38
Brain Research, 582 (1992) 38-46 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17774
Age-related changes in cortico-releasing factor, somatostatin, neuropeptide Y, methionine enkephalin and fl-endorphin in specific rat brain areas C. Kowalski a, J. Micheau b, R. Corder c, R.
Gaillard c and
B.
Conte-Devolx
a
aLaboratoire de Neuroendrocinologie Expdrimentale, 1NSERM U 297, Facultd de Mddecine Nord, Marseille (France), bCentre de Recherche Delalande, Rueil-Malmaison, (France) and CDepartment of Medicine, University Hospital Micheli-Du-Crest, Geneva (Switzerland) (Accepted 21 January 1992) Key words: Cortico-releasing factor; Somatostatin, Neuropeptide Y; Methionine enkephalin; fl-Endorphin, Aging
We investigated the age-related changes in the tissular protein, cortico-releasing factor (CRF), somatostatin (SOM), neuropeptide Y(NPY), methionine enkephalin (M-ENK) and fl-endorphin (r-END) levels in frontal cortex, hippocampus, striatum and hypothalamus of young (4month-old), mature (18-month-old) and senescent (26-month-old) Wistar male rats, bred in a specific pathogen free environment. Between the age of 4 and 18 months, the tissular protein levels increased in all 4 structures studied. The CRF and SOM levels increased in the hippocampus, while the NPY levels decreased. During this time, the NPY content increased in the striatum, whereas the SOM and M-Enk striatal levels decreased. Concomitantly, the NPY and r-End levels decreased in the hypothalamus. Interestingly, no significant variations were found to occur in the frontal cortex whatever the neuropeptide studied. Between the age of 18 and 26 months, no significant changes in the tissular protein levels were detected, except in the hippocampus. The changes in the neuropeptide concentrations observed during this period depended on the neuropeptide and the brain structure studied. The CRF and r-End levels decreased in the frontal cortex and the hypothalamus, respectively. The NPY peptidergic systems seem to be preferentially affected by aging processes since 3 out of the 4 structures studied -- the frontal cortex, the striatum and the hypothalamus -- showed a decrease in their tissular NPY content. During the same period, none of the 5 neuropeptides studied were affected in the hippocampus. INTRODUCTION The alterations observed in the aging brain have been described as a progressive decline in the activities of the neuronal system, sometimes accompanied by neuronal loss 33. For example, age-related deficits in cognitive functions have been observed along with a decrease in the cholinergic transmission in the neocortex ~7, while deficits in extrapyramidal motor function have been associated with an impairment of the striatal dopaminergic transmission37. The cholinergic and monoaminergic systems have therefore been described up to now as the main central systems to be altered with aging. In view of the close relationships between these systems and the central neuropeptidergic systems, which are sometimes expressed in terms of intraneuronal colocalization 13'22'29, the age-related changes in these n e u r o m o d u l a t o r y peptides within the brain may also been decisive, however. Despite the increasing a m o u n t of information being published at present about the distribution, regulation and putative functions of these neuropeptides, few data are available about the age-related changes in the central
peptidergic activities. The present study was designed to investigate the issue of cortico-releasing factor (CRF, fl-endorphin ( r - E N D ) , methionine enkephalin (MENK), neuropeptide Y (NPY) and somatostatin (SOM) tissular levels in the aging brain, these 5 neuropeptides being present in large amounts throughout the adult brain 1'11'16'27'36. Putative changes in neuropeptide concentrations have been thought to parallel changes in tissular protein content possibly occurring with age in the brain. Age-related changes in protein and neuropeptide tissular concentrations were therefore simultaneously measured at hypothalamic, striatal, hippocampal and frontal cortex levels in young (4-month-old), mature (18month-old) and senescent (26-month-old) male Wistar rats so as to build up an overall picture of the changes liable to occur with aging. MATERIAL AND METHODS Tissue preparation Male Wistar rats 4 (n = 20), 18 (n = 18) and 26 (n = 16) months of age were purchased together from IFFA-CREDO, where they were bred in specific pathogen free (SPF) facilities. The animals were housed in animal care facilities under a 12-h light-dark cycle
Correspondence: C. Kowalski, Laboratoire de Neuroendocrinologie Exp6rimentale, INSERM U 297, Facult6 de M6decine Nord, Bd. P. Dramard, 13326 Marseille Cedex 15, France.
