (Chouanière etal., 2002). Chronic toluene exposure, either occupationallyand/or intentionally, can lead to neurotoxic CNS damage suchas brain atrophy, white matter impairments, multifocalhemodynamic changes and various neuropsychological defi-cits (Aydin et al., 2009; Deleu andHanssens, 2000; Fornazzari etal., 1983; Hormes et al., 1986; Küçük et al., 2000; Kurtzman etal., 2001). Because the primary pathophysiological mechanismof toluene-induced neurotoxicity is oxidative/nitrosativestress (Burmistrov et al., 2001; Mattia et al., 1993), severalstudies have successfully demonstrated that melatonin canoffset the neurotoxic effects of toluene. In fact, this indola-mine can counteract toluene-induced lipid peroxidation andgliosis in the cerebral cortex, hippocampus and cerebellum(Baydas et al., 2003). Furthermore, we showed in a recent studythat the total basilar dendritic length and branching impair-ment induced by toluene inhalation was significantly amelio-rated by melatonin administration (Pascual et al., 2010).Because the basilar dendrites of cortical pyramidal cells followa proximal-to-distal developmental time course, i.e., dendritesemerging directly from the soma (1st order dendrites) matureearlier than their 2nd order counterparts, and because distaldendrites appear to be more sensitive to various epigeneticcues (Hickmott and Ethel, 2006), it is possible that either thetoxic effect of toluene or the protective-trophic-like action ofmelatonin can affect distinct dendritic loci. Because thisphenomenon has not been studied, we took advantage ofthe multilevel centrifugal approach developed by Colemanand Riesen (1968) to evaluate the neurotoxic effect of tolueneand the rescue effect of melatonin administration on thecomplexity of basilar dendritic branching of layer II/III frontal,parietal and occipital pyramidal cells (Fig. 1).

2. Results

The number of dendritic branches per neuron in the 3rd, 4th,and 5th order of frontal pyramidal cells was significantlyreduced in animals exposed to toluene fumes (Fig. 2;*p<0.05, **p<0.01; ANOVA). In contrast, toluene-exposedanimals that were treated with 5 mg kg−1 melatonin showeda significant restorative effect on dendritic segments com-pared to the corresponding controls. In addition, as shownin the 3rd–6th orders, melatonin not only offset thedeterioration caused by toluene but promoted dendriticbranching above the level found in the control group. Incontrast, toluene produced dendritic impairments only in

proximal orders (2nd and 3rd) of parietal cortical neurons.Furthermore, while melatonin induced recovery followingtoluene, its trophic-like effect on dendritic branching wasless (4th and 5th orders) than that observed in frontalneurons (Fig. 3; *p<0.05, **p<0.01; ANOVA). Finally, exposureto toluene altered occipital pyramidal cells only at theirmost distal dendritic branches. Despite rescue by melatoninadministration, the branching overgrowth observed in thefrontal and parietal cortices was not detected (Fig. 4;**p<0.01; ANOVA). The neurotoxic effect of toluene and theability of melatonin to recover/promote dendritic branchingover control levels are shown in the representative photo-micrographs accompanying the graphics. There were nodifferences in body weight between control and solvent/melatonin-exposed animals (Fig. 5).

3. Discussion

In the present study, we demonstrate that toluene inhalationin juvenile rats significantly impaired basilar dendritic treeramifications and that melatonin not only protected neuronsfrom toluene neurotoxicity but also increased dendriticcomplexity in two of the three regions studied (frontal and

Fig. 2 – Frontal photomicrographs of representative Golgi-stained cortical neurons of control animals (A), toluene-exposedanimals (B), and toluene-exposed animals treated with 5 mg kg−1 melatonin (C). (D) Number of basilar dendrites per cell as afunction of dendritic order. T/M: toluene+melatonin. Data are means±SEM; *p<0.05, **p<0.01, ANOVA. Bar=40 μm.

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parietal cortices). These results are similar to those observedin our previous studies (Pascual et al., 2010), however, in thisstudy, we were able to demonstrate that the dendritic locimost vulnerable to toluene inhalation were the distalbranches in the frontal and occipital pyramidal cells. Incontrast, the proximal dendritic segments were the mostaffected in parietal cells (the 2nd and 3rd orders). Similarly, themelatonin-induced overgrowth was most dramatic in thefrontal neurons (the 3rd–6th orders) but was less marked inthe parietal neurons and not detected in occipital neurons.

