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

Neurobiology of Aging 34 (2013) 1988e1995

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Nerve growth factor retrieves neuropeptide Y and cholinergic immunoreactivity in the nucleus accumbens of old rats Pedro A. Pereira*, Diana Santos, João Neves, M. Dulce Madeira, Manuel M. Paula-Barbosa Department 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

a b s t r a c t

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

The nucleus accumbens (NAc) contains high levels of neuropeptide Y (NPY), which is involved in the regulation of functions and behaviors that deteriorate with aging. We sought to determine if aging alters NPY expression in this nucleus and, in the affirmative, if those changes are attributable to the cholinergic innervation of the NAc. The total number and the somatic volume of NPY- and choline acetyltransferaseimmunoreactive 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 by 20% and their size was unaltered. The number of cholinergic neurons and the density of the cholinergic varicosities were unchanged, but their somas were hypertrophied. Nerve growth factor administration to aged rats further increased the volume of cholinergic neurons, augmented the density of the cholinergic varicosities, and reversed the age-related decrease in the number of NPY neurons. Our data show that the age-related changes in NPY levels in the NAc cannot be solely ascribed to the cholinergic innervation of the nucleus. Ó 2013 Elsevier Inc. All rights reserved.

Keywords: Stereology Nucleus accumbens Aging Neuropeptide Y Acetylcholine Nerve growth factor

1. Introduction The extensive use of magnetic resonance imaging, particularly during the past decade, allowed the demonstration of variations in the size of numerous regions of the human brain during the normal process of aging. Most of these studies agree that the volume or thickness of the gray matter decreases as age increases and that this variation, although with marked regional heterogeneity, occurs in the cortical gray matter and in deep subcortical telencephalic regions (for a review, see Walhovd et al., 2011). The nucleus accumbens (NAc) is such a region. Its volume is negatively correlated with age (Jernigan et al., 2001; Long et al., 2012; Walhovd et al., 2011) and, according to recently published data (Walhovd et al., 2011), is the brain structure that shows the largest estimated percentage of age difference. Another recent study (de Jong et al., 2012) has also revealed that the volume of the NAc is closely associated with the occurrence of dementia and predicts cognitive decline in older people. In the rat, the NAc is located in the rostroventral part of the striatum and is regarded as a functional interface between limbic and motor systems (for a review, see Groenewegen and Trimble, * Corresponding author at: Department of Anatomy, Faculty of Medicine, University of Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal. Tel.: þ351 22 5513616; fax: þ351 22 5513617. E-mail address: [email protected] (P.A. Pereira). 0197-4580/$ e see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2013.02.011

2007; Morgane et al., 2005). Approximately 90% of its neurons are densely spiny projection neurons, and the remaining are interneurons almost or completely devoid of spines that produce either gamma-aminobutyric acid or acetylcholine (Meredith, 1999). Gamma-aminobutyric acid interneurons costore various neuropeptides, including neuropeptide Y (NPY; Meredith, 1999) and cholinergic interneurons, represent approximately 1.7% of the total neuronal population of the striatum (Phelps et al., 1985), and are the only source of the dense cholinergic innervation of the NAc (Meredith, 1999; Pennartz et al., 1994). Despite the relatively small number of NPY and cholinergic neurons in the striatum, it contains one of the highest concentrations of NPY and acetylcholine in the brain (Hoover et al., 1978; Wettstein et al., 1995). NPY plays a crucial role in functions and behaviors that are frequently altered by aging, such as cognition, circadian rhythms and sleep, feeding, and cardiovascular regulation (for review, see Thorsell and Ehlers, 2006; Wettstein et al., 1995). There is evidence that aging is associated with reduced NPY levels in several regions of the brain, namely the brainstem, hypothalamus, hippocampal formation, and neocortex (Cadacio et al., 2003; Cardoso et al., 2006; Cha et al., 1997; Huguet et al., 1993; Huh et al., 1997; Kowalski et al., 1992; Zhang et al., 1998). However, data about age-related effects on the NAc are scarce and controversial, with one study (Huh et al., 1997) reporting unchanged, and another (Cha et al., 1997) slightly decreased numbers of NPY-immunoreactive neurons in old relative to adult rats. Acetylcholine has been likewise implicated in modulating