39 at 24 + 2°C and provided with water and food at libitum for one week before the experiment was carried out. The animals were killed by decapitation and the brains rapidly dissected out from the skull before being rinsed in a phosphate buffered saline solution (PBS: 100 Mm, pH 7.4). The hypothalami, hippocampi, striata and frontal cortex were then quickly removed and dropped into cryogenic tubes filled with liquid nitrogen. The brain structures were kept at -80°C until neuropeptide extraction.
Neuropeptide extraction Neuropeptide extractions were performed at the same time on each structure of interest in all 3 groups of rats. Brain structures were sonicated in HC1 0.1 N, and centrifuged at 6,000 rpm. The pellets were kept aside and their protein content measured using the BIORAD assay. The supernatants were collected, and protease denaturation was performed by heating for 10 min at 80°C followed by cooling for 15 rain at 4°C. Supernatants were then divided into 5 aliquots (one for each type of neuropeptide assay) and lyophilised. The lyophilates were kept at -80°C until the specific neuropeptide measurements were performed. Using this methodology, the extraction recovery rate ranged from 72 to 85% with all 5 neuropeptides, as determined by spiking some separate samples with iodinated or tritiated tracer peptides. Measurement of tissular neuropeptide levels The concentration of each neuropeptide was determined in each of the 4 brain structures in all 3 groups of rats at a single experimental session. Aliquoted samples were dissolved in assay buffer at an appropriate dilution for obtaining 20-80% displacement of radioactive tracer by the sample. The CRF 7, SOM21, d-END 2s and M-ENK5"9 tissular contents were measured at our laboratory by specific radio-immunoassay (RIA) as previously described. One series of samples was sent on dry ice to R. Corder in Geneva (Switzerland) to have the NPY concentration measured by immunoradiometric assays. Results Protein and neuropeptide tissular levels were expressed in mg and ng per structure, respectively (paired structures were pooled). Significance among groups of data was evaluated by performing one way ANOVA and post hoc Scheffe F-test. RESULTS
Frontal cortex The tissular p r o t e i n and n e u r o p e p t i d e concentrations m e a s u r e d in the frontal cortex of 4-, 18- and 26-monthold rats are given in Fig. 1. B e t w e e n 4 and 18 months, the protein levels increased by 26% ( P < 0.01) from 0.76 + 0.03 mg to 0.96 + 0.04 mg. N o significant differences were o b s e r v e d in the protein concentration b e t w e e n 18and 26-month-old rat frontal cortices. N o r was any significant variation found to have occurred b e t w e e n 4- and 18-month-old rats whatever the n e u r o p e p t i d e studied. B e t w e e n the age of 18 and 26 months, the S O M , d - E N D and M - E N K levels did not vary significantly. O n the contrary, during this time, the C R F and N P Y levels decreased ( P < 0.01) from 0.29 ___ 0.02 ng to 0.2 + 0.01 ng (-35.5%) and from 0.83 + 0.06 to 0.59 + 0.05 ng (-36%), respectively. Hippocampus The protein, CRF, S O M , NPY, d - E N D and M - E N K
h i p p o c a m p a l levels are given in Fig. 2. The hippocampal protein levels showed a biphasic profile d e p e n d i n g on the animals' age. B e t w e e n 4 and 18 months, the protein levels increased by 30% ( P < 0.01), but between 18 and 26 months they decreased to a value similar to that obtained at 4 months ( P < 0.01). The d - E N D and M - E N K levels were not found to change significantly with age (Fig. 2). Interestingly, the C R F and S O M levels increased by 94% and 70% ( P < 0.01) b e t w e e n 4 and 18 months, from 0.17 + 0.01 ng to 0.33 + 0.03 ng in the case of C R F and from 2.73 + 0.13 ng to 4.63 + 0.5 ng in the case of SOM. During this time, the N P Y decreased by 34% ( P < 0.01) from 2.26 + 0.15 ng to 1.49 _+ 0.16 ng. No significant variations were detected in any of these n e u r o p e p t i d e levels b e t w e e n 18- and 26-monthold rat hippocampi.
