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Dopaminergic mesencephalic systems and behavioral performance in very old rats
Neuroscience 154 (2008) 1598 –1606
DOPAMINERGIC MESENCEPHALIC SYSTEMS AND BEHAVIORAL PERFORMANCE IN VERY OLD RATS H. L. SANCHEZ,a* L. B. SILVA,a E. L. PORTIANSKY,b C. B. HERENU,c R. G. GOYAc AND G. O. ZUCCOLILLIa
mainly located within the substantia nigra (SN) and the ventral tegmental area (VTA) which correspond to the A9 and A10 catecholaminergic areas described by Dahlström and Fuxe (1964) (Tillet and Kitahama, 1998), respectively (Fig. 1). They form two long-length DA systems, the nigrostriatal and meso-cortico-limbic systems. The former is represented by the nigral A9 neurons projecting mainly to the neostriatum (caudate nucleus and putamen). The meso-cortico-limbic system is formed by the A10 neurons, which spread out axons from the VTA to striatal, limbic and cortical areas (Bentivoglio and Morelli, 2005). This has long been recognized as an oversimplification because SN contains not only neurons projecting to the striatum, but also neurons that innervate cortical and limbic areas (Björklund and Dunnett, 2007). Thus, dense projections of the ventral and intermediate sheets of the substantia nigra pars compacta (SNpc) and ventro-lateral VTA innervate the caudate-putamen (Gerfen, 1992). DA neurons located within medial SNpc and middorsal VTA project to the nucleus accumbens and olfactory tubercle, whereas dopamine projections to the amygdaloid nuclei arise from lateral SNpc and SN lateral (Fallon and Loughlin, 1995). VTA DA cell bodies distributed in a loosely topographical fashion and nigral DA neurons of the dorsal-most sheet of SNpc innervate the prefrontal, cingulated, perirhinal and entorhinal cortices (Fallon, 1988). In addition, sparse DA projections have been reported arising from SN and VTA to connect with neurons of other neocortical fields (Berger et al., 1991), cerebellum (Ikai et al., 1992), hypothalamus, hippocampus, pallidum and locus coeruleus (Fallon and Moore, 1978a,b). In the adult rat, the total number of mesencephalic DA cells bilaterally is 40,000 – 45,000, with about half of the neurons located in the SN (German and Manaye, 1993). Morphologic and functional studies in laboratory animals describing the impact of aging on mesencephalic DA neurons are rather scanty and inconclusive. In squirrel monkeys, it has been reported that aging causes either a significant loss of tyrosine hydroxylase immunoreactive (TH-ir; i.e. DA neurons) cells in the SN (McCormack et al., 2004) or no change (Irwin et al., 1994). Despite this discrepancy both studies reported a marked reduction of motor activity in the aged monkeys. In rhesus monkeys, a significant (50.3%) age-related loss of TH-ir neurons which correlated with a marked fall in motor performance was reported (Emborg et al., 1998). The only stereological study in aging mice we are aware of reported a significant loss of TH-ir neurons in the SN of 104-week-old animals as compared with 8-week-old counterparts (Tatton et al., 1991). We are unaware of stereological studies on the mesencephalic DA neurons of aging rats and have only detected
a
Institute of Anatomy, Faculty of Veterinary Sciences, National University of La Plata, CC296, Calle 60 y 118, 1900 La Plata, Argentina
b
Institute of Pathology, School of Veterinary Sciences, National University of La Plata, La Plata, Argentina
c
INIBIOLP-Histology “B,” School of Medicine, National University of La Plata, La Plata, Argentina
Abstract—Morphologic and functional studies describing the impact of aging on mesencephalic dopaminergic (DA) neurons in laboratory animals are rather scanty and inconclusive. In rats, stereological studies characterizing age changes in the mesencephalic DA neurons have not been documented. In order to fill this information gap and to determine whether the very old rat may serve as a suitable animal model of Parkinson’s disease, we performed a stereological assessment of the mesencephalic tyrosine hydroxylase immunoreactive (TH-ir) neurons in young-adult (4 – 6 months), old (22–24 months) and senile (30 –32 months) Sprague–Dawley female rats. Morphometric analysis of the TH-ir neurons of the substantia nigra (SN) and ventral tegmental area (VTA) was performed using an appropriate image analysis system. Age changes in motor performance were assessed measuring the endurance of rats to hang from a wire mesh pole or to remain on a ramp set at different angles to the floor. Age changes in locomotion and exploratory activity were evaluated by the open field test. We observed a significant age-related reduction in TH-ir neuron numbers in the SN (17 and 33% reduction in old and senile rats, respectively compared with young counterparts) but not in the VTA. The size of the TH-ir cells increased significantly in both the SN and VTA of the senescent animals but TH labeling intensity fell. Motor, locomotor and exploratory performance deteriorated markedly in the old and senile rats as compared with young animals. These findings reveal the existence of a moderate but significant vulnerability of mesencephalic DA neurons to aging in rats. This phenomenon, which is particularly marked in the SN of very old rats, may contribute to the age-related decline in motor and exploratory performance recorded in this species. © 2008 Published by Elsevier Ltd on behalf of IBRO. Key words: aging, mesencephalic, tyrosine hydroxylase, morphometry, motor tests, open field.
Mesencephalic dopaminergic (DA) neurons account for 80% of brain DA neurons. The perikarya of these cells are *Corresponding author. Tel: ⫹54-221-425-6735; fax: ⫹54-221-425-3276. E-mail address: [email protected] (please cc rgoya@netverk. com.ar) (H. L. Sanchez). Abbreviations: ANOVA, analysis of variance; DA, dopaminergic; DAB, 3,3-diaminobenzidine tetrahydrochloride; IA, interaural; ml, medial lemniscus; mp, mammillary peduncle; SN, substantia nigra; SNpc, substantia nigra pars compacta; TH-ir, tyrosine hydroxylase immunoreactive; VTA, ventral tegmental area. 0306-4522/08$32.00⫹0.00 © 2008 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2008.04.016
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Fig. 1. Schematic representation of coronal sections of the midbrain showing the distribution of TH-ir neuron bodies. (a) Coronal section at the level of the mammillary body (MB) showing the distribution of TH-ir neuron bodies in the SN correspond to the A9, cerebral peduncle (cp), mp, periaqueductal gray (PAG), third ventricle (3V), posterior commissure (pc). (b) Coronal section at the level of the interpeduncular nucleus (ip) showing the distribution of TH-ir neuron bodies in the VTA correspond to the A10, cerebral peduncle (cp), ml, central linear nucleus raphé (CL), red nucleus (R), aqueduct (Aq), commissure superior colliculus (CSC).
a study reporting neurochemical but not morphologic changes (histologically assessed) in nigrostriatal TH-ir neurons of 24 to 25-month-old male rats as compared with 5 to 6-month-old counterparts (Emerich et al., 1993). The authors documented a significant reduction in locomotor activity and motor performance in the aged animals. In the present study we employed an unbiased stereological approach to quantitate the impact of old and very old age on the number and morphology of mesencephalic TH-ir neurons. Since direct evaluation of nigral DA function by the apomorphine- or amphetamine-induced rotation tests can only be performed in unilaterally lesioned animals (not in intact animals with symmetric nigral DA neuron loss), we assessed age-changes in motor performance and exploratory activity in an attempt to gain a general idea on the correlation between morphometric changes in nigral DA neurons and those in behavioral parameters.
