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Neurobiology of Aging 34 (2013) 1988e1995

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Neurobiology of Aging

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Nerve growth factor retrieves neuropeptide Y and cholinergic immunoreactivityin the nucleus accumbens of old rats

Pedro A. Pereira*, Diana Santos, João Neves, M. Dulce Madeira, Manuel M. Paula-BarbosaDepartment of Anatomy, Faculty of Medicine, University of Porto, Alameda Professor Hernâni Monteiro, Porto, Portugal

a r t i c l e i n f o

Article history:Received 27 July 2012Received in revised form 1 February 2013Accepted 15 February 2013Available online 26 March 2013

Keywords:StereologyNucleus accumbensAgingNeuropeptide YAcetylcholineNerve growth factor

* Corresponding author at: Department of AnaUniversity of Porto, Alameda Professor Hernâni MonteTel.: þ351 22 5513616; fax: þ351 22 5513617.

E-mail address: pedroper@med.up.pt (P.A. Pereira)

0197-4580/$ e see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.neurobiolaging.2013.02.011

a b s t r a c t

The nucleus accumbens (NAc) contains high levels of neuropeptide Y (NPY), which is involved in theregulation of functions and behaviors that deteriorate with aging. We sought to determine if aging altersNPY expression in this nucleus and, in the affirmative, if those changes are attributable to the cholinergicinnervation of the NAc. The total number and the somatic volume of NPY- and choline acetyltransferase-immunoreactive neurons, and the density of cholinergic varicosities were estimated in the NAc of adult(6 months old) and aged (24 months old) rats. In aged rats, the number of NPY neurons was reduced by20% and their size was unaltered. The number of cholinergic neurons and the density of the cholinergicvaricosities were unchanged, but their somas were hypertrophied. Nerve growth factor administration toaged rats further increased the volume of cholinergic neurons, augmented the density of the cholinergicvaricosities, and reversed the age-related decrease in the number of NPY neurons. Our data show that theage-related changes in NPY levels in the NAc cannot be solely ascribed to the cholinergic innervation ofthe nucleus.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

The extensive use of magnetic resonance imaging, particularlyduring the past decade, allowed the demonstration of variations inthe size of numerous regions of the human brain during the normalprocess of aging. Most of these studies agree that the volume orthickness of the gray matter decreases as age increases and that thisvariation, although with marked regional heterogeneity, occurs inthe cortical gray matter and in deep subcortical telencephalicregions (for a review, see Walhovd et al., 2011). The nucleusaccumbens (NAc) is such a region. Its volume is negatively corre-lated with age (Jernigan et al., 2001; Long et al., 2012; Walhovdet al., 2011) and, according to recently published data (Walhovdet al., 2011), is the brain structure that shows the largest esti-mated percentage of age difference. Another recent study (de Jonget al., 2012) has also revealed that the volume of the NAc is closelyassociated with the occurrence of dementia and predicts cognitivedecline in older people.

In the rat, the NAc is located in the rostroventral part of thestriatum and is regarded as a functional interface between limbicand motor systems (for a review, see Groenewegen and Trimble,

tomy, Faculty of Medicine,iro, 4200-319 Porto, Portugal.

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2007; Morgane et al., 2005). Approximately 90% of its neuronsare densely spiny projection neurons, and the remaining areinterneurons almost or completely devoid of spines that produceeither gamma-aminobutyric acid or acetylcholine (Meredith, 1999).Gamma-aminobutyric acid interneurons costore various neuro-peptides, including neuropeptide Y (NPY; Meredith, 1999) andcholinergic interneurons, represent approximately 1.7% of the totalneuronal population of the striatum (Phelps et al., 1985), and arethe only source of the dense cholinergic innervation of the NAc(Meredith, 1999; Pennartz et al., 1994). Despite the relatively smallnumber of NPY and cholinergic neurons in the striatum, it containsone of the highest concentrations of NPY and acetylcholine in thebrain (Hoover et al., 1978;Wettstein et al., 1995). NPYplays a crucialrole in functions and behaviors that are frequently altered by aging,such as cognition, circadian rhythms and sleep, feeding, andcardiovascular regulation (for review, see Thorsell and Ehlers, 2006;Wettstein et al., 1995). There is evidence that aging is associatedwith reduced NPY levels in several regions of the brain, namely thebrainstem, hypothalamus, hippocampal formation, and neocortex(Cadacio et al., 2003; Cardoso et al., 2006; Cha et al., 1997; Huguetet al., 1993; Huh et al., 1997; Kowalski et al., 1992; Zhang et al.,1998). However, data about age-related effects on the NAc arescarce and controversial, with one study (Huh et al., 1997) reportingunchanged, and another (Cha et al., 1997) slightly decreasednumbers of NPY-immunoreactive neurons in old relative to adultrats. Acetylcholine has been likewise implicated in modulating