Striatum Fig. 3 shows the striatal protein and n e u r o p e p t i d e contents in the 3 groups of rats. A s already observed in the frontal cortex and the hippocampi, a slight (18,5%) but significant ( P < 0.01) increase in the striatal protein content occurred between the age of 4 and 18 months. No significant changes in the striatal protein levels were m e a s u r e d after the age of 18 months. N o further significant variations in the C R F or d - E N D striatal levels were observed whatever the group studied (Fig. 3). B e t w e e n the age of 4 and 18 months, the striatal M - E N K and S O M decreased by 43% and 48%, from 8.28 + 0.54 ng to 4.76 + 0.32 ng in the case of M - E N K ; from 11.23 + 0.74 ng to 5.88 + 0.61 ng in the case of S O M ( P < 0.01). The most noteworthy finding was that during this time, the N P Y levels m a r k e d l y increased (566%) from 2.5 + 0.17 ng to 14.16 + 0.79 ng ( P < 0.01). A t the age of 26 months, the N P Y striatal levels were still significantly ( P < 0.01) higher than the striatal N P Y content at 4 months, but lower ( P < 0.01) than the 18-month levels (Fig. 3). No changes in the striatal tissular M - E N K or S O M contents were observed between the age of 18 and 26 months. Hypothalamus Protein, CRF, S O M , NPY, d - E N D and M - E N K hypothalamic tissular contents versus age are given in Fig. 4. The hypothalamic protein levels increased by 32% between the age of 4 and 18 months ( P < 0.01). N o difference was then d e t e c t e d b e t w e e n the age of 18 and 26 months. N o r were any significant differences observed between the respective concentrations of CRF, S O M and M - E N K in the 3 groups of rats. The N P Y and d - E N D hypothalamic contents gradually decreased, however, with time (Fig. 4). The N P Y content first decreased by 28% (from 21.85 + 1.63 ng to 15.75 + 0.6 ng, P < 0.01)
40
PROTEIN
CRF 0,5.
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Fig. 1. Age-related changes in frontal cortex tissular protein, corticotropin releasing factor (CRF), somatostatin (SOM), neuropeptide Y (NPY), fl-endorphin (B-END) and methionine enkephalin (M-ENK) levels in 4 (n = 20), 18 (n = 18) and 26 (n = 16) month-old rats. Brain structures were acid extracted, boiled, centrifuged and lyophilised before neuropeptides were measured by means of specific radioimmuno(CRF, SOM, B-END and M-ENK) or immunoradiometric- (NPY) assays (for details, see text). Scale bars represent means and S.E.M. Statistical analysis was carried out using one way ANOVA followed by Scheffe F-test comparisons. ** Significantly different from 4-monthold rat levels, P < 0.01. "'Significantly different from 18-month-old rat levels, P < 0.01.
between 4 and 18 months, and then decreased by 26% (15.75 + 0.6 ng to 11.67 + 1.26 ng, P < 0.01) between 18 and 26 months. The fl-END content decreased by 41% (from 18.7 + 1.94 ng to 11.08 ___ 0.75 ng, P < 0.01)
between 4 and 18 months, and then again by 30% (from 11.08 + 0.75 ng to 7.84 ___ 0.62 ng, P < 0.01) between 18 and 26 months (Fig. 4).
41
PROTEIN
CRF
lfl
0,5.
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Fig. 2. Age-related changes in hippocampal tissular protein, corticotropin releasing factor (CRF), somatostatin (SOM), neuropeptide Y (NPY), fl-endorphin (B-END) and methionine enkephalin (M-ENK) levels in 4- (n = 20), 18- (n = 18) and 26- (n = 16) month-old rats. Brain structures were acid extracted, boiled, centrifuged and lyophilised before neuropeptides were measured by means of specific radioimmuno- (CRF, SOM, B-END and M-ENK) or immunoradiometric- (NPY) assays (for details, see text). Scale bars represent means and S.E.M. Statistical analysis was carried out using one way ANOVA followed by Scheffe F-test comparisons. ** Significantly different from 4-monthold rat levels, P < 0.01. " Significantly different from 18-month-old rat levels, P < 0.01.