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functions that deteriorate with aging, such as sleepewake cycles, cognitive performance, learning, and memory (Bartus, 2000; Everitt and Robbins, 1997; Hasselmo, 2006; Jones, 2008; Sarter et al., 2003; Schliebs and Arendt, 2011; Steriade, 2004). In particular, striatal cholinergic neurotransmission is involved in ageassociated cognitive impairment (Lazaris et al., 2003; Ragozzino et al., 2009; Stemmelin et al., 2000) and, via interaction with dopamine, in motor control (Umegaki et al., 2008), both of which are altered by aging. Although there are a few studies showing ageassociated changes in the striatal cholinergic system, namely reductions 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; Wu et al., 1988) and cholinesterase activity (Das et al., 2001) in aged rats compared with young rats, no investigations have examined the effects of aging on the cholinergic neurons of the NAc. The present study was designed to investigate, using stereological methods, whether there are age-related changes in the total number and somatic volume of NPY and cholinergic neurons in the NAc of male rats and, in the affirmative, if those changes can be ascribed to the age-associated disruption of nerve growth factor (NGF) trophic support (Bruno and Cuello, 2012; Sofroniew et al., 2001; Williams et al., 2006). Notably, age-related atrophy of basal forebrain cholinergic neurons can be reversed by administration of NGF, resulting in amelioration of age-related cognitive 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 can revert the age-related changes in the number of NPYimmunoreactive neurons in the somatosensory cortex (Cardoso et al., 2006), and of vasopressin- and vasoactive intestinal polypeptide-producing neurons in the suprachiasmatic nucleus (Pereira et al., 2005) of old rats. Thus, to examine if the levels of NPY and acetylcholine in the NAc are dependent on NGF trophic support we have estimated the total number and the somatic size of neurons producing NPY and acetylcholine, and the density of cholinergic varicosities in the NAc of old rats that were treated, during the last 12 days of the experiment, with NGF delivered intracerebroventricularly. 2. Methods 2.1. Animals and treatments A total of 15 male Wistar rats, 5 young (6 months old) and 10 aged (24 months old), were used in the present study. Animals were housed in a temperature controlled room (22  C) in 12-hour light/dark cycles (lights on at 7:00 AM) with solid diet (4RF21/C; Mucedola, Milan, Italy) and water available ad libitum. Half of the aged rats (n ¼ 5) were randomly selected and infused with 2.5S NGF (Prince Laboratories, Toronto, Ontario, Canada) during 12 days before death (see details in section 2.2.). Because there is evidence that this surgical procedure does not interfere with the central NPY-ergic (Cardoso et al., 2006) and cholinergic (Cadete-Leite et al., 2003) systems, vehicle-treated rats were not included in this study. The experiments were performed in accordance with European Communities Council Directive (2010/63/EU) of 22 September 2010 and Portuguese Act n 129/92. All efforts were made to minimize the number of animals used, and their discomfort and suffering. 2.2. Surgical procedures and drug treatment For intracerebroventricular administration of NGF, rats were anesthetized by sequentially injecting, at intervals of 10 minutes,