P.A. Pereira et al. / Neurobiology of Aging 34 (2013) 1988e1995 1989

functions that deteriorate with aging, such as sleepewake cycles,cognitive performance, learning, and memory (Bartus, 2000;Everitt and Robbins, 1997; Hasselmo, 2006; Jones, 2008; Sarteret al., 2003; Schliebs and Arendt, 2011; Steriade, 2004). In partic-ular, striatal cholinergic neurotransmission is involved in age-associated cognitive impairment (Lazaris et al., 2003; Ragozzinoet al., 2009; Stemmelin et al., 2000) and, via interaction withdopamine, in motor control (Umegaki et al., 2008), both of whichare altered by aging. Although there are a few studies showing age-associated changes in the striatal cholinergic system, namelyreductions in neuronal size (Altavista et al., 1988; Fischer et al.,1987) and density (Altavista et al., 1988; Stemmelin et al., 2000),and in baseline release of acetylcholine (Wang et al., 2007; Wuet al., 1988) and cholinesterase activity (Das et al., 2001) in agedrats compared with young rats, no investigations have examinedthe effects of aging on the cholinergic neurons of the NAc.

The present study was designed to investigate, using stereo-logical methods, whether there are age-related changes in thetotal number and somatic volume of NPY and cholinergic neuronsin the NAc of male rats and, in the affirmative, if those changes canbe ascribed to the age-associated disruption of nerve growthfactor (NGF) trophic support (Bruno and Cuello, 2012; Sofroniewet al., 2001; Williams et al., 2006). Notably, age-related atrophyof basal forebrain cholinergic neurons can be reversed byadministration of NGF, resulting in amelioration of age-relatedcognitive deficits (Fischer et al., 1987; Markowska et al., 1994;Nagahara et al., 2009; Niewiadomska et al., 2002; Smith et al.,1999). There is also evidence that NGF delivered to the brain canrevert the age-related changes in the number of NPY-immunoreactive neurons in the somatosensory cortex (Cardosoet al., 2006), and of vasopressin- and vasoactive intestinalpolypeptide-producing neurons in the suprachiasmatic nucleus(Pereira et al., 2005) of old rats. Thus, to examine if the levels ofNPY and acetylcholine in the NAc are dependent on NGF trophicsupport we have estimated the total number and the somatic sizeof neurons producing NPY and acetylcholine, and the density ofcholinergic varicosities in the NAc of old rats that were treated,during the last 12 days of the experiment, with NGF deliveredintracerebroventricularly.

2. Methods

2.1. Animals and treatments

A total of 15 male Wistar rats, 5 young (6 months old) and 10aged (24 months old), were used in the present study. Animalswere housed in a temperature controlled room (22 �C) in 12-hourlight/dark cycles (lights on at 7:00 AM) with solid diet (4RF21/C;Mucedola, Milan, Italy) and water available ad libitum. Half of theaged rats (n ¼ 5) were randomly selected and infused with 2.5SNGF (Prince Laboratories, Toronto, Ontario, Canada) during 12 daysbefore death (see details in section 2.2.). Because there is evidencethat this surgical procedure does not interfere with the centralNPY-ergic (Cardoso et al., 2006) and cholinergic (Cadete-Leiteet al., 2003) systems, vehicle-treated rats were not included inthis study.

The experiments were performed in accordance with EuropeanCommunities Council Directive (2010/63/EU) of 22 September 2010and Portuguese Act n�129/92. All efforts were made to minimizethe number of animals used, and their discomfort and suffering.