National Institutes of Health (INIBIOLP’s Animal Welfare Assurance No A5647-01). The minimum number of rats compatible with detecting reasonable (⬎25%) differences in the different parameters studied was used and every effort was made to avoid or at least minimize the suffering of animals during the experiments performed.
Killing and specimen collection Animals were anesthetized with ketamine hydrochloride (40 mg/kg; i.p.) plus xylazine (8 mg/kg; i.m.) and perfused transcardially with buffered saline– 4% formaldehyde solution. The brain was carefully removed from the cranium, equilibrated in a cryoprotectant solution containing 30% sucrose, 0.1 M PB (0.1 M Na2HPO4 buffer) in H2O and stored at ⫺20 °C until processing. Each brain was trimmed down to a block containing the whole mesencephalon, from the rostral level of mammillary body (interaural (IA) line 4.20 mm) to the caudal level of the pontomesencephalic grove (IA line 2.28 mm) (Paxinos and Watson, 1998). The block was then serially cut into coronal sections 40 m thick on a freezing microtome.
Immunohistochemistry
EXPERIMENTAL PROCEDURES Animals and specimen collection Young (5– 6 months), old (22–24 months), and senile (30 –32 months) Sprague–Dawley female rats, raised in our aging rat colony, were used for morphological studies. For motor and open field tests, slightly different age groups were employed. Animals were housed in a temperature-controlled room (21⫾2 °C) on a 12-h light/dark cycle. Rats were housed in groups of four in stainless steel cages (32⫻32⫻18 cm, L⫻W⫻H). Food and water were available ad libitum. In our rat colony, the average 50% survival time for females, studied in groups of 50 – 60 animals, is 31 months (range, 30 –32 months). Animal experiments were done following the Guidelines on the Use of Animals in Neuroscience Research (the Society of Neuroscience) using IACUC approved procedures (approval date 06/01/04) and Animal Welfare Guidelines of the
Stereological assessment of brain structures was carried out using a simplified variant of the optical fractionator method (West et al., 1991). In each animal, one of every four mesencephalic serial sections was sampled throughout this structure. The sections were submitted to free floating immunohistochemistry using an anti-TH monoclonal antibody (Calbiochem, Inc., La Jolla, CA, USA). As a detection system, the Vectastain Universal ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA) was used, with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO, USA) used as chromogen. Sections were dehydrated and mounted for light microscopy assessment.
Image analysis The images of the hemimesencephalon (objective 1.25⫻), the whole SN and VTA (objective 4⫻) and the TH-ir neurons (objective 20⫻) were captured with a digital color video camera (EvolutionVF, QIm-
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aging, Surrey, BC, Canada) attached to a microscope (Olympus BX50, Olympus Ltd., Tokyo, Japan) and connected to a high performance computer loaded with the Image Pro Plus v6.2 (Media Cybernetics, Silver Spring, MD, USA) image analysis software. Images were captured with a pixel depth of 24 bits, RGB and TIFF format. The hemimesencephalon and the SN and VTA were digitally circumscribed and their respective TH-ir and mesencephalic areas determined. The sections from different animals corresponding to the same level were aligned using neuroanatomical markers. Measurements were thus recorded for the overall structure and for different rostrocaudal levels. The recognition of forebrain structures in co-stained sections was accomplished by inspection of TH-ir adjacent sections and the Paxinos and Watson (1998) atlas of the rat brain. The VTA and SN were outlined for each section and analyzed according to TH-ir adjacent sections. In order to delimit the SN from the VTA, the following tracts of fibers were used from rostral to caudal: mammillary peduncle (mp), emergence of the III cranial pair (3n) and medial lemniscus (ml) (Fig. 1). Immunoreactive perikarya showing clearly shaped nuclei were characterized using the following parameters: cellular area, major and minor cell axis, perimeter and cell roundness. THimmunoreactive neuronal bodies were counted. The intensity of labeling, as determined by DAB staining (optical density), was also recorded. The total number of TH-ir perikarya was estimated using the following equation: n
1 N ⫽ ssf
兺x
Open field test Rats were individually placed in a Plexiglas square arena (45⫻45⫻35 cm) divided into nine equal sectors on the floor. The number of sector crossings (with all four paws), episodes of selfgrooming and rearing was recorded for 5 min.
Statistical analysis The analysis of variance (ANOVA) followed by the Bonferroni post hoc test was used to evaluate intra- and inter-group differences in morphometric variables (Zar, 1984), while the non-parametric Kruskal-Wallis one-way ANOVA followed by the Dunn’s test was used to analyze behavioral data. Significant differences between age groups for each parameter were defined as those with P⬍0.05. Highly significant differences were defined as those with a P⬍0.01.
RESULTS Morphologic age changes in TH-ir mesencephalic neurons There was a macroscopically detectable age-related reduction in the TH-ir staining in coronal sections of the SN but not of the VTA (Figs. 2 and 3, respectively, left panels). A significant age-related reduction in neuronal density was observed in both regions (Tables 1 and 2).
t⫺1
where, N⫽total estimated number of TH-ir perikarya per region; ssf⫽section sampling fraction (1/4); n⫽number of sections sampled per region; x⫽number of TH-ir perikarya counted per sampled section x (from sampled section # 1 to # n). Each sampled section was divided into frames, images of all frames captured and TH-ir perikarya counted. Therefore, the number of objects in each sampled section was not estimated (from an area sampling fraction or asf) but determined by full counting. Similarly, objects were counted throughout the height (thickness) of sampling sections instead of being estimated from a height sampling fraction or hsf, as proposed by the original optical fractionator method (West et al., 1991). The entire SN and VTA areas were used for counting cells and neuronal density was expressed as the number of counted neuronal bodies per mm2.
Motor coordination tests Female rats of different ages were submitted to a battery of motor coordination and strength tests adapted in part from Wallace et al. (1980). Suspension from a horizontal wire mesh pole. The time during which the rats could sustain their own weight was determined by placing the animals on a horizontal wire mesh pole and immediately rotating the pole so that the animals were left suspended from the wire mesh 70 cm over a water tank. The latency for the animals to fall was recorded as the average of three consecutive tests. Performance on a wire mesh ramp set at different angles to the floor. A 90 cm long by 42 cm wide metal ramp set at different angles to the floor was used. The ramp was covered by a central strip of wire mesh (65⫻20 cm) to offer the animals a grip and the base of the ramp was submerged into water up to 15 cm to prevent the animals from descending to the floor. Animals were placed on the central strip and the latency for them to fall to the water was recorded as the average of three consecutive tests. If an animal lasted up to 120 s, it was removed from the ramp and its performance was recorded as maximal.
Fig. 2. TH-ir neurons corresponding to the A9 nigral area in female rats of different ages. Left panels show a low magnification view of the SN in coronal midbrain sections. An age-related reduction in TH-ir nigral cross-sectional area can be observed. Right panels show higher magnification views of the same sections. A reduction in TH-ir neuron density and labeling intensity is evident in senile animals. Y: young (5 months). O: old (24 months). S: senile (32 months). For further technical details see Materials and Methods. Left and right scale bars⫽0.3 mm and 20 m, respectively.