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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 Company Ltd, 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 and lambda in the same horizontal plane. After a midline skin incision, the calvaria were exposed. For intracerebroventricular delivery of NGF, permanent stainless steel cannulae (Alzet brain infusion kit; Alza Corporation, Palo Alto, CA, USA) were stereotaxically placed in the right lateral ventricle, 1.1 mm posterior to the bregma, 1.7 mm lateral to the midline, and 4.0 mm below the surface of the skull (Paxinos and Watson, 1998). The cannulae were connected to methylene blue (0.01%; Sigma) filled Alzet osmotic minipumps (model 2002) via sterile coiled polyethylene tubing (PE-60; Intramedic, Becton Dickinson, Sparks, MD, USA). This tubing was filled with aireoil spacer at the pump end and with NGF (150 mg diluted in 150 mL of vehicle composed of artificial cerebrospinal fluid supplemented with 0.1% bovine serum albumin; Sigma). Osmotic minipumps were pretested to confirm their delivery rate, and implanted subcutaneously in the neck. Skin incisions were closed with surgical stitches and treated with local antiseptic. After surgery, rats were individually housed and maintained in a warm place until they woke up. Postoperative care consisted of subcutaneous injections of 0.9% saline (2 mL), during the 48 hours after surgery, to prevent dehydration and weight loss. Twelve days after the beginning of NGF infusion, rats were killed and the total infusion volume was calculated. The mean volume of NGF injected per rat was 117.27  29.35 mL and the mean flow rate of the pumps was 0.41  0.10 mL/h. 2.3. Tissue preparation Rats were deeply anesthetized by intraperitoneal injection of a solution (3 mL/kg body weight) containing 1% sodium pentobarbital and 4% chloral hydrate in physiological saline. They were then perfused transcardially with 150 mL of 0.1 M phosphate buffer (PB; pH 7.6) for vascular rinse, followed by 250 mL of a fixative solution containing 4% paraformaldehyde in PB, at pH 7.6. The brains were removed from the skulls, coded, immersed for 1 hour in the same fixative, and maintained overnight in a solution of 10% sucrose in PB, at 4  C. After trimming away the occipital poles, the blocks were placed on a vibratome and serially sectioned in the coronal plane at 40 mm through the NAc. The sections were collected in phosphate-buffered saline (PBS). From the entire set of sections obtained from each brain, 4 series were formed using a systematic, random sampling procedure (Gundersen et al., 1999). Accordingly, the first section was randomly selected from the first group of 4 collected sections, and the remaining were sampled, along the entire rostrocaudal extent of the NAc, at regular intervals of 160 mm (i.e., 1 out of 4 sections). The first, second, and third series were used for NPY, choline acetyltransferase (ChAT) and vesicular acetylcholine 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), sections were washed twice in PBS, treated with 3% H2O2 for 10 minutes to inactivate 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 antirabbit antibody (Vector Laboratories, Burlingame, CA, USA; 1:400 dilution in PBS) was used as the secondary antibody. For visualization of ChAT-immunoreactive neurons (Figs. 1 and 2), sections were

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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 for neuropeptide 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.

pretreated as described above in section 2.4. and incubated, for 48 hours at 4  C, with the primary antiserum against ChAT (AB144P; Chemicon, Millipore Corporation, Billerica, MA, USA; 1:2000 dilution in PBS). Biotinylated rabbit anti-goat antibody (Vector Laboratories; 1:400 dilution in PBS) was used as the secondary antibody. For VAChT immunohistochemistry (Fig. 2), sections were immersed in a 5% solution of rabbit normal serum (Vector Laboratories) in PBS, for 30 minutes at room temperature. Thereafter, they were incubated, for 72 hours at 4  C, with the primary antiserum against VAChT (AB1578, Chemicon, Millipore Corporation; 1:15000 dilution in 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 were treated with avidinebiotin peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories; 1:800 dilution in PBS). In the last 2 steps, the incubation was carried out for at least 1 hour at room temperature. After treatment with peroxidase complex, sections were incubated for 10 minutes in 0.05% diaminobenzidine (Sigma)

to which H2O2 was added to a final concentration of 0.01%. Sections were rinsed with PBS for at least 15 minutes between each step. To increase tissue penetration, 0.5% Triton X-100 was added to the PBS used in all immunoreactions and washes. All procedures were performed on a rocking table. Immunostained sections were mounted on gelatin-coated slides and air-dried. Then, they were dehydrated in a series of ethanol solutions (50%, 70%, 90%, and 100%), cleared in xylol, and coverslipped using Histomount (National Diagnostics, Atlanta, GA, USA). To prevent variability in staining, sections from all groups analyzed were processed in parallel at the same time. The same procedure was followed for control sections, which were incubated without antiserum; no immunostaining was observed in these sections (data not shown). The Nissl-stained sections (Fig. 1) were used for estimating the total number of NAc neurons and for help in identifying the boundaries of the NAc in immunostained sections. The sections were mounted serially on gelatin-coated slides. After air-drying overnight at room temperature, they were stained with Cresyl