2.2. Surgical procedures and drug treatment

For intracerebroventricular administration of NGF, rats wereanesthetized by sequentially injecting, at intervals of 10 minutes,

solutions of promethazine (10 mg/kg body weight, subcutaneous;Laboratórios Vitória, Amadora, Portugal), followed by xylazine(2.6 mg/kg body weight, intramuscular; Sigma-Aldrich CompanyLtd, Madrid, Spain) and, finally, ketamine (50 mg/kg body weight,intramuscular; Merial Portuguesa, Rio de Mouro, Portugal). Then,they were placed on a stereotaxic apparatus with bregma andlambda in the same horizontal plane. After a midline skin incision,the calvaria were exposed. For intracerebroventricular delivery ofNGF, permanent stainless steel cannulae (Alzet brain infusion kit;Alza Corporation, Palo Alto, CA, USA) were stereotaxically placed inthe right lateral ventricle, 1.1 mm posterior to the bregma, 1.7 mmlateral to the midline, and 4.0 mm below the surface of the skull(Paxinos and Watson, 1998). The cannulae were connected tomethylene blue (0.01%; Sigma) filled Alzet osmotic minipumps(model 2002) via sterile coiled polyethylene tubing (PE-60; Intra-medic, Becton Dickinson, Sparks, MD, USA). This tubing was filledwith aireoil spacer at the pump end and with NGF (150 mg dilutedin 150 mL of vehicle composed of artificial cerebrospinal fluid sup-plemented with 0.1% bovine serum albumin; Sigma). Osmoticminipumps were pretested to confirm their delivery rate, andimplanted subcutaneously in the neck. Skin incisions were closedwith surgical stitches and treated with local antiseptic. Aftersurgery, rats were individually housed and maintained in a warmplace until they woke up. Postoperative care consisted of subcuta-neous injections of 0.9% saline (2 mL), during the 48 hours aftersurgery, to prevent dehydration and weight loss. Twelve days afterthe beginning of NGF infusion, rats were killed and the total infu-sion volume was calculated. The mean volume of NGF injected perrat was 117.27 � 29.35 mL and the mean flow rate of the pumps was0.41 � 0.10 mL/h.

2.3. Tissue preparation

Rats were deeply anesthetized by intraperitoneal injection ofa solution (3 mL/kg body weight) containing 1% sodium pento-barbital and 4% chloral hydrate in physiological saline. They werethen perfused transcardially with 150 mL of 0.1 M phosphate buffer(PB; pH 7.6) for vascular rinse, followed by 250 mL of a fixativesolution containing 4% paraformaldehyde in PB, at pH 7.6. Thebrainswere removed from the skulls, coded, immersed for 1 hour inthe same fixative, and maintained overnight in a solution of 10%sucrose in PB, at 4 �C. After trimming away the occipital poles, theblocks were placed on a vibratome and serially sectioned in thecoronal plane at 40 mm through the NAc. The sections werecollected in phosphate-buffered saline (PBS). From the entire set ofsections obtained from each brain, 4 series were formed usinga systematic, random sampling procedure (Gundersen et al., 1999).Accordingly, the first section was randomly selected from the firstgroup of 4 collected sections, and the remaining were sampled,along the entire rostrocaudal extent of the NAc, at regular intervalsof 160 mm (i.e., 1 out of 4 sections). The first, second, and third serieswere used for NPY, choline acetyltransferase (ChAT) and vesicularacetylcholine transporter (VAChT) immunostaining, respectively,and the fourth was used for Nissl staining.

2.4. Immunohistochemistry and Nissl staining

For detection of NPY-immunoreactive neurons (Fig. 1), sectionswere washed twice in PBS, treated with 3% H2O2 for 10 minutes toinactivate endogenous peroxidase, and incubated overnight, at 4 �C,with the primary antiserum against NPY (T-4070; Bachem Ltd,Merseyside, UK; 1:10000 dilution in PBS). Biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA; 1:400dilution in PBS) was used as the secondary antibody. For visualiza-tion of ChAT-immunoreactive neurons (Figs. 1 and 2), sections were

Fig. 1. Photomicrographs of coronal sections through approximately the mid-level of the NAc of an adult rat. Adjacent sections stained with Cresyl Violet (A) and immunostained forneuropeptide Y (B) and for ChAT (C). NAc neurons immunoreactive for neuropeptide Y (D) and ChAT (E). Scale bars: 400 mm in (A), (B), and (C), and 10 mm in (D) and (E).Abbreviations: ac, anterior commissure; ChAT, choline acetyltransferase; lv, lateral ventricle; NAc, nucleus accumbens.