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Table 2. Morphometric parameters corresponding to THir neuronal bodies in the VTA of rats of different age Parameter
Young (n⫽7)
Old (n⫽6)
Senile (n⫽7)
Cell area (m2) Aspect Axis (major) (m) Axis (minor) (m) Perimeter (m) Roundness Neuronal density/ (mm2)
148.86⫾0.77 1.76⫾0.07 18.26⫾0.51 10.61⫾0.12 51.77⫾0.40 1.48⫾0.01 449.12⫾14.03
174.86⫾5.19 2.04⫾0.07 21.16⫾0.02 10.95⫾0.37 54.32⫾0.55 1.61⫾0.03 379.06⫾10.09
177.51⫾2.46* 2.08⫾0.02 22.61⫾1.05 10.99⫾0.46 58.66⫾0.84* 1.51⫾0.03 233.98⫾11.53*
Data are expressed as mean⫾S.E.M.; numbers in parentheses indicate n values for the corresponding group. Asterisks represent the level of significance of the differences between the indicated age group and the corresponding young group. * P⬍0.05.
Fig. 3. TH-ir neurons corresponding to the A10 VTA in female rats of different ages. Left panels show a low magnification view of the VTA in coronal midbrain sections. TH-ir VTA cross-sectional area is comparable in the three sections shown. Right panels show higher magnification views of the same sections. As in the SN, a reduction in TH-ir neuron density and labeling intensity is evident in senile animals. Other details are as in Fig. 2. Left and right scale bars⫽0.3 mm and 20 m, respectively.
(Figs. 2 and 3, respectively, right panels and Tables 1 and 2, respectively). The number of TH-ir neurons declined progressively with age in the SN, showing a 17% (NS) and 33% (P⬍0.05) reduction in the old and senile rats, respectively as compared with the young animals (Fig. 4). In contrast, the number of TH-ir neurons in the VTA was not significantly affected by age (Fig. 4). Consistent with the TH-ir neuron numbers recorded in the SN and VTA of the three age groups, the mean coronal nigral, but not VTA, TH-ir area decreased significantly with age (Fig. 5, main panel). It is of interest that the mean coronal TH-ir area of the whole mesencephalon increased with age (Fig. 5, inset). Age changes in motor and behavioral performance In the horizontal mesh pole suspension test a highly significant age-related decline in performance was observed even between young and adult animals (Fig. 6, lower panel). The same age-related decline in performance was
Histologically, the more conspicuous age change was an increase in the size of TH-ir neurons in both the SN and VTA (Figs. 2 and 3, respectively, right panels and Tables 1 and 2, respectively). In the senile rats, nigral but not VTA TH-ir neurons showed a significant increase in roundness Table 1. Morphometric parameters corresponding to TH-ir neuronal bodies in the SN of rats of different age Parameter
Young (n⫽7)
Old (n⫽6)
Senile (n⫽7)
160.98⫾13.48 162.11⫾12.03 230.27⫾17.01* Cell area (m2) Aspect 2.17⫾0.02 2.08⫾0.20 1.77⫾0.06 Axis (major) (m) 20.75⫾0.63 21.48⫾1.38 22.74⫾0.65 Axis (minor) (m) 10.34⫾0.60 10.97⫾0.72 13.17⫾0.75* Perimeter (m) 55.65⫾2.79 54.93⫾3.07 63.75⫾2.44* Roundness 1.60⫾0.03 1.55⫾0.09 1.43⫾0.01* Neuronal density/ 716.58⫾39.63 549.50⫾15.09* 517.90⫾14.26* (mm2) Data are expressed as mean⫾S.E.M.; numbers in parentheses indicate n values for the corresponding group. Asterisks represent the level of significance of the differences between the indicated age group and the corresponding young group. * P⬍0.05.
Fig. 4. Effect of age on mesencephalic TH-ir neurons numbers. The graph shows the quantitation of TH-ir neurons in the SN and VTA of young, old and senile female rats. TH-ir neuron counting was performed both manually and automatically using an appropriate image analysis software (see Materials and Methods for further details). Bars over columns represent S.E.M. values. Numbers over bars indicate n values for the corresponding group. An asterisk over a column indicates a significant (P⬍0.05) difference respective to the corresponding young group.
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Fig. 5. Effect of age on TH-ir mesencephalic cross-sectional areas in rats. The main panel shows the average TH-ir cross-sectional area of the SN and VTA in young, old and senile female rats. Average areas were estimated by means of an appropriate image analysis software (see Materials and Methods for further details). Inset, overall mesencephalic area in the same animal groups. * P⬍0.05; ** P⬍0.01 respective to young counterpart. Other details are as in Fig. 4.
recorded in the ramp test when the angle of the ramp was set at 88° (Fig. 6 upper panel); however when the angle of the ramp was set at 70°, the 2- and 7-month-old rats consistently achieved maximal performance (120 s) and showed no difficulties for climbing and descending the ramp (down to the water level). In contrast the old and senescent animals did not show a dramatic improvement in their performance as compared with that on the 88° ramp (Fig. 6, inset of upper panel). In the open field test, rearing, sector crossing and self-grooming activity was recorded in 2-, 9-, 20- and 26month-old females. Rearing and sector crossing frequencies showed a marked age-related decline which attained high significance in 20 and 26-month-old animals (Fig. 7, upper and lower panel, respectively). Self-grooming activity also showed an age-associated decline but this change was more gradual than those recorded for sector-crossing and rearing (Fig. 7, inset).
DISCUSSION For several years the aging female Sprague–Dawley rat has been our model for studies related to tuberoinfundibular DA neuron aging in the hypothalamus and for the implementation of restorative interventions on these neurons in aging animals (Goya et al., 1990; Sanchez et al.,
2003; Herenu et al., 2007). The present results extend our studies to the mesencephalic DA neurons of this animal model. The data reveal significant age changes in the TH-ir neurons of the female rat mesencephalon, particularly in the SN. As expected, the changes were more conspicuous in the senile animals. Our morphologic data in the young rats are in agreement with those of the literature. Thus, the number of TH-ir neurons recorded in the SN and VTA of adult animals is in agreement with those reported by German and Manaye (1993). Also, the cell area of the mesencephalic TH-ir neurons of young rats is in general agreement with those reported by Poirier et al. (1983) who recorded neuron diameters ranging from 7.5–35 m in the SN of adult rats. In aging rats, Emerich et al. (1993) failed to detect age changes in number, area or length of A8, A9 or A10 TH-ir neurons in 24 to 25-month-old male Sprague–Dawley rats as compared with 5 to 6-month-old counterparts. These results are at variance with ours in the same age groups. The authors did not perform a stereological evaluation of the animals but a histological analysis in three coronal mesencephalic sections per animal. This discrepancy might reflect a sex-related difference in the vulnerability of DA neurons to aging. However, it seems more likely that the disagreement between our morphometric data and
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Fig. 6. Effect of age on hanging endurance and ramp test performance. Lower panel shows the resistance of rats of different ages to fall from a horizontal mesh pole. The upper panel shows the ability of rats (expressed in seconds to fall to the water at the base of ramp) to remain on a ramp set at 88° to the horizontal. Inset, shows the same test using a ramp set at a 70° angle. Error bars represent S.E.M. whereas numbers over columns represent number of animals per group. Double asterisks indicate a highly significant difference (P⬍0.01) between the indicated age group and the corresponding 2-month-old control.