Fig. 2. ChAT and VAChT immunoreactivity in the NAc of adult (A) and (D), aged (B) and (E), and NGF-treated aged (C) and (F) rats. Photomicrographs show coronal sections through approximately the mid-level of the NAc. The smaller size of the ChAT-positive neuronal cell body of the adult rat (A) relative to those of old (B) and NGF-treated old (C) rats is evident. In the latter animal (C), the size of the ChAT-immunoreactive neuronal profile is obviously larger than in the old rat (B).The density of VAChT-immunoreactive varicosities in the core (DeF) and in the shell (not shown) of the NAc is similar in adult and aged rats and markedly higher in NGF-infused old rats. Scale bars: 10 mm. Abbreviations: ChAT, choline acetyltransferase; NAc, nucleus accumbens; NGF, nerve growth factor; VAChT, vesicular acetylcholine transporter.

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Violet, dehydrated, and coverslipped with Histomount (National Diagnostics). 2.5. Stereological analyses The optical fractionator (Madeira et al., 1995; West et al., 1991) was used to estimate total neuron numbers. Cell counting was carried out on blind-coded slides using an Olympus C.A.S.T.-Grid system (Olympus DK A/S, version 2.00) and a Hiedenhain MT-12 microcator. As a general rule, neurons were counted, at final magnification 2000, using neuronal nuclei as the counting unit; neuronal nuclei touching the left or bottom sides of the counting frame were discarded. All Nissl-stained sections and all sections immunostained for NPY and ChAT that contained the NAc were used, which provided an average of 13 sections per nucleus analyzed. In each Nissl-stained section, the fields of view were systematically sampled using an interframe distance of 600 mm (xand y-axes). Actual cell counting was done using a dissector height of 10 mm and a counting frame area of 791 mm2 at the tissue level. On average, 157 neurons were counted per nucleus; the mean coefficient of error (CE) of the estimates was 0.09. The sampling scheme used for estimating the total number of NPY- and ChATimmunoreactive cells in the NAc was as described in section 2.5., with the following modifications: microscope fields were sampled using a step size of 250 mm (x- and y-axes) for NPY neurons and of 200 mm (x- and y-axes) for ChAT neurons. The area of the counting frames was 8436 mm2 and 8419 mm2, respectively, and the height of the dissector was 10 mm for both neuronal populations. By applying this sampling scheme, an average of 186 NPY- and 128 ChATimmunoreactive cells was counted per nucleus. The mean CE of the estimates was 0.08 and 0.10, respectively. The mean somatic volume of NPY- and ChAT-immunostained neurons was estimated by applying the optical rotator (Leal et al., 1998; Tandrup et al., 1997). Neurons used for measurements were selected with optical dissectors, as described in section 2.5. These procedures were implemented with the C.A.S.T.-Grid system software (version 2.00), which allows the estimation of the mean somatic volume using a spatial line grid. Measurements of intersections between the cell membrane and the spatial line grid were performed using a 2-grid line and 2 focal planes per each cell, at magnification 2000. The mean CE of the estimates was 0.07 and 0.09, respectively. The density of the cholinergic varicosities (Fig. 2) was estimated from VAChT-immunostained sections (n ¼ 5 per animal) selected at mid-NAc levels. Varicosities were counted using a computerassisted image analyzer (Leica QWin) fitted with a Leica DMR microscope and a Leica DC 300F video camera, at final magnification 1000. Considering the obvious difference in the density of the cholinergic 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 core and 2 for shell, were randomly selected. Because the variations in the core and in the shell were of the same type and magnitude data were pooled. The varicosities were defined as darkly stained axonal dilations with size greater than 0.25 mm2 (Cardoso et al., 2006). A sample frame (1.52  106 mm2) was laid over each field of view and the number of varicosities falling within it was counted. Results were expressed as areal densities (n/mm2).