P.A. Pereira et al. / Neurobiology of Aging 34 (2013) 1988e19951990

pretreated as described above in section 2.4. and incubated, for 48hours at 4 �C, with the primary antiserum against ChAT (AB144P;Chemicon, Millipore Corporation, Billerica, MA, USA; 1:2000 dilu-tion in PBS). Biotinylated rabbit anti-goat antibody (Vector Labora-tories; 1:400 dilution in PBS) was used as the secondary antibody.For VAChT immunohistochemistry (Fig. 2), sections were immersedin a 5% solution of rabbit normal serum (Vector Laboratories) in PBS,for 30 minutes at room temperature. Thereafter, they were incu-bated, for 72 hours at 4 �C, with the primary antiserum againstVAChT (AB1578, Chemicon, Millipore Corporation; 1:15000 dilutionin PBS). Biotinylated rabbit anti-goat antibody (Vector Laboratories;1:400 dilution in PBS) was used as the secondary antibody.

After incubation with the secondary antibodies, sections weretreated with avidinebiotin peroxidase complex (Vectastain EliteABC kit; Vector Laboratories; 1:800 dilution in PBS). In the last 2steps, the incubation was carried out for at least 1 hour at roomtemperature. After treatment with peroxidase complex, sectionswere incubated for 10 minutes in 0.05% diaminobenzidine (Sigma)

Fig. 2. ChAT and VAChT immunoreactivity in the NAc of adult (A) and (D), aged (B) and (E), aapproximately the mid-level of the NAc. The smaller size of the ChAT-positive neuronal ceevident. In the latter animal (C), the size of the ChAT-immunoreactive neuronal profile is obvthe core (DeF) and in the shell (not shown) of the NAc is similar in adult and aged rats and macetyltransferase; NAc, nucleus accumbens; NGF, nerve growth factor; VAChT, vesicular ace

to which H2O2 was added to a final concentration of 0.01%. Sectionswere rinsed with PBS for at least 15 minutes between each step. Toincrease tissue penetration, 0.5% Triton X-100 was added to the PBSused in all immunoreactions and washes. All procedures wereperformed on a rocking table. Immunostained sections weremounted on gelatin-coated slides and air-dried. Then, they weredehydrated in a series of ethanol solutions (50%, 70%, 90%, and100%), cleared in xylol, and coverslipped using Histomount(National Diagnostics, Atlanta, GA, USA). To prevent variability instaining, sections from all groups analyzed were processed inparallel at the same time. The same procedure was followed forcontrol sections, which were incubated without antiserum; noimmunostaining was observed in these sections (data not shown).

The Nissl-stained sections (Fig. 1) were used for estimating thetotal number of NAc neurons and for help in identifying theboundaries of the NAc in immunostained sections. The sectionswere mounted serially on gelatin-coated slides. After air-dryingovernight at room temperature, they were stained with Cresyl

nd NGF-treated aged (C) and (F) rats. Photomicrographs show coronal sections throughll body of the adult rat (A) relative to those of old (B) and NGF-treated old (C) rats isiously larger than in the old rat (B).The density of VAChT-immunoreactive varicosities inarkedly higher in NGF-infused old rats. Scale bars: 10 mm. Abbreviations: ChAT, cholinetylcholine transporter.

P.A. Pereira et al. / Neurobiology of Aging 34 (2013) 1988e1995 1991

Violet, dehydrated, and coverslipped with Histomount (NationalDiagnostics).

2.5. Stereological analyses

The optical fractionator (Madeira et al., 1995; West et al., 1991)was used to estimate total neuron numbers. Cell counting wascarried out on blind-coded slides using an Olympus C.A.S.T.-Gridsystem (Olympus DK A/S, version 2.00) and a Hiedenhain MT-12microcator. As a general rule, neurons were counted, at final mag-nification �2000, using neuronal nuclei as the counting unit;neuronal nuclei touching the left or bottom sides of the countingframe were discarded. All Nissl-stained sections and all sectionsimmunostained for NPY and ChAT that contained the NAc wereused, which provided an average of 13 sections per nucleusanalyzed. In each Nissl-stained section, the fields of view weresystematically sampled using an interframe distance of 600 mm (x-and y-axes). Actual cell counting was done using a dissector heightof 10 mm and a counting frame area of 791 mm2 at the tissue level.On average, 157 neurons were counted per nucleus; the meancoefficient of error (CE) of the estimates was 0.09. The samplingscheme used for estimating the total number of NPY- and ChAT-immunoreactive cells in the NAc was as described in section 2.5.,with the following modifications: microscope fields were sampledusing a step size of 250 mm (x- and y-axes) for NPY neurons and of200 mm (x- and y-axes) for ChAT neurons. The area of the countingframes was 8436 mm2 and 8419 mm2, respectively, and the height ofthe dissector was 10 mm for both neuronal populations. By applyingthis sampling scheme, an average of 186 NPY- and 128 ChAT-immunoreactive cells was counted per nucleus. The mean CE ofthe estimates was 0.08 and 0.10, respectively.