those of these workers stems from methodological differences. Despite the lack of agreement in morphological data, our results agree with those of Emerich et al. (1993) concerning the marked reduction in motor performance, locomotion and exploratory activity recorded in the old animals. In a study employing histofluorescence to characterize catecholaminergic nigral neurons in 3- and 26month-old Fischer 344 male rats, a significant reduction in the number of nigral fluorescent neurons was reported in the aged animals (Felten et al., 1992). To our knowledge, there are no morphometric data documented for DA mesencephalic neurons in senile (⬎28 months) rats nor are there morphometric rat studies assessing sex-differences in the impact of aging on mesencephalic DA neurons. The limited biochemical data available suggest that from 2 to
24 –26 months of age, male rats show more severe deficits in striatal TH activity and D2 binding, while female rats show a more excessive decrease in striatal DA along with a substantial increase in their DOPAC/DA ratios (Fernandez-Ruiz et al., 1992). The age-related reduction observed here in the TH-ir cross-sectional area of the SN but not the VTA, is consistent with the age-related decrease in nigral but not VTA TH-ir neuron numbers recorded in the same animals. Since we did not determine the expression of TH in the target sites, i.e. basal ganglia, a correlation could not be established between TH expression in the VTA and SN versus TH expression in the corresponding target regions. The significant increase of the total (TH-ir and non TH-ir) cross-sectional mesencephalic area in the senile rats sug-
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Fig. 7. Effect of age on open field activities. The lower panel shows the frequency of sector crossings in the same animals. Upper panel shows the frequency of rearing events in animals of different ages. Inset, shows the frequency of self-grooming episodes the same age groups. Other details are as in Fig. 6.
gests that gliosis may have developed in the mesencephalon of these animals. The presence of reactive gliosis has been reported in animal models of Parkinson’s disease (Wang et al., 2006) as well as in the aging brain (Conde and Streit, 2006). Although there is controversy as to whether aging is associated with a loss of nigral DA neurons, there is general agreement that motor performance decreases significantly during aging in laboratory animals (McCormack et al., 2004; Irwin et al., 1994; Emborg et al., 1998; Emerich et al., 1993). The present results agree with the latter view and reveal that a fall in motor performance is already detectable between 2 and 7 months of age in rats. Clearly, the deterioration of motor performance observed in the aged rats is the result of age-related alterations that occur at different levels of the neuromuscular unit. Most likely, the dysfunctionality or loss of DA neurons in the SN of senile rats accounts for a fraction of their decline in motor
performance, with sarcopenia being a major contributing factor in this decline (Doherty, 2003). Although a direct assessment of nigral DA function deterioration with age by the apomorphine- or amphetamine-induced rotation test could not be performed in our animal model (intact rats with bilateral DA neuron loss), in a previous study on the impact of very old age on the hypothalamic DA neuron population in female rats we could demonstrate a marked age-related deterioration of hypothalamic DA function (regulation of serum prolactin levels) without a substantial loss of hypothalamic TH⫹ neurons (Sanchez et al., 2003; Herenu et al., 2007). Therefore, the possibility exists that part of the mesencephalic TH-ir neurons present in the old and very old female rats studied here were dysfunctional DA neurons. On the other hand, it is also possible that during aging, part of the mesencephalic DA neurons remain viable but undergo a downregulation in the expression of TH thus becoming TH negative. This is the case in monkeys,
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where the number of TH-positive neurons in the SN declines with age. This effect, which amounts to 40 –50% in aged animals, is due at least in part, to a downregulation of TH in surviving DA neurons, as detected by DA transporter immunostaining and neuromelanin content (Emborg et al., 1998; Chu et al., 2002; McCormack et al., 2004). Interestingly, insulin-like growth factor I gene therapy in the hypothalamus of aging female rats increased tuberoinfundibular DA neuron numbers and corrected the chronic hyperprolactinemia of the animals (Herenu et al., 2007). A similar gene therapy approach in the SN of aging rats might improve their motor performance. The present study documents for the first time, the impact of very old age on the mesencephalic DA neuron populations of rats in absence of neurological diseases by using the criteria of modern cell-counting methods. It should be pointed out that most in vivo models developed for the assessment of therapeutic strategies for Parkinson’s disease are based on the use of neurotoxins to lesion nigral DA neurons in young animals. The neurological lesions studied in these models are caused by experimental manipulations rather than by aging, the only unequivocal risk factor for this pathology (de Rijk et al., 1995; Mayeux et al., 1995). In this context, our findings point to the senile female rat as an interesting model of spontaneous TH-ir neuron loss and/or dysfunction as well as of age-related deterioration of motor and exploratory performance. Acknowledgments—The authors thank Ms. Yolanda E. Sosa for technical help. This work was supported in part by grant #R21TW6665 from the National Institutes of Health and grant #11-V142 from the National University of La Plata.
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Emerich DF, McDermont P, Krueger P, Banks M, Zhao J, Marszalkowski J, Frydel B, Winn SR, Sanberg PR (1993) Locomotion of aged rats: relationship to neurochemical but not morphological changes in nigrostriatal dopaminergic neurons. Brain Res Bull 32:477– 486. Fallon JH (1988) Topographic organization of ascending dopaminergic projections. In: The mesocorticolimbic dopamine system (Kalivas PW, Nemeroff CB, eds), pp 1–9. New York: New York Academy of Sciences. Fallon JA, Loughlin SE (1995) Substantia nigra. In: The rat nervous system (Paxinos G, ed), pp 215–239. New York: Academic Press. Fallon JH, Moore RY (1978a) Catecholamine innervation of the basal forebrain. III. Olfactory bulb, anterior olfactory nuclei, olfactory tubercle and piriform cortex. J Comp Neurol 180:533–544. Fallon JH, Moore RY (1978b) Catecholamine innervation of the basal forebrain. IV. Topography of dopamine projection to the basal forebrain and neostriatum. J Comp Neurol 180:545–580. Felten DL, Felten SY, Steece-Collier K, Date I, Clemens JA (1992) Age-related decline in the dopaminergic nigrostriatal system: the oxidative hypothesis and protective strategies. Ann Neurol 32:133–136. Fernandez-Ruiz J, De Miguel R, Hernandez ML, Cebeira M, Ramos JA (1992) Comparisons between brain dopaminergic neurons of juvenile and age rats: sex-related differences. Mech Age Dev 63:45–55. Gerfen CR (1992) The neostriatal mosaic: Multiple levels of compartmental organization in the basal ganglia. Annu Rev Neurosci 15:285–320. German DC, Manaye KF (1993) Midbrain dopaminergic neurons (nuclei A8, A9, and A10): three-dimensional reconstruction in the rat. J Comp Neurol 331:297–309. Goya RG, Lu JKH, Meites J (1990) Gonadal function in aging rats and its relation to pituitary and mammary pathology. Mech Age Dev 56:77– 88. Herenu CB, Cristina C, Rimoldi OJ, Becú-Villalobos D, Cambiaggi V, Portiansky EL, Goya RG (2007) Restorative effect of insulin-like growth factor-I gene therapy in the hypothalamus of senile rats with dopaminergic dysfunction. Gene Ther 14:237–245. Ikai Y, Takada M, Ahinonaga Y, Mizuno N (1992) Dopaminergic and non dopaminergic neurons in the ventral tegmental area of the rat project, respectively, to the cerebellar cortex and deep cerebellar nuclei. Neuroscience 51:719 –728. Irwin I, DeLanney LE, McNeill T, Chan P, Forno LS, Murphy GM, Di Monte DA, Sandy MS, Langston JW (1994) Aging and the nigrostriatal dopamine system: a non-human primate study. Neurodegeneration 3:251–265. Mayeux R, Marder K, Cote LJ, Denaro J, Hemenegildo N, Mejia H, Tang MX, Lantigua R, Wilder D, Gurland B (1995) The frequency of idiopathic Parkinson’s disease by age, ethnic group and sex in northern Manhattan, 1988 –1993. Am J Epidemiol 142:820 – 827. McCormack AL, Di Monte DA, Delfani K, Irwin I, Delanney LE, Langston WJ, Janson AM (2004) Aging of the nigrostriatal system in the squirrel monkey. J Comp Neurol 471:387–395. Paxinos G, Watson CH (1998) The rat brain in stereotaxic coordinates. San Diego, CA: Academic Press. Poirier LJ, Giguere M, Marchand R (1983) Comparative morphology of the substantia nigra and ventral tegmental area in the monkey, cat and rat. Brain Res Bull 11:371–397. Sanchez HL, Silva LB, Portiansky EL, Goya RG, Zuccolilli GO (2003) Impact of very old age on hypothalamic dopaminergic neurons in the female rat: A morphometric study. J Comp Neurol 458: 319 –325. Tatton WG, Greenwood CE, Salo PT, Seniuk NA (1991) Transmitter synthesis increases in substantia nigra neurons of the aged mouse. Neurosci Lett 131:179 –182. Tillet Y, Kitahama K (1998) Distribution of central catecholaminergic neurons: a comparison between ungulates, humans and other species. Histol Histopathol 13:1163–1177.