DISCUSSION A l t h o u g h measuring tissular n e u r o p e p t i d e concentrations does not provide any particular information a b o u t the biosynthesis, m a t u r a t i o n or release of a given neur o p e p t i d e , evaluating tissular n e u r o p e p t i d e concentrations, is actually a valuable tool for assessing the state
of a given n e u r o p e p t i d e r g i c system. O n the o t h e r hand, in o r d e r to minimize the effects of putative changes in aged brain weight (in particular with the shrinkage problem) or those of age-related changes in the brain protein concentration, we decided to express the tissular neur o p e p t i d e levels in mg p e r structure on the basis of a significant n u m b e r of rats (minimum 16 animals p e r age
42
PROTEIN
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II
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6. 4.
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Fig. 3. Age-related changes in striatal tissular protein, corticotropin releasing factor (CRF), somatostatin (SOM), neuropeptide Y (NPY), /~-endorphin (B-END) and methionine enkephalin (M-ENK) levels in 4- (n = 20), 18- (n = 18) and 26- (n = 16) month-old rats. Brain structures were acid extracted, boiled, centrifuged and lyophilised before neuropeptides were measured by means of specific radioimmuno(CRF, SOM, B-END and M-ENK) or immunoradiometric- (NPY) assays (for details, see text). Scale bars represent means and S.E.M. Statistical analysis was carried out using one way ANOVA followed by Scheffe F-test comparisons. **Significantly different from 4-month-old rat levels, P < 0.01. "*Significantly different from 18-month-old rat levels, P < 0.01.
groups).
Under
present
experiment
these
conditions, revealed
the
results of the
complex
studied.
age-related
changes in the C R F , S O M , N P Y , E - E N D and M - E N K c o n c e n t r a t i o n s which d e p e n d e d o n the brain structure
Age-related changes in brain protein levels A g e n e r a l increase in the tissular p r o t e i n levels was
43
CRF
PROTEIN
I
r~
I
O~
4
18 AGE
18 AGE
26
26
NPY
SOM 3o]
1
II
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•*., 21
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30
20
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I
II
I ,,t,,t
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4
18 AGE
26
Fig. 4. Age-related changes in hypothalamic tissular protein, corticotropin releasing factor (CRF), somatostatin (SOM), neuropeptide Y (NPY), fl-endorphin (B-END) and methionine enkephalin (M-ENK) levels in 4- (n = 20), 18- (n = 18) and 26- (n = 16) month-old rats. Brain structures were acid extracted, boiled, centrifuged and lyophilised before neuropeptides were measured by means of specific radioimmuno- (CRF, SOM, B-END and M-ENK) or immunoradiometric- (NPY) assays (for details, see text). Scale bars represent means and S.E.M. Statistical analysis was carried out using one way ANOVA followed by Scheffe F-test comparisons. ** Significantly different from 4-month-old rat levels, P < 0.01. "" Significantly different from 18-month-old rat levels, P < 0.01.
found to occur between 4- and 18-month-old rat groups whatever the brain structure studied. These findings can be compared with previous data on the rate of protein synthesis, which showed the occurrence of age-related
variations in the rate of protein synthesis between young and middle-aged rats 14. This increase in protein levels could be linked to the increasing weight of the brain in the young rat. A p a r t from the hippocampus, we did not
44 observe any significant differences in the protein content between 18- and 26-month-old rats which is in agreement with a previous report showing no significant differences in the rate of protein synthesis between old and middleaged rat brains TM. Since aging is frequently associated with cellular death, measuring protein levels throughout the brain helps to approximately quantify this putative cellular loss. The fact that we did not observe any significant decrease in the tissular protein levels in 3 out of the 4 structures studied argues against this idea, although the glial cell proliferation which occurs in response to neural loss may explain why the protein levels were preserved. Furthermore, despite the drop in the protein levels observed in the hippocampus, no significant differences in any of the neuropeptides studied were detected within the hippocampus, Since no direct relationship exists between protein decrease and neuropeptide levels, aging may therefore not involve neuronal loss of the peptidergic system studied here, at least in the hippocampus.