The mean somatic volume of NPY- and ChAT-immunostainedneurons was estimated by applying the optical rotator (Leal et al.,1998; Tandrup et al., 1997). Neurons used for measurements wereselected with optical dissectors, as described in section 2.5. Theseprocedures were implemented with the C.A.S.T.-Grid system soft-ware (version 2.00), which allows the estimation of the meansomatic volume using a spatial line grid. Measurements of inter-sections between the cell membrane and the spatial line grid wereperformed using a 2-grid line and 2 focal planes per each cell, atmagnification �2000. The mean CE of the estimates was 0.07 and0.09, respectively.

The density of the cholinergic varicosities (Fig. 2) was estimatedfrom VAChT-immunostained sections (n¼ 5 per animal) selected atmid-NAc levels. Varicosities were counted using a computer-assisted image analyzer (Leica QWin) fitted with a Leica DMRmicroscope and a Leica DC 300F video camera, at final magnifica-tion�1000. Considering the obvious difference in the density of thecholinergic varicosities between the core and shell of the NAc,measurements were performed separately in these 2 regions.Within each section, 4 different placements of the frame, 2 for coreand 2 for shell, were randomly selected. Because the variations inthe core and in the shell were of the same type and magnitude datawere pooled. The varicosities were defined as darkly stained axonaldilations with size greater than 0.25 mm2 (Cardoso et al., 2006). Asample frame (1.52 � 106 mm2) was laid over each field of view andthe number of varicosities falling within it was counted. Resultswere expressed as areal densities (n/mm2).

2.6. Statistical analyses

The precision of individual estimates of neuron numbers wasevaluated as the CE (Gundersen et al., 1999). The precision ofindividual estimates of mean somatic volumes was calculated byapplying the equation: CE2 ¼ CV2/n (Gundersen and Jensen, 1987),

in which CV denotes the intraindividual coefficient of variation(CV ¼ SD/mean) and n is the number of observations in one animal.The mean CE was calculated from estimates for an individual, asdescribed by West et al. (1991). Data were analyzed by one-wayanalysis of variance (ANOVA) followed by pairwise post hoccomparisons using the Tukey Honest Significant Difference test.Differences were considered significant if p < 0.05.

3. Results

3.1. Qualitative observations

As shown in Fig. 1, NPY-immunoreactive neurons are abun-dant in the NAc whereas ChAT-immunoreactive neurons arerelatively sparse. A moderately dense NPY-immunoreactive fiberplexus was seen in the shell and core of the NAc (Fig. 1B and D).In agreement with earlier observations (Meredith, 1999; Vuilletet al., 1992), neurons immunoreactive for NPY are medium-sized and neurons immunoreactive for ChAT have larger cellbodies (Fig. 1D and E). Aging was associated with hypertrophy ofChAT-immunoreactive neuronal cell bodies, but did not changethe density of VAChT-immunoreactive fibers in the NAc (Fig. 2).As can be seen in Fig. 2, the administration of NGF to aged ratsleads to a further enlargement of ChAT neuronal cell bodies anddistinctly increases the density of the VAChT-immunoreactivefibers in the NAc.

3.2. Total number of NAc neurons

As showed using ANOVA, aging and NGF administration did notproduce significant variations (F(2,12) ¼ 0.22; p ¼ 0.80) in the totalnumber of NAc neurons (Fig. 3).

3.3. Total number and somatic size of NPY-immunoreactive neurons

The NAc of adult male rats contains, on average, 10,262 NPY-immunoreactive neurons (Fig. 4A). ANOVA revealed that agingand NGF administration significantly influence the total number ofNPY-immunoreactive neurons (F(2,12) ¼ 14.85; p < 0.001). In agedrats the total number of neurons was reduced by approximately20%, a difference that is statistically significant. Treatment of agedrats with NGF was associated with an increase in the total numberof NPY-immunoreactive neurons to values similar to those of adultrats. Conversely, the mean somatic volume of NPY-immunoreactiveneurons (Fig. 4B) was unaltered in aged rats and did not change inresponse to NGF administration (F(2,12) ¼ 0.69; p ¼ 0.52).