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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, as described by West et al. (1991). Data were analyzed by one-way analysis of variance (ANOVA) followed by pairwise post hoc comparisons 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 abundant in the NAc whereas ChAT-immunoreactive neurons are relatively sparse. A moderately dense NPY-immunoreactive fiber plexus was seen in the shell and core of the NAc (Fig. 1B and D). In agreement with earlier observations (Meredith, 1999; Vuillet et al., 1992), neurons immunoreactive for NPY are mediumsized and neurons immunoreactive for ChAT have larger cell bodies (Fig. 1D and E). Aging was associated with hypertrophy of ChAT-immunoreactive neuronal cell bodies, but did not change the density of VAChT-immunoreactive fibers in the NAc (Fig. 2). As can be seen in Fig. 2, the administration of NGF to aged rats leads to a further enlargement of ChAT neuronal cell bodies and distinctly increases the density of the VAChT-immunoreactive fibers in the NAc. 3.2. Total number of NAc neurons As showed using ANOVA, aging and NGF administration did not produce significant variations (F(2,12) ¼ 0.22; p ¼ 0.80) in the total number 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 NPYimmunoreactive neurons (Fig. 4A). ANOVA revealed that aging and NGF administration significantly influence the total number of NPY-immunoreactive neurons (F(2,12) ¼ 14.85; p < 0.001). In aged rats the total number of neurons was reduced by approximately 20%, a difference that is statistically significant. Treatment of aged rats with NGF was associated with an increase in the total number of NPY-immunoreactive neurons to values similar to those of adult rats. Conversely, the mean somatic volume of NPY-immunoreactive neurons (Fig. 4B) was unaltered in aged rats and did not change in response 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 4426 cholinergic neurons in adult rats, and that this number is not altered 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 administration (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 in adult rats, and the administration of NGF to old rats caused an additional increase in the somatic volume of these neurons.

2.6. Statistical analyses

3.5. Density of cholinergic varicosities

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

The density of cholinergic varicosities was significantly influenced by aging and NGF administration (F(2,12) ¼ 39.89; p < 0.00001). Specifically, the density of VAChT-positive fiber varicosities was similar in adult and aged rats, and the administration of

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

NGF to old rats increased, by approximately 47%, the density of the varicosities in the NAc (Figs. 2 and 5C). 4. Discussion Thus far, the question of whether aging interferes with the content of NPY in the NAc has been addressed in only two studies that, however, yielded different conclusions. Specifically, Huh et al. (1997) found no changes in the density of NPY-immunoreactive neurons, and Cha et al. (1997) detected a mild (<15%) reduction in the number of NPY-immunoreactive neurons counted from a series of level-matched sections of the nucleus. Our data, estimated by applying stereological methods, not only support the last conclusion, because we found a 20% reduction in the total number of NPY-immunoreactive neurons in the NAc of old rats, but also extends it by showing that these neurons do not undergo agerelated changes in their somatic size. The reduction in the number of NPY-immunoreactive neurons is not because of cell death because we have also established that the total number of NAc neurons is identical in adult and in old rats. It is thus very probable that the decrease in neuron numbers that we have observed might be a consequence of reduced NPY synthesis, a hypothesis that is in line with data from studies showing lower NPY messenger RNA levels in the hypothalamus of old relative to adult rats (reviewed in Kmiec, 2011). Previous studies have revealed that striatal NPY neurons do not express NGF receptors (Barrett et al., 2005; Sobreviela et al., 1994;