Age-related changes in the neuropeptide concentrations between 4- and 18-month-old rats Both increases and decreases in the tissular concentrations occurred in the rat brain between 4 and 18 months of life in the case of all the neuropeptides studied. The CRF and SOM contents increased in the hippocampus, while the NPY levels decreased in this structure. The NPY increased, on the contrary, in the striatum, whereas the SOM and M-ENK levels decreased during this time. On the other hand, as previously reported 4, we observed no changes in the SOM concentrations in the hypothalamus or the frontal cortices of young and mature rats. However, the above study reported a significant decrease in the rat striatal SOM levels between the age of 11 and 28 months, while we observed a decrease in the striatal SOM levels between 4 and 18 months but no significant changes between 18 and 26 months. The discrepancy between these two sets of data suggests that changes in the striatal SOM levels probably occurred between 11 and 18 months in the rat. Although the physiological significance of these variations cannot as yet be specified, these data taken together with the present observation of increased protein levels would lead to the conclusion that the variations measured between 4 and 18 months of age in the rat brain might be due to metabolic changes in the adult brain rather than being specifically associated with aging. The changes in neuropeptide concentrations observed during aging depend on the neuropeptide and the brain region investigated Interestingly, the changes in the neuropeptide concen-
trations observed between the age of 18 and 26 months involved only a few of the neuropeptides studied. The tissular concentrations of 3 neuropeptides were selectively affected depending on the brain structure examined. The CRF levels decreased in the frontal cortex, the fl-END contents decreased specifically in the hypothalami, and the tissular NPY concentrations decreased in 3 out of the 4 structures studied. In the mammalian CNS, CRF is widely distributed and in rodents, CRF nerve cells have been immunocytochemically identified in cortical layers II and III and CRF terminal fields in laminae I and IV - areas with high densities of CRF receptors 12'38. The significance of the CRF cortical concentrations has not yet been elucidated, however. There exists, nevertheless, neuropharmacological evidence that complex interactions occur between the cholinergic system and CRF (for review see ref. 15). The decrease in the cortical CRF concentrations with age might be linked with the concomitant decreases in the activity of the cortical cholinergic neurons observed in aging animals. The authors of several reports in the literature have referred to the decrease in the hypothalamic fl-END levels t°'31. Furthermore not only is less fl-END to be found in the hypothalamus of aging rats, but a marked alteration in the post-translational processing of this peptide occurs during aging 41. Among the other pro-opiomelanocortin (POMC)-derived peptides, the concentrations of a-melanostimulating hormone (a-MSH), adreno-corticotropin hormone (ACTH) and fl-lipotropin have been found to decrease in the hypothalamus of aged male rats (for review see ref. 35). The decrease in the amounts of the 4 major POMC-derived peptides may be attributable to a decrease in POMC gene transcription, a reduced or inappropriate post-translational processing or an increase in the rate of release of these 4 peptides. CRF is known to be one of the two main hypothalamic stimulatory factors of the biosynthesis and the release of POMC-derived peptides (for review see ref. 3). Although, as mentioned above, measuring tissular n e u r o p e p t i d e concentrations does not provide any particular information about the peptide biosynthesis, maturation or release, it is worth noting that there is no change in hypothalamic CRF tissular concentrations occurring during aging which could explain the decrease observed in B-END hypothalamic level. Further studies will be necessary to solve the mechanisms at the origin of the decrease in hypothalamic POMC-derived peptides occurring during aging. As previously mentioned, we observed a decrease in NPY levels in 3 out of the 4 structures studied. Recently, Fuxe et al. demonstrated the vulnerability of the NPY peptidergic system to aging processes 19. In agreement