3.4. Total number and somatic size of cholinergic neurons

Our estimates show that the NAc contains approximately 4426cholinergic neurons in adult rats, and that this number is notaltered by aging or by NGF treatment (F(2,12) ¼ 0.50; p ¼ 0.62;Fig. 5A). Conversely, the mean somatic volume of these neurons(Fig. 5B) was significantly influenced by aging and NGF adminis-tration (F(2,12) ¼ 56.81; p < 0.000005). As shown in Figs. 2 and 5B,the somatic volume of cholinergic neurons was larger in old than inadult rats, and the administration of NGF to old rats caused anadditional increase in the somatic volume of these neurons.

3.5. Density of cholinergic varicosities

The density of cholinergic varicosities was significantly influ-enced by aging and NGF administration (F(2,12) ¼ 39.89; p <

0.00001). Specifically, the density of VAChT-positive fiber varicos-ities was similar in adult and aged rats, and the administration of

Fig. 3. Graphic representation of the total number of Nissl-stained neurons in the NAcof adult (adult), old (old), and NGF-treated old (oldþNGF) rats. Columns representmeans and vertical bars � 1 SD. Abbreviations: NAc, nucleus accumbens; NGF, nervegrowth factor.

P.A. Pereira et al. / Neurobiology of Aging 34 (2013) 1988e19951992

NGF to old rats increased, by approximately 47%, the density of thevaricosities in the NAc (Figs. 2 and 5C).

4. Discussion

Thus far, the question of whether aging interferes with thecontent of NPY in the NAc has been addressed in only two studiesthat, however, yielded different conclusions. Specifically, Huh et al.(1997) found no changes in the density of NPY-immunoreactiveneurons, and Cha et al. (1997) detected a mild (<15%) reductionin the number of NPY-immunoreactive neurons counted froma series of level-matched sections of the nucleus. Our data, esti-mated by applying stereological methods, not only support the lastconclusion, because we found a 20% reduction in the total numberof NPY-immunoreactive neurons in the NAc of old rats, but alsoextends it by showing that these neurons do not undergo age-related changes in their somatic size. The reduction in thenumber of NPY-immunoreactive neurons is not because of celldeath because we have also established that the total number ofNAc neurons is identical in adult and in old rats. It is thus veryprobable that the decrease in neuron numbers that we haveobserved might be a consequence of reduced NPY synthesis,a hypothesis that is in line with data from studies showing lowerNPY messenger RNA levels in the hypothalamus of old relative toadult rats (reviewed in Kmiec, 2011).

Previous studies have revealed that striatal NPY neurons do notexpress NGF receptors (Barrett et al., 2005; Sobreviela et al., 1994;

Venero et al., 1994). Therefore, the NGF-induced increase in thenumber of NPY-immunoreactive neurons in the NAc of aged ratscan only be explained by an indirect action of this neurotrophin.Considering our observations and the fact that cholinergic neuronsof the NAc express trkA receptors (Sobreviela et al., 1994;Sofroniew et al., 2001; Venero et al., 1994), we have hypothesizedthat the age-related reduction in the total number of NPY neuronsmight be caused by insufficient trophic support provided by theircholinergic afferents. Indeed, it is known that NPY neurons of theNAc receive synaptic contacts from cholinergic fibers (Vuillet et al.,1992) and it has been shown that the loss of NPY neurons in theneocortex of aged rats occurs in parallel with reductions in thelevels of acetylcholinesterase (Zhang et al., 1998) and in the densityof cortical cholinergic varicosities (Cardoso et al., 2006). Thehypothesis that acetylcholine might act as a neurotrophic factoralso derives from studies showing that lesions of basal forebraincholinergic neurons led to a decrease in the number of peptidergicneurons in the hippocampal formation (Milner et al., 1997),neocortex (Zhang et al., 1998), and hypothalamus (Madeira et al.,2004). However, our study revealed that the total number ofcholinergic neurons in the NAc, which are the only recognizedsource of the cholinergic innervation of this nucleus (Meredith,1999; Pennartz et al., 1994), does not differ between adult andaged rats and that the density of cholinergic varicosities is likewisenot altered by aging. These findings were surprising because earlierstudies that have examined the whole striatum have shown thatthe density of acetylcholinesterase-immunoreactive neurons inmale rats (Altavista et al., 1988), and the number of acetylcholin-esterase- (Fischer et al., 1987) and ChAT-immunoreactive(Stemmelin et al., 2000) neurons in female rats decrease withaging. Even though the discrepancy between these and our owndata might be ascribed to differences in the quantitative methodsused (Altavista et al., 1988; Stemmelin et al., 2000) and/or the sex ofthe animals studied (Fischer et al., 1987; Stemmelin et al., 2000), itis probable that they might point toward the existence of regionspecificity in the effects of age on the brain cholinergic system(Allard et al., 2012; Bartus et al., 1982; Baskerville et al., 2006).