Venero et al., 1994). Therefore, the NGF-induced increase in the number of NPY-immunoreactive neurons in the NAc of aged rats can only be explained by an indirect action of this neurotrophin. Considering our observations and the fact that cholinergic neurons of the NAc express trkA receptors (Sobreviela et al., 1994; Sofroniew et al., 2001; Venero et al., 1994), we have hypothesized that the age-related reduction in the total number of NPY neurons might be caused by insufficient trophic support provided by their cholinergic afferents. Indeed, it is known that NPY neurons of the NAc receive synaptic contacts from cholinergic fibers (Vuillet et al., 1992) and it has been shown that the loss of NPY neurons in the neocortex of aged rats occurs in parallel with reductions in the levels of acetylcholinesterase (Zhang et al., 1998) and in the density of cortical cholinergic varicosities (Cardoso et al., 2006). The hypothesis that acetylcholine might act as a neurotrophic factor also derives from studies showing that lesions of basal forebrain cholinergic neurons led to a decrease in the number of peptidergic neurons 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 of cholinergic neurons in the NAc, which are the only recognized source of the cholinergic innervation of this nucleus (Meredith, 1999; Pennartz et al., 1994), does not differ between adult and aged rats and that the density of cholinergic varicosities is likewise not altered by aging. These findings were surprising because earlier studies that have examined the whole striatum have shown that the density of acetylcholinesterase-immunoreactive neurons in male rats (Altavista et al., 1988), and the number of acetylcholinesterase- (Fischer et al., 1987) and ChAT-immunoreactive (Stemmelin et al., 2000) neurons in female rats decrease with aging. Even though the discrepancy between these and our own data might be ascribed to differences in the quantitative methods used (Altavista et al., 1988; Stemmelin et al., 2000) and/or the sex of the animals studied (Fischer et al., 1987; Stemmelin et al., 2000), it is probable that they might point toward the existence of region specificity 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 been observed in the neostriatum of male (Altavista et al., 1988) and in the whole striatum of female (Fischer et al., 1987) rats, cholinergic neurons of the NAc are hypertrophied in old relative to adult male rats. In the brain of aged rats, neuronal hypertrophy has thus far been detected in regions such as the basal forebrain (Armstrong et al., 1993) and hypothalamus (Madeira et al., 2000, 2001), and there are reports of similar alterations in the brain of aged human (Cabello et al., 2002; de Lacalle et al., 1991; Rudow et al., 2008) and nonhuman primates (Stroessner-Johnson et al., 1992; Voytko et al., 1995). In the particular case of the NAc, the age-related increase in neuronal size is not accompanied by an increase in the density of cholinergic varicosities, which shows that the hypertrophied cholinergic neurons are not engaged in the synthesis of higher amounts of acetylcholine and suggests that the age-induced changes in the somatic size of these neurons might merely reflect neuronal dysfunction. Despite this fact, the cholinergic neurons of the NAc of aged rats are able to increase their activity in response to the administration of NGF, as revealed by the simultaneous increase in the density of the cholinergic varicosities and the additional enlargement in their somatic volume. The effect of NGF that we have noticed in neuronal size is not unique because it was already observed in other cholinergic and noncholinergic neuronal populations of adult (Cadete-Leite et al., 2003; Hagg et al., 1989; Kordower et al., 1996; Paula-Barbosa et al., 2001) and old (Fischer et al., 1987; Niewiadomska et al., 2002; Pereira et al., 2005) male and female rats. Concerning the cholinergic varicosities, it was also demonstrated that exogenous NGF increases their number to

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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 and vertical 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 total number of NPY-immunostained neurons does not differ from that of adult rats. (B) Somatic volume of NPY-immunopositive neurons. The somatic size of NPY-immunostained neurons 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, nucleus accumbens; NGF, nerve growth factor; NPY, neuropeptide Y; NPY-ir, NPY-immunoreactive.

greater than control values in the cerebral cortex of rats submitted to unilateral devascularizing cortical lesions (Garofalo et al., 1992). The observation that, in aged rats, there is no strict causal relationship between the number of NPY neurons and the cholinergic innervation of the NAc associated with the finding that, after NGF administration, there is a parallel increase in the number of NPY neurons and in the density of cholinergic varicosities unveils the complexity of the regulation of NPY levels in this region of the brain.