45 with our results, these authors have shown that a m a r k e d loss of N P Y immunoreactivity occurs in the nerve terminals of the paraventricular hypothalamic nuclei as well as a d r o p in the N P Y concentrations in many hypothalamic nuclei in the 24-month-old rat. F u r t h e r m o r e , in the same study, a loss of N P Y immunoreactive p e r i k a r y a (but not of the other p e p t i d e perikarya) was o b s e r v e d during aging within the striatum. It is worth noting that the N P Y and the S O M , known to be co-localized in the same neuronal population 6'24'39, were not affected in the same way during this time, since we did not observe any significant changes between 18 and 26 months in S O M levels whatever the brain region studied. These decreases in N P Y concentration in central brain structures are of particular interest in view of the recent evidence that central N P Y peptidergic systems are involved in the neur o m o d u l a t i o n of the sensori-motor axes 23 as well as in cognitive functions TM, these two neuronal systems being preferentially i m p a i r e d during aging. F u r t h e r m o r e , previous studies 25 have d e m o n s t r a t e d the close relationship as well as at the synaptological features 4° and the biochemical interaction levels 26'34 between the N P Y peptidergic and m o n o a m i n e r g i c systems - - in particular with the dopaminergic system. These studies r e p o r t e d that a decrease in N P Y m R N A expression, and in the n u m b e r of N P Y positive immunostaining p e r i k a r y a , occurs in the striatum of adult Wistar rats when the disruption of the dopaminergic nigrostriatal transmission is induced by a d o p a m i n e ( D A ) r e c e p t o r antagonist or a blocker of D A synthesis. Since it is known nowadays that the central dopaminergic systems are particularly strongly affected in the aged brain, as shown by the m a r k e d decrease in
REFERENCES 1 Akil, H., Watson, S.J., Young, E., Lewis, M.E., Khachaturian, H. and Walker, J.M., Endogenous opioids: biology and function, Annu. Rev. Neurosci., 7 (1984) 223-255. 2 Amenta, E, Cavalloti, C., Collier, W.L., De Michele, M. and Ricci, A., Age-related changes of dopamine sensitive cyclic AMP generation in the rat frontal cortex, Mech. Ageing Dev., 54 (1990) 63-73. 3 Antoni, EA., Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41- residu corticotropin-releasing factor, Endocrine Rev., 7 (1986) 351-378. 4 Buck, S.H., Deshmukh, P.P., Burks, T.E and Yamamura, H.I., A survey of substance P, somatostatin, and neurotensin levels in aging in the rat and human central nervous system, Neurobiol. Aging, 2 (1981) 257-264. 5 Castanas, E., Giraud, E, Drissi, R., Chabrier, EE., Conte-Devolx, B., Boudouresque, E, Cantau, P., Cesselin, E, Cupo, A., Eiden, L.E. and Oliver, C., Characterization of enkephalins and related peptides in rat hypophysial portal blood, Brain Res., 310 (1984) 1-6. 6 Chronwall, B.M., Chase, T.N. and O'Donohue, T.L., Coexistence of neuropeptide Y and somatostatin in rat and human cortical and rat hypothalamic neurons, Neurosci. Lett., 52 (1984) 213-217.
striatal and nigral tyrosine-hydroxylase immunoreactivity (TH) 33, D A concentrations 37, dopaminergic receptors numbers 32 (and recently D 2 mRNA3°), as well as in the D 1 d e p e n d e n t adenylate-cyclase activity 2, it is tempting to correlate the decreased N P Y concentrations with a decrease in D A transmission occurring during aging. In conclusion
Changes in n e u r o p e p t i d e concentration resulting from aging processes can be said to occur between 18 and 26 months of age in rat brain structures rather than between 4 and 18 months. The changes in n e u r o p e p t i d e concentrations observed during aging were selective, depending on the n e u r o p e p t i d e and the brain region investigated. CRF, B - E N D and N P Y peptidergic systems were differentially affected, the most vulnerable to aging being the N P Y systems. The decrease in the N P Y concentrations observed with aging might be linked to the decrease in the dopaminergic activity known to occur in the aged rat brain. It is worth noting that the N P Y peptidergic systems, which are assumed to be spared in the cortex of A l z h e i m e r patients 2°, were altered in the cortex during aging, while the S O M peptidergic systems, which have been r e p o r t e d to be d a m a g e d in this p a t h o l o g y 2°, were not affected by aging.