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(Accepted 9 April 2008) (Available online 16 April 2008)
DOPAMINERGIC MESENCEPHALIC SYSTEMS AND BEHAVIORAL PERFORMANCE IN VERY OLD RATS H. L. SANCHEZ,a* L. B. SILVA,a E. L. PORTIANSKY,b C. B. HERENU,c R. G. GOYAc AND G. O. ZUCCOLILLIa
mainly located within the substantia nigra (SN) and the ventral tegmental area (VTA) which correspond to the A9 and A10 catecholaminergic areas described by Dahlström and Fuxe (1964) (Tillet and Kitahama, 1998), respectively (Fig. 1). They form two long-length DA systems, the nigrostriatal and meso-cortico-limbic systems. The former is represented by the nigral A9 neurons projecting mainly to the neostriatum (caudate nucleus and putamen). The meso-cortico-limbic system is formed by the A10 neurons, which spread out axons from the VTA to striatal, limbic and cortical areas (Bentivoglio and Morelli, 2005). This has long been recognized as an oversimplification because SN contains not only neurons projecting to the striatum, but also neurons that innervate cortical and limbic areas (Björklund and Dunnett, 2007). Thus, dense projections of the ventral and intermediate sheets of the substantia nigra pars compacta (SNpc) and ventro-lateral VTA innervate the caudate-putamen (Gerfen, 1992). DA neurons located within medial SNpc and middorsal VTA project to the nucleus accumbens and olfactory tubercle, whereas dopamine projections to the amygdaloid nuclei arise from lateral SNpc and SN lateral (Fallon and Loughlin, 1995). VTA DA cell bodies distributed in a loosely topographical fashion and nigral DA neurons of the dorsal-most sheet of SNpc innervate the prefrontal, cingulated, perirhinal and entorhinal cortices (Fallon, 1988). In addition, sparse DA projections have been reported arising from SN and VTA to connect with neurons of other neocortical fields (Berger et al., 1991), cerebellum (Ikai et al., 1992), hypothalamus, hippocampus, pallidum and locus coeruleus (Fallon and Moore, 1978a,b). In the adult rat, the total number of mesencephalic DA cells bilaterally is 40,000 – 45,000, with about half of the neurons located in the SN (German and Manaye, 1993). Morphologic and functional studies in laboratory animals describing the impact of aging on mesencephalic DA neurons are rather scanty and inconclusive. In squirrel monkeys, it has been reported that aging causes either a significant loss of tyrosine hydroxylase immunoreactive (TH-ir; i.e. DA neurons) cells in the SN (McCormack et al., 2004) or no change (Irwin et al., 1994). Despite this discrepancy both studies reported a marked reduction of motor activity in the aged monkeys. In rhesus monkeys, a significant (50.3%) age-related loss of TH-ir neurons which correlated with a marked fall in motor performance was reported (Emborg et al., 1998). The only stereological study in aging mice we are aware of reported a significant loss of TH-ir neurons in the SN of 104-week-old animals as compared with 8-week-old counterparts (Tatton et al., 1991). We are unaware of stereological studies on the mesencephalic DA neurons of aging rats and have only detected
a
Institute of Anatomy, Faculty of Veterinary Sciences, National University of La Plata, CC296, Calle 60 y 118, 1900 La Plata, Argentina
b
Institute of Pathology, School of Veterinary Sciences, National University of La Plata, La Plata, Argentina
c
INIBIOLP-Histology “B,” School of Medicine, National University of La Plata, La Plata, Argentina
Abstract—Morphologic and functional studies describing the impact of aging on mesencephalic dopaminergic (DA) neurons in laboratory animals are rather scanty and inconclusive. In rats, stereological studies characterizing age changes in the mesencephalic DA neurons have not been documented. In order to fill this information gap and to determine whether the very old rat may serve as a suitable animal model of Parkinson’s disease, we performed a stereological assessment of the mesencephalic tyrosine hydroxylase immunoreactive (TH-ir) neurons in young-adult (4 – 6 months), old (22–24 months) and senile (30 –32 months) Sprague–Dawley female rats. Morphometric analysis of the TH-ir neurons of the substantia nigra (SN) and ventral tegmental area (VTA) was performed using an appropriate image analysis system. Age changes in motor performance were assessed measuring the endurance of rats to hang from a wire mesh pole or to remain on a ramp set at different angles to the floor. Age changes in locomotion and exploratory activity were evaluated by the open field test. We observed a significant age-related reduction in TH-ir neuron numbers in the SN (17 and 33% reduction in old and senile rats, respectively compared with young counterparts) but not in the VTA. The size of the TH-ir cells increased significantly in both the SN and VTA of the senescent animals but TH labeling intensity fell. Motor, locomotor and exploratory performance deteriorated markedly in the old and senile rats as compared with young animals. These findings reveal the existence of a moderate but significant vulnerability of mesencephalic DA neurons to aging in rats. This phenomenon, which is particularly marked in the SN of very old rats, may contribute to the age-related decline in motor and exploratory performance recorded in this species. © 2008 Published by Elsevier Ltd on behalf of IBRO. Key words: aging, mesencephalic, tyrosine hydroxylase, morphometry, motor tests, open field.