The beneficial effects of melatonin reported in this studyare consistent with the results obtained by Baydas et al. (2003),which showed that a mixture of volatile solvents (3000 ppm)administered to adults rats for 1 h/day for 45 days produced asignificant increase in lipid peroxidation as shown by in-creased levels of malondialdehyde (MDA) and 4-hydroxyalk-enals (4-HDA) in the cerebral cortex, hippocampus, andcerebellum. Moreover, the concomitant administration ofmelatonin (10 mg/kg/day) to solvent-exposed animals re-duced the levels of MDA/4-HAD, thus highlighting thepowerful antioxidant action of melatonin. The authors also

showed that melatonin acted as a free radical scavenger andincreased the levels of antioxidant enzymes such as glutathi-one (GSH).Whilewe did not evaluate oxidative stressmarkers,it is possible that the rescue effect of melatonin observed inthis study could be associated with reductions in reactiveoxygen species and/or the increased expression of protectiveenzymes. Furthermore, Baydas' study showed that toluene-exposed, but not melatonin-treated, animals showed higherlevels of glial fibrillary acidic protein, suggesting that toluene-induced neural damage was similarly mitigated by melatoninadministration and thus providing evidence in support ofmelatonin's neuroprotective effect. In addition, the currentfindings are consistent with studies performed in animalssubjected to ischemic brain injury that were treated witha continuous intravenous administration of melatonin(10 mg kg−1). These studies showed that, 90 days after therats experienced 15 min of global cerebral ischemia, melato-nin significantly reduced the loss of pyramidal neurons inboth the hippocampus and in the cerebral cortex (Letechipía-Vallejo et al., 2007). In addition, consistent with our data,melatonin (5 or 10 mg kg−1) was shown to aid in the recovery

Fig. 3 – Parietal photomicrographs of control animals (A), toluene-exposed animals (B), and toluene-exposed animals treatedwith 5 mg kg−1 melatonin (C). (D) Number of basilar dendrites per cell as a function of dendritic order. T/M: toluene+melatonin.Data are means±SEM; *p<0.05, **p<0.01, ANOVA. Bar=40 μm.

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of both dendritic branching and spine density in prefrontalpyramidal neurons of rats subjected to the same ischemicbrain damage (Chen et al., 2009; García-Chávez et al., 2008).

In the present study, the toluene- or melatonin-inducedneuronal changes were not homogeneous between thecortical regions studied.While the neurotoxic effect of toluenein frontal and occipital pyramidal cells impaired primarily themiddle and distal orders (the 3rd, 4th, and 5th), the mostaffected dendritic segments in the parietal cortex were thoselocated in proximal branches (the 2nd and 3rd orders). Thesedifferent responses may be attributed to the different devel-opmental programs of cortical neurons in each of the threebrain areas, resulting in neurons becoming more or lesssensitive to diverse epigenetic cues. In fact, as was shownsome decades ago, there is an anterior-to-posterior gradient ofcorticalmaturation (Bayer andAltman, 1991;Miller and Peters,1981). We found a similar pattern of neuronal response tomelatonin. Relative to controls, melatonin-treated animalsexhibited a greater trophic response in frontal neurons (at the3rd, 4th, 5th, and 6th dendritic orders), a lower response in theparietal cortex (at the 4th and 5th dendritic orders), and no

response in occipital neurons. While the neurobiologicalmechanisms underlying this difference in the regional patternof melatonin response are still unknown, it is likely thatdifferent degrees of plasticity may be related to the differentcytodifferentiation rates of neocortical cells. As stated above,the timing of neural development varies among the threecortical regions studied, following a frontal-to-occipital gradi-ent of cytodifferentiation.

Melatonin could have produced its neuroprotective effectsvia two primary mechanisms. Either (i) by directly scavengingthe excess free radicals formed by toluene exposure (Manda etal., 2008) or (ii) through signaling induced by the binding ofmelatonin to MT1/MT2 receptors. In the former case, (i.e.,oxidative stress) it has been shown that melatonin can bothincrease the levels of the anti-apoptotic protein Bcl-2 andreduce the levels of the pro-apoptotic protein Bax in themitochondria. In the latter scenario, it has been shown thatmelatonin can re-localize the anti-apoptotic protein Bcl-2 onthe mitochondria via a cellular signal transduction system(through the activation of MT1/MT2 receptors), and thusincrease the protein's anti-apoptotic activity (Radogna et al.,

Fig. 4 – Occipital photomicrographs of control animals (A), toluene-exposed animals (B), and toluene-exposed animals treatedwith 5 mg kg−1 melatonin (C). (D) Number of basilar dendrites per cell as a function of dendritic order. T/M: toluene+melatonin.Data are means±SEM; **p<0.01, ANOVA. Bar=40 μm.

Fig. 5 – Mean body weight (g) measured in experimentalanimals. T/M: toluene+melatonin.