Acknowledgements. This study was supported by a grant from Minist6re Frangais de la Recherche et de la Technologie. We thank Dr. A. Cupo (CNRS, Marseille) for providing us with anti-M-ENK antibody and M. Renard for her technical help. We are grateful to Prof. A. Nieoullon (CNRS, Marseille) for his critical reading and helpful contributions and to Dr. J. Blanc for correcting the English version of the manuscript.
7 Conte-Devolx, B., Grino, M., Nieoullon, A., Javoy-Agid, E, Castanas, E., Guillaume, V., Tonon, M.C., Vaudry, M. and Oliver, C., Corticoliberin, somatocrinin and amin contents in normal and Parkinsonian human hypothalamus, Neurosci. Lett., 35 (1985) 217-222. 8 Corder, R. and Lowry, P.J., An Immunoradiometric assay for the measurement of neuropeptide Y in plasma, Peptides, 6 (1985) 1195-1200. 9 Cupo, A. and Jarry, T., Detection of methionine enkephalin at the 10-16 mole level, J. Neuroimmunol., 8 (1985) 57-67. 10 Dax, E.M., Reichman, C., Fullerton, M., Wallace, C., Smith, A.I. and Funder, J.W., Beta endorphin and dynorphin levels in rat pituitary and hypothalamus: age studies, Neuroendocrinology, 47 (1988) 241-248. 11 De Quidt, M.E. and Emson, P.C., Distribution of neuropeptide Y-like immunoreactivity in the rat central nervous system-II immunohistochemical analysis, Neuroscience, 18 (1986) 545-618. 12 De Souza, E.B., Perrin, M.H., Insel, T.R., Rivier, J., Vale, W.W. and Kuhar, M.J., Corticotropin releasing factor receptors in rat forebrain: autoradiographic identification, Science, 224 (1984) 1449-1451. 13 Everitt, B.J., H6kfelt, T., Terenius, L., Tatemoto, K., Mutt, V. and Goldstein, M., Differential co-existence of neuropeptide Y (NPY)-like immunoreactivity with cathecolamines in the central
46 nervous system of the rat, Neuroscience, 11 (1984) 443-462. 14 Fando, J.L., Salinas, M. and Wasterlain, C.G., Age-dependent changes in brain protein synthesis in the rat, Neurochem. Res., 5 (1980) 373-383. 15 Ferrier, I.N. and Leake, A., Peptides in the neocortex in Alzheimer's disease and ageing, Phychoneuroendocrinology, 15 (1990) 89-95. 16 Finley, J.C.W., Maderbrut, J.L., Roger, L.J. and Petrusz, P., The immunocytochemical localization of somatostatin-containing neurons in the rat central nervous system, Neuroscience, 6 (1981) 2173-2192. 17 Fischer, W., Wictorin, K., Bj6rklund, A., Williams, L.R., Varon, S. and Gage, F.H., Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor, Nature, 329 (1987) 65-68. 18 Flood, J.E, Hernandez, E.N. and Morley, J.E., Modulation of memory processing by neuropeptide Y, Brain Res., 421 (1987) 280-290. 19 Fuxe, K., Agnati, L.F., H~irfstrand, A., Zoli, M., Von Euler, G., Grimaldi, R., Merlo Pich, E., Bjelke, B., Eneroth, P., Benfenati, E, Cintra, A., Zini, I. and Martire, M., On the role of neuropeptide Y in information handling in the central nervous system in normal and physiopathological states, Ann. N.Y. Acad. Sci., 579 (1990) 28-67. 20 Gaspar, P., Duyckaerts, C., Febvret, A., Benoit, R., Beck, B. and Berger, B., Subpopulations of somatostatin 28-immunoreactive neurons display different vulnerability in senile dementia of the Alzheimer type, Brain Res., 490 (1989) 1-13. 21 Gillioz, P., Giraud, P., Conte-