Our results also show that, in contrast to what has beenobserved in the neostriatum of male (Altavista et al., 1988) and inthe whole striatum of female (Fischer et al., 1987) rats, cholinergicneurons of the NAc are hypertrophied in old relative to adult malerats. In the brain of aged rats, neuronal hypertrophy has thus farbeen detected in regions such as the basal forebrain (Armstronget al., 1993) and hypothalamus (Madeira et al., 2000, 2001), andthere are reports of similar alterations in the brain of aged human(Cabello et al., 2002; de Lacalle et al., 1991; Rudow et al., 2008) andnonhuman primates (Stroessner-Johnson et al., 1992; Voytko et al.,1995). In the particular case of the NAc, the age-related increase inneuronal size is not accompanied by an increase in the density ofcholinergic varicosities, which shows that the hypertrophiedcholinergic neurons are not engaged in the synthesis of higheramounts of acetylcholine and suggests that the age-inducedchanges in the somatic size of these neurons might merely reflectneuronal dysfunction. Despite this fact, the cholinergic neurons ofthe NAc of aged rats are able to increase their activity in response tothe administration of NGF, as revealed by the simultaneous increasein the density of the cholinergic varicosities and the additionalenlargement in their somatic volume. The effect of NGF that wehave noticed in neuronal size is not unique because it was alreadyobserved in other cholinergic and noncholinergic neuronal pop-ulations of adult (Cadete-Leite et al., 2003; Hagg et al., 1989;Kordower et al., 1996; Paula-Barbosa et al., 2001) and old (Fischeret al., 1987; Niewiadomska et al., 2002; Pereira et al., 2005) maleand female rats. Concerning the cholinergic varicosities, it was alsodemonstrated that exogenous NGF increases their number to

Fig. 4. Graphic representation of the morphometric data obtained from the NAc of adult (adult), old (old), and NGF-treated old (oldþNGF) rats. Columns represent means andvertical bars � 1 SD. (A) Total number of NPY-ir neurons. The number of NPY-positive neurons is significantly reduced in old relative to adult rats. In NGF-treated old rats, the totalnumber of NPY-immunostained neurons does not differ from that of adult rats. (B) Somatic volume of NPY-immunopositive neurons. The somatic size of NPY-immunostainedneurons is similar in all groups analyzed. Tukey post hoc tests: * p < 0.005, compared with adult rats; þ p < 0.001, compared with aged rats. Abbreviations: NAc, nucleusaccumbens; NGF, nerve growth factor; NPY, neuropeptide Y; NPY-ir, NPY-immunoreactive.

P.A. Pereira et al. / Neurobiology of Aging 34 (2013) 1988e1995 1993

greater than control values in the cerebral cortex of rats submittedto unilateral devascularizing cortical lesions (Garofalo et al., 1992).

The observation that, in aged rats, there is no strict causal rela-tionship between the number of NPY neurons and the cholinergicinnervation of the NAc associated with the finding that, after NGFadministration, there is a parallel increase in the number of NPYneurons and in the density of cholinergic varicosities unveils thecomplexity of the regulation of NPY levels in this region of the brain.