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2008). Furthermore, it is also possible that MT1/2 signaling canregulate gene expression to activate the machinery fordendritic growth (Scott and Lou, 2000). Because the MT1receptor has been primarily detected in the cerebral cortex(Uz et al., 2005), it is possible that the remarkable ability ofmelatonin to promote branching is associated with thesynergistic action of melatonin acting either via the MT1receptor or as amajor oxygen- and nitrogen-reactivemoleculescavenger (Reiter et al., 2003). Alternatively, it is possible thatmelatonin acts directly on dendritic cytoskeletal proteins, in-creasing microtubule polymerization andmicrofilament arraysthat normally occur during dendritic outgrowth (Benítez-King,2006).

While we cannot determine the functional consequencesof these dendritic alterations from our analysis of the Golgi-stained specimens, it is highly probable that changes in theneuronal structure can affect the spatial distribution ofsynaptic contacts and thus alter the amplitude and theshape of local excitatory postsynaptic potentials, resulting inchanges to the computational properties of the neuronsinvolved (Magee, 2000).

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In summary, the current data confirm and extend ourprevious reports showing that (i) toluene significantlyimpairs basilar dendritic ramifications in layer II/IIIcortical pyramidal cells, either in the proximal (parietal)or distal (frontal/occipital) dendritic orders; and (ii) mela-tonin exerts a rescue/trophic-like effect, primarily in thedistal dendritic orders of frontal and parietal pyramidalcells.

4. Experimental procedures

Adult female Sprague–Dawley rats were mated withcolony-breeder male rats. One week before birth, damswere individually housed in laboratory cages under con-trolled conditions (12:12 h light/dark cycle; 21±2 C°). Theanimals had free access to food and water. Approximately12 h after delivery (postnatal day 0, P0), litters were culledto 8 pups/dam (5 males and 3 females) and housedtogether with a mother in standard laboratory cages.Pups were weaned at P21 and randomly assigned to eithercontrol (C; n=10,) or toluene (T; n=18) groups. BetweenP22–P32, male rats were gently placed into a whole-bodyinhalation glass chamber with a vent in the floor, andexposed to either toluene vapors or clean air during10 min/day. Toluene fumes were generated by placingtoluene embedded filter paper at the bottom of the glasschamber and vaporized by a fan. Due to the chamber volumeand the physicochemical properties of toluene, animals wereexposed to 5000–6,000 ppm toluene (Shelton, 2007). Whentoluene exposure ended (P32), the animals were furtherassigned to the following experimental groups: (a) control/saline (C/S; n=10), (b) toluene/saline (T/S; n=10), and (c)toluene/melatonin 5 mg kg−1 (T/M; n=8). Melatonin (Sigma-Aldrich) was diluted in 10% ethanol-saline (0.9%NaCl and 0.1%ethanol; Merck) and injected intraperitoneally (0.1 ml). Controlanimals received saline in a similar volume. Melatonin orvehicle solutions were administered daily (17.00 h) betweenP32 and P38.

Forty-eight hours after the final toluene exposure, theanimals were weighed, deeply anesthetized with ether andsacrificed. To determine the impact of toluene inhalation andthe potential neurorescue effect of melatonin on corticalneuron differentiation, brains were stained using the Golgi-Cox procedure, modified by Sholl (Sholl, 1953). After 30 daysof impregnation, brains were dehydrated in ethanol-acetoneand ethanol-ether solutions (50/50%; Merck), embedded inceloidin (Merck), cut into coronal sections (120 μm thick), andmounted on previously coded slides with cover slips. TheGolgi-Cox method provides substantial dendritic impregna-tion, permitting adequate morphological measurements.Selected neocortical neurons required an adequate stainingof the soma and dendrites, well-defined pyramidal shape,and no extensive processes overlapping neighboring neu-rons; cells also needed to be located in a cortical stripbetween 200–600 μm from the pial surface (cortical layers II/III). Selected neurons were analyzed under an Olympus CX-3light microscope (400×), captured with a digital cameraattached to the microscope (Olympus CCD 5.0) and analyzedusing Micrometrics SE Premium V-2.8 software. A total of 672

pyramidal cells were sampled from the frontal (C/S=80; T/S=80; T/M=64), parietal (C/S=80; T/S=80; T/M=64) andoccipital (C/S=80; T/S=80; T/M=64) cortices, as delimited bythe coordinates provided in the Stereotaxic Atlas of the Rat(Paxinos andWatson, 1998). The number of dendritic brancheswas counted using the method described by Coleman andRiesen (1968): dendrites leaving the cell soma were defined asfirst order, direct branches from first-order dendrites weredesignated as second order and so on (Fig. 1). Neuronal datawere statistically analyzed using one-way ANOVA, comple-mented with the Scheffé test for post hoc analysis (STATA 9.1software).

Animals were treated and housed in accordance with the“Principles of Laboratory Animal Care” (NIH publication No 86-23, revised 1985), and experimental protocols received ap-proval from the local animal ethics committee.

Acknowledgments

This research was supported by grant PUCV-VRIEA 127.706/2008.

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