Mesencephalic dopaminergic (DA) neurons account for 80% of brain DA neurons. The perikarya of these cells are *Corresponding author. Tel: ⫹54-221-425-6735; fax: ⫹54-221-425-3276. E-mail address: [email protected] (please cc rgoya@netverk. com.ar) (H. L. Sanchez). Abbreviations: ANOVA, analysis of variance; DA, dopaminergic; DAB, 3,3-diaminobenzidine tetrahydrochloride; IA, interaural; ml, medial lemniscus; mp, mammillary peduncle; SN, substantia nigra; SNpc, substantia nigra pars compacta; TH-ir, tyrosine hydroxylase immunoreactive; VTA, ventral tegmental area. 0306-4522/08$32.00⫹0.00 © 2008 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2008.04.016
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Fig. 1. Schematic representation of coronal sections of the midbrain showing the distribution of TH-ir neuron bodies. (a) Coronal section at the level of the mammillary body (MB) showing the distribution of TH-ir neuron bodies in the SN correspond to the A9, cerebral peduncle (cp), mp, periaqueductal gray (PAG), third ventricle (3V), posterior commissure (pc). (b) Coronal section at the level of the interpeduncular nucleus (ip) showing the distribution of TH-ir neuron bodies in the VTA correspond to the A10, cerebral peduncle (cp), ml, central linear nucleus raphé (CL), red nucleus (R), aqueduct (Aq), commissure superior colliculus (CSC).
a study reporting neurochemical but not morphologic changes (histologically assessed) in nigrostriatal TH-ir neurons of 24 to 25-month-old male rats as compared with 5 to 6-month-old counterparts (Emerich et al., 1993). The authors documented a significant reduction in locomotor activity and motor performance in the aged animals. In the present study we employed an unbiased stereological approach to quantitate the impact of old and very old age on the number and morphology of mesencephalic TH-ir neurons. Since direct evaluation of nigral DA function by the apomorphine- or amphetamine-induced rotation tests can only be performed in unilaterally lesioned animals (not in intact animals with symmetric nigral DA neuron loss), we assessed age-changes in motor performance and exploratory activity in an attempt to gain a general idea on the correlation between morphometric changes in nigral DA neurons and those in behavioral parameters.
National Institutes of Health (INIBIOLP’s Animal Welfare Assurance No A5647-01). The minimum number of rats compatible with detecting reasonable (⬎25%) differences in the different parameters studied was used and every effort was made to avoid or at least minimize the suffering of animals during the experiments performed.
Killing and specimen collection Animals were anesthetized with ketamine hydrochloride (40 mg/kg; i.p.) plus xylazine (8 mg/kg; i.m.) and perfused transcardially with buffered saline– 4% formaldehyde solution. The brain was carefully removed from the cranium, equilibrated in a cryoprotectant solution containing 30% sucrose, 0.1 M PB (0.1 M Na2HPO4 buffer) in H2O and stored at ⫺20 °C until processing. Each brain was trimmed down to a block containing the whole mesencephalon, from the rostral level of mammillary body (interaural (IA) line 4.20 mm) to the caudal level of the pontomesencephalic grove (IA line 2.28 mm) (Paxinos and Watson, 1998). The block was then serially cut into coronal sections 40 m thick on a freezing microtome.
Immunohistochemistry
EXPERIMENTAL PROCEDURES Animals and specimen collection Young (5– 6 months), old (22–24 months), and senile (30 –32 months) Sprague–Dawley female rats, raised in our aging rat colony, were used for morphological studies. For motor and open field tests, slightly different age groups were employed. Animals were housed in a temperature-controlled room (21⫾2 °C) on a 12-h light/dark cycle. Rats were housed in groups of four in stainless steel cages (32⫻32⫻18 cm, L⫻W⫻H). Food and water were available ad libitum. In our rat colony, the average 50% survival time for females, studied in groups of 50 – 60 animals, is 31 months (range, 30 –32 months). Animal experiments were done following the Guidelines on the Use of Animals in Neuroscience Research (the Society of Neuroscience) using IACUC approved procedures (approval date 06/01/04) and Animal Welfare Guidelines of the
Stereological assessment of brain structures was carried out using a simplified variant of the optical fractionator method (West et al., 1991). In each animal, one of every four mesencephalic serial sections was sampled throughout this structure. The sections were submitted to free floating immunohistochemistry using an anti-TH monoclonal antibody (Calbiochem, Inc., La Jolla, CA, USA). As a detection system, the Vectastain Universal ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA) was used, with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO, USA) used as chromogen. Sections were dehydrated and mounted for light microscopy assessment.
Image analysis The images of the hemimesencephalon (objective 1.25⫻), the whole SN and VTA (objective 4⫻) and the TH-ir neurons (objective 20⫻) were captured with a digital color video camera (EvolutionVF, QIm-
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aging, Surrey, BC, Canada) attached to a microscope (Olympus BX50, Olympus Ltd., Tokyo, Japan) and connected to a high performance computer loaded with the Image Pro Plus v6.2 (Media Cybernetics, Silver Spring, MD, USA) image analysis software. Images were captured with a pixel depth of 24 bits, RGB and TIFF format. The hemimesencephalon and the SN and VTA were digitally circumscribed and their respective TH-ir and mesencephalic areas determined. The sections from different animals corresponding to the same level were aligned using neuroanatomical markers. Measurements were thus recorded for the overall structure and for different rostrocaudal levels. The recognition of forebrain structures in co-stained sections was accomplished by inspection of TH-ir adjacent sections and the Paxinos and Watson (1998) atlas of the rat brain. The VTA and SN were outlined for each section and analyzed according to TH-ir adjacent sections. In order to delimit the SN from the VTA, the following tracts of fibers were used from rostral to caudal: mammillary peduncle (mp), emergence of the III cranial pair (3n) and medial lemniscus (ml) (Fig. 1). Immunoreactive perikarya showing clearly shaped nuclei were characterized using the following parameters: cellular area, major and minor cell axis, perimeter and cell roundness. THimmunoreactive neuronal bodies were counted. The intensity of labeling, as determined by DAB staining (optical density), was also recorded. The total number of TH-ir perikarya was estimated using the following equation: n
1 N ⫽ ssf
兺x
Open field test Rats were individually placed in a Plexiglas square arena (45⫻45⫻35 cm) divided into nine equal sectors on the floor. The number of sector crossings (with all four paws), episodes of selfgrooming and rearing was recorded for 5 min.
Statistical analysis The analysis of variance (ANOVA) followed by the Bonferroni post hoc test was used to evaluate intra- and inter-group differences in morphometric variables (Zar, 1984), while the non-parametric Kruskal-Wallis one-way ANOVA followed by the Dunn’s test was used to analyze behavioral data. Significant differences between age groups for each parameter were defined as those with P⬍0.05. Highly significant differences were defined as those with a P⬍0.01.