In addition to acetylcholine (Milner et al., 1997; Zhang et al., 1998), several other neurotransmitters including dopamine (Lindefors et al., 1990; Obuchowicz et al., 2005; Salin et al., 1990, 1994; Smialowska,1995) seem to be involved in the regulation of brain NPY levels. Yet, despite evidence indicating that dopaminergic neurotransmission influences NPY levels, it is still not clear if its effects are stimulatory or inhibitory. In fact, whereas one study reported a decrease in the number of NPY neurons in the ipsilateral

Fig. 5. Graphic representation of the morphometric data obtained from the NAc of adult (adult), old (old), and NGF-treated old (oldþNGF) rats. Columns represent means and vertical bars  1 SD. (A) Total number of ChAT-ir neurons. The total number of ChAT-positive neurons does not differ between groups. (B) Somatic volume of ChAT-immunostained neurons. The somatic size of NAc cholinergic neurons is significantly larger in old than in adult rats. NGF treatment of old rats leads to an additional increase in the somatic volume of these neurons. (C) Density of VAChT-positive varicosities. The graph shows pooled data obtained from measurements in the core and in the shell of the NAc. No significant differences were found in the density of cholinergic varicosities between adult and old rats. The density of VAChT-immunoreactive varicosities is significantly higher in the NAc of NGF-infused aged rats than in the NAc of adult and old rats. Tukey post hoc tests: * p < 0.005, ** p < 0.0005, compared with adult rats; þ p < 0.0005, compared with aged rats. Abbreviations: ChAT, choline acetyltransferase; ChAT-ir, ChAT-immunoreactive; NAc, nucleus accumbens; NGF, nerve growth factor; VAChT, vesicular acetylcholine transporter.

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frontoparietal cortex after unilateral lesions of the ventral tegmental area (Lindefors et al., 1990), another showed that the pharmacological blockade of dopaminergic receptors causes a significant increase in NPY immunoreactivity in several cortical regions (Smialowska, 1995). It is known that striatal NPY neurons are innervated by dopaminergic afferents originating in the ventral tegmental area and substantia nigra pars compacta (Groenewegen and Trimble, 2007; Threlfell and Cragg, 2011; Vuillet et al., 1989), and there is morphological and neurochemical evidence that dopamine differently regulates the metabolic activity of neurons in the dorsal and in the ventral striatum. In particular, it was shown that unilateral lesions of midbrain dopaminergic neurons cause an increase in the numerical density of caudate-putamen neurons that express NPY messenger RNA or protein (Lindefors et al., 1990; Salin et al., 1994), whereas selective lesions of the nigral dopaminergic neurons 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 dopaminergic innervation, or in the balance between dopamine and acetylcholine, might contribute to the reduction in the total number of NPY neurons in the NAc of old rats inasmuch as there is evidence that the basal levels of dopamine in this nucleus (Yoshimoto et al., 2001), and the concentration and binding potential of dopamine receptors in striatal interneurons are markedly reduced in old relative to adult rats (Umegaki et al., 2008). It is also conceivable that the effect of NGF on the expression of NPY in the NAc of old rats might rely on its influence on dopaminergic afferents to the NAc. Actually, it was demonstrated that this neurotrophin can increase the levels of striatal dopamine in mice (Garcia et al., 1992) and the release of dopamine in neuronal cultures (Blöchl and Sirrenberg, 1996). In conclusion, the present data show that aging causes a reduction in the total number of NPY-immunoreactive neurons that is reversed by NGF. They also show that the age-associated changes in NPY neurons do not result from cholinergic dysfunction because aging does not alter the number of cholinergic neurons and the cholinergic innervation of the NAc. It is however possible that the enhanced availability of acetylcholine, consequent to the administration of NGF, might contribute, possibly associated with other neurotransmitters, for the increase in NPY expression observed in aged rats. Our results might be of importance for understanding the still cryptic role of the NPY-ergic and cholinergic systems of the striatum in several age-associated functional and behavioral alterations and the potential therapeutic role of NGF in the treatment of these age-related changes. Disclosure statement The authors declare that there are no actual or potential conflicts of interest. The experiments were performed in accordance with European Communities Council Directive (2010/63/EU) of 22 September 2010 and Portuguese Act n 129/92. All efforts were made to minimize the 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 the Strategic Project Centro de Morfologia Experimental (CME/FM/UP) 2011-2012 and Project PEst-OE/SAU/UI0121/2011. References Allard, S., Scardochio, T., Cuello, A.C., Ribeiro-da-Silva, A., 2012. Correlation of cognitive performance and morphological changes in neocortical pyramidal neurons in aging. Neurobiol. Aging 33, 1466e1480.

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