Fig. 5. Graphic representation of the morphometric data obtained from the NAc of adultvertical bars � 1 SD. (A) Total number of ChAT-ir neurons. The total number of ChAT-positivneurons. The somatic size of NAc cholinergic neurons is significantly larger in old than in aduthese neurons. (C) Density of VAChT-positive varicosities. The graph shows pooled datadifferences were found in the density of cholinergic varicosities between adult and old rats.NGF-infused aged rats than in the NAc of adult and old rats. Tukey post hoc tests: * p < 0.0Abbreviations: ChAT, choline acetyltransferase; ChAT-ir, ChAT-immunoreactive; NAc, nucleu

In addition to acetylcholine (Milner et al., 1997; Zhang et al., 1998),several other neurotransmitters including dopamine (Lindeforset al., 1990; Obuchowicz et al., 2005; Salin et al., 1990, 1994;Smialowska,1995) seemtobe involved in the regulationofbrainNPYlevels. Yet, despite evidence indicating that dopaminergic neuro-transmission influences NPY levels, it is still not clear if its effects arestimulatory or inhibitory. In fact, whereas one study reporteda decrease in the number of NPY neurons in the ipsilateral

(adult), old (old), and NGF-treated old (oldþNGF) rats. Columns represent means ande neurons does not differ between groups. (B) Somatic volume of ChAT-immunostainedlt rats. NGF treatment of old rats leads to an additional increase in the somatic volume ofobtained from measurements in the core and in the shell of the NAc. No significantThe density of VAChT-immunoreactive varicosities is significantly higher in the NAc of05, ** p < 0.0005, compared with adult rats; þ p < 0.0005, compared with aged rats.s accumbens; NGF, nerve growth factor; VAChT, vesicular acetylcholine transporter.

P.A. Pereira et al. / Neurobiology of Aging 34 (2013) 1988e19951994

frontoparietal cortex after unilateral lesions of the ventral tegmentalarea (Lindefors et al., 1990), another showed that the pharmaco-logical blockade of dopaminergic receptors causes a significantincrease in NPY immunoreactivity in several cortical regions(Smialowska, 1995). It is known that striatal NPY neurons areinnervated by dopaminergic afferents originating in the ventraltegmental area and substantia nigra pars compacta (Groenewegenand Trimble, 2007; Threlfell and Cragg, 2011; Vuillet et al., 1989),and there is morphological and neurochemical evidence thatdopamine differently regulates the metabolic activity of neurons inthedorsal and in theventral striatum. Inparticular, itwas shown thatunilateral lesions of midbrain dopaminergic neurons cause anincrease in the numerical density of caudate-putamen neurons thatexpress NPY messenger RNA or protein (Lindefors et al., 1990; Salinet al., 1994), whereas selective lesions of the nigral dopaminergicneurons led to a decrease in the density of NPY neurons in the NAc(Salin et al., 1990). It is therefore likely that changes in the dopami-nergic innervation, or in the balance between dopamine andacetylcholine, might contribute to the reduction in the total numberof NPY neurons in the NAc of old rats inasmuch as there is evidencethat the basal levels of dopamine in this nucleus (Yoshimoto et al.,2001), and the concentration and binding potential of dopaminereceptors in striatal interneurons are markedly reduced in old rela-tive to adult rats (Umegaki et al., 2008). It is also conceivable that theeffect of NGF on the expression of NPY in the NAc of old rats mightrelyon its influenceondopaminergic afferents to theNAc. Actually, itwas demonstrated that this neurotrophin can increase the levels ofstriatal dopamine in mice (Garcia et al., 1992) and the release ofdopamine in neuronal cultures (Blöchl and Sirrenberg, 1996).

In conclusion, the present data show that aging causes a reduc-tion in the total number of NPY-immunoreactive neurons that isreversed by NGF. They also show that the age-associated changes inNPY neurons do not result from cholinergic dysfunction becauseaging does not alter the number of cholinergic neurons and thecholinergic innervation of the NAc. It is however possible that theenhanced availability of acetylcholine, consequent to the adminis-tration of NGF, might contribute, possibly associated with otherneurotransmitters, for the increase in NPY expression observed inaged rats. Our results might be of importance for understanding thestill cryptic role of the NPY-ergic and cholinergic systems of thestriatum in several age-associated functional and behavioral alter-ations and the potential therapeutic role of NGF in the treatment ofthese age-related changes.

Disclosure statement

The authors declare that there are no actual or potential conflictsof interest.

The experiments were performed in accordance with EuropeanCommunities Council Directive (2010/63/EU) of 22 September 2010and Portuguese Act n�129/92. All efforts were made to minimizethe number of animals used, and their discomfort and suffering.

Acknowledgements

This work was supported by National Funds through FCT -Fundação para a Ciência e a Tecnologia within the scope of theStrategic Project Centro deMorfologia Experimental (CME/FM/UP) -2011-2012 and Project PEst-OE/SAU/UI0121/2011.

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