RESULTS Morphologic age changes in TH-ir mesencephalic neurons There was a macroscopically detectable age-related reduction in the TH-ir staining in coronal sections of the SN but not of the VTA (Figs. 2 and 3, respectively, left panels). A significant age-related reduction in neuronal density was observed in both regions (Tables 1 and 2).
t⫺1
where, N⫽total estimated number of TH-ir perikarya per region; ssf⫽section sampling fraction (1/4); n⫽number of sections sampled per region; x⫽number of TH-ir perikarya counted per sampled section x (from sampled section # 1 to # n). Each sampled section was divided into frames, images of all frames captured and TH-ir perikarya counted. Therefore, the number of objects in each sampled section was not estimated (from an area sampling fraction or asf) but determined by full counting. Similarly, objects were counted throughout the height (thickness) of sampling sections instead of being estimated from a height sampling fraction or hsf, as proposed by the original optical fractionator method (West et al., 1991). The entire SN and VTA areas were used for counting cells and neuronal density was expressed as the number of counted neuronal bodies per mm2.
Motor coordination tests Female rats of different ages were submitted to a battery of motor coordination and strength tests adapted in part from Wallace et al. (1980). Suspension from a horizontal wire mesh pole. The time during which the rats could sustain their own weight was determined by placing the animals on a horizontal wire mesh pole and immediately rotating the pole so that the animals were left suspended from the wire mesh 70 cm over a water tank. The latency for the animals to fall was recorded as the average of three consecutive tests. Performance on a wire mesh ramp set at different angles to the floor. A 90 cm long by 42 cm wide metal ramp set at different angles to the floor was used. The ramp was covered by a central strip of wire mesh (65⫻20 cm) to offer the animals a grip and the base of the ramp was submerged into water up to 15 cm to prevent the animals from descending to the floor. Animals were placed on the central strip and the latency for them to fall to the water was recorded as the average of three consecutive tests. If an animal lasted up to 120 s, it was removed from the ramp and its performance was recorded as maximal.
Fig. 2. TH-ir neurons corresponding to the A9 nigral area in female rats of different ages. Left panels show a low magnification view of the SN in coronal midbrain sections. An age-related reduction in TH-ir nigral cross-sectional area can be observed. Right panels show higher magnification views of the same sections. A reduction in TH-ir neuron density and labeling intensity is evident in senile animals. Y: young (5 months). O: old (24 months). S: senile (32 months). For further technical details see Materials and Methods. Left and right scale bars⫽0.3 mm and 20 m, respectively.
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Table 2. Morphometric parameters corresponding to THir neuronal bodies in the VTA of rats of different age Parameter
Young (n⫽7)
Old (n⫽6)
Senile (n⫽7)
Cell area (m2) Aspect Axis (major) (m) Axis (minor) (m) Perimeter (m) Roundness Neuronal density/ (mm2)
148.86⫾0.77 1.76⫾0.07 18.26⫾0.51 10.61⫾0.12 51.77⫾0.40 1.48⫾0.01 449.12⫾14.03
174.86⫾5.19 2.04⫾0.07 21.16⫾0.02 10.95⫾0.37 54.32⫾0.55 1.61⫾0.03 379.06⫾10.09
177.51⫾2.46* 2.08⫾0.02 22.61⫾1.05 10.99⫾0.46 58.66⫾0.84* 1.51⫾0.03 233.98⫾11.53*
Data are expressed as mean⫾S.E.M.; numbers in parentheses indicate n values for the corresponding group. Asterisks represent the level of significance of the differences between the indicated age group and the corresponding young group. * P⬍0.05.
Fig. 3. TH-ir neurons corresponding to the A10 VTA in female rats of different ages. Left panels show a low magnification view of the VTA in coronal midbrain sections. TH-ir VTA cross-sectional area is comparable in the three sections shown. Right panels show higher magnification views of the same sections. As in the SN, a reduction in TH-ir neuron density and labeling intensity is evident in senile animals. Other details are as in Fig. 2. Left and right scale bars⫽0.3 mm and 20 m, respectively.
(Figs. 2 and 3, respectively, right panels and Tables 1 and 2, respectively). The number of TH-ir neurons declined progressively with age in the SN, showing a 17% (NS) and 33% (P⬍0.05) reduction in the old and senile rats, respectively as compared with the young animals (Fig. 4). In contrast, the number of TH-ir neurons in the VTA was not significantly affected by age (Fig. 4). Consistent with the TH-ir neuron numbers recorded in the SN and VTA of the three age groups, the mean coronal nigral, but not VTA, TH-ir area decreased significantly with age (Fig. 5, main panel). It is of interest that the mean coronal TH-ir area of the whole mesencephalon increased with age (Fig. 5, inset). Age changes in motor and behavioral performance In the horizontal mesh pole suspension test a highly significant age-related decline in performance was observed even between young and adult animals (Fig. 6, lower panel). The same age-related decline in performance was
Histologically, the more conspicuous age change was an increase in the size of TH-ir neurons in both the SN and VTA (Figs. 2 and 3, respectively, right panels and Tables 1 and 2, respectively). In the senile rats, nigral but not VTA TH-ir neurons showed a significant increase in roundness Table 1. Morphometric parameters corresponding to TH-ir neuronal bodies in the SN of rats of different age Parameter
Young (n⫽7)
Old (n⫽6)
Senile (n⫽7)
160.98⫾13.48 162.11⫾12.03 230.27⫾17.01* Cell area (m2) Aspect 2.17⫾0.02 2.08⫾0.20 1.77⫾0.06 Axis (major) (m) 20.75⫾0.63 21.48⫾1.38 22.74⫾0.65 Axis (minor) (m) 10.34⫾0.60 10.97⫾0.72 13.17⫾0.75* Perimeter (m) 55.65⫾2.79 54.93⫾3.07 63.75⫾2.44* Roundness 1.60⫾0.03 1.55⫾0.09 1.43⫾0.01* Neuronal density/ 716.58⫾39.63 549.50⫾15.09* 517.90⫾14.26* (mm2) Data are expressed as mean⫾S.E.M.; numbers in parentheses indicate n values for the corresponding group. Asterisks represent the level of significance of the differences between the indicated age group and the corresponding young group. * P⬍0.05.
Fig. 4. Effect of age on mesencephalic TH-ir neurons numbers. The graph shows the quantitation of TH-ir neurons in the SN and VTA of young, old and senile female rats. TH-ir neuron counting was performed both manually and automatically using an appropriate image analysis software (see Materials and Methods for further details). Bars over columns represent S.E.M. values. Numbers over bars indicate n values for the corresponding group. An asterisk over a column indicates a significant (P⬍0.05) difference respective to the corresponding young group.
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Fig. 5. Effect of age on TH-ir mesencephalic cross-sectional areas in rats. The main panel shows the average TH-ir cross-sectional area of the SN and VTA in young, old and senile female rats. Average areas were estimated by means of an appropriate image analysis software (see Materials and Methods for further details). Inset, overall mesencephalic area in the same animal groups. * P⬍0.05; ** P⬍0.01 respective to young counterpart. Other details are as in Fig. 4.
recorded in the ramp test when the angle of the ramp was set at 88° (Fig. 6 upper panel); however when the angle of the ramp was set at 70°, the 2- and 7-month-old rats consistently achieved maximal performance (120 s) and showed no difficulties for climbing and descending the ramp (down to the water level). In contrast the old and senescent animals did not show a dramatic improvement in their performance as compared with that on the 88° ramp (Fig. 6, inset of upper panel). In the open field test, rearing, sector crossing and self-grooming activity was recorded in 2-, 9-, 20- and 26month-old females. Rearing and sector crossing frequencies showed a marked age-related decline which attained high significance in 20 and 26-month-old animals (Fig. 7, upper and lower panel, respectively). Self-grooming activity also showed an age-associated decline but this change was more gradual than those recorded for sector-crossing and rearing (Fig. 7, inset).
DISCUSSION For several years the aging female Sprague–Dawley rat has been our model for studies related to tuberoinfundibular DA neuron aging in the hypothalamus and for the implementation of restorative interventions on these neurons in aging animals (Goya et al., 1990; Sanchez et al.,
2003; Herenu et al., 2007). The present results extend our studies to the mesencephalic DA neurons of this animal model. The data reveal significant age changes in the TH-ir neurons of the female rat mesencephalon, particularly in the SN. As expected, the changes were more conspicuous in the senile animals. Our morphologic data in the young rats are in agreement with those of the literature. Thus, the number of TH-ir neurons recorded in the SN and VTA of adult animals is in agreement with those reported by German and Manaye (1993). Also, the cell area of the mesencephalic TH-ir neurons of young rats is in general agreement with those reported by Poirier et al. (1983) who recorded neuron diameters ranging from 7.5–35 m in the SN of adult rats. In aging rats, Emerich et al. (1993) failed to detect age changes in number, area or length of A8, A9 or A10 TH-ir neurons in 24 to 25-month-old male Sprague–Dawley rats as compared with 5 to 6-month-old counterparts. These results are at variance with ours in the same age groups. The authors did not perform a stereological evaluation of the animals but a histological analysis in three coronal mesencephalic sections per animal. This discrepancy might reflect a sex-related difference in the vulnerability of DA neurons to aging. However, it seems more likely that the disagreement between our morphometric data and
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Fig. 6. Effect of age on hanging endurance and ramp test performance. Lower panel shows the resistance of rats of different ages to fall from a horizontal mesh pole. The upper panel shows the ability of rats (expressed in seconds to fall to the water at the base of ramp) to remain on a ramp set at 88° to the horizontal. Inset, shows the same test using a ramp set at a 70° angle. Error bars represent S.E.M. whereas numbers over columns represent number of animals per group. Double asterisks indicate a highly significant difference (P⬍0.01) between the indicated age group and the corresponding 2-month-old control.
those of these workers stems from methodological differences. Despite the lack of agreement in morphological data, our results agree with those of Emerich et al. (1993) concerning the marked reduction in motor performance, locomotion and exploratory activity recorded in the old animals. In a study employing histofluorescence to characterize catecholaminergic nigral neurons in 3- and 26month-old Fischer 344 male rats, a significant reduction in the number of nigral fluorescent neurons was reported in the aged animals (Felten et al., 1992). To our knowledge, there are no morphometric data documented for DA mesencephalic neurons in senile (⬎28 months) rats nor are there morphometric rat studies assessing sex-differences in the impact of aging on mesencephalic DA neurons. The limited biochemical data available suggest that from 2 to
24 –26 months of age, male rats show more severe deficits in striatal TH activity and D2 binding, while female rats show a more excessive decrease in striatal DA along with a substantial increase in their DOPAC/DA ratios (Fernandez-Ruiz et al., 1992). The age-related reduction observed here in the TH-ir cross-sectional area of the SN but not the VTA, is consistent with the age-related decrease in nigral but not VTA TH-ir neuron numbers recorded in the same animals. Since we did not determine the expression of TH in the target sites, i.e. basal ganglia, a correlation could not be established between TH expression in the VTA and SN versus TH expression in the corresponding target regions. The significant increase of the total (TH-ir and non TH-ir) cross-sectional mesencephalic area in the senile rats sug-
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Fig. 7. Effect of age on open field activities. The lower panel shows the frequency of sector crossings in the same animals. Upper panel shows the frequency of rearing events in animals of different ages. Inset, shows the frequency of self-grooming episodes the same age groups. Other details are as in Fig. 6.
gests that gliosis may have developed in the mesencephalon of these animals. The presence of reactive gliosis has been reported in animal models of Parkinson’s disease (Wang et al., 2006) as well as in the aging brain (Conde and Streit, 2006). Although there is controversy as to whether aging is associated with a loss of nigral DA neurons, there is general agreement that motor performance decreases significantly during aging in laboratory animals (McCormack et al., 2004; Irwin et al., 1994; Emborg et al., 1998; Emerich et al., 1993). The present results agree with the latter view and reveal that a fall in motor performance is already detectable between 2 and 7 months of age in rats. Clearly, the deterioration of motor performance observed in the aged rats is the result of age-related alterations that occur at different levels of the neuromuscular unit. Most likely, the dysfunctionality or loss of DA neurons in the SN of senile rats accounts for a fraction of their decline in motor
performance, with sarcopenia being a major contributing factor in this decline (Doherty, 2003). Although a direct assessment of nigral DA function deterioration with age by the apomorphine- or amphetamine-induced rotation test could not be performed in our animal model (intact rats with bilateral DA neuron loss), in a previous study on the impact of very old age on the hypothalamic DA neuron population in female rats we could demonstrate a marked age-related deterioration of hypothalamic DA function (regulation of serum prolactin levels) without a substantial loss of hypothalamic TH⫹ neurons (Sanchez et al., 2003; Herenu et al., 2007). Therefore, the possibility exists that part of the mesencephalic TH-ir neurons present in the old and very old female rats studied here were dysfunctional DA neurons. On the other hand, it is also possible that during aging, part of the mesencephalic DA neurons remain viable but undergo a downregulation in the expression of TH thus becoming TH negative. This is the case in monkeys,
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where the number of TH-positive neurons in the SN declines with age. This effect, which amounts to 40 –50% in aged animals, is due at least in part, to a downregulation of TH in surviving DA neurons, as detected by DA transporter immunostaining and neuromelanin content (Emborg et al., 1998; Chu et al., 2002; McCormack et al., 2004). Interestingly, insulin-like growth factor I gene therapy in the hypothalamus of aging female rats increased tuberoinfundibular DA neuron numbers and corrected the chronic hyperprolactinemia of the animals (Herenu et al., 2007). A similar gene therapy approach in the SN of aging rats might improve their motor performance. The present study documents for the first time, the impact of very old age on the mesencephalic DA neuron populations of rats in absence of neurological diseases by using the criteria of modern cell-counting methods. It should be pointed out that most in vivo models developed for the assessment of therapeutic strategies for Parkinson’s disease are based on the use of neurotoxins to lesion nigral DA neurons in young animals. The neurological lesions studied in these models are caused by experimental manipulations rather than by aging, the only unequivocal risk factor for this pathology (de Rijk et al., 1995; Mayeux et al., 1995). In this context, our findings point to the senile female rat as an interesting model of spontaneous TH-ir neuron loss and/or dysfunction as well as of age-related deterioration of motor and exploratory performance. Acknowledgments—The authors thank Ms. Yolanda E. Sosa for technical help. This work was supported in part by grant #R21TW6665 from the National Institutes of Health and grant #11-V142 from the National University of La Plata.
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(Accepted 9 April 2008) (Available online 16 April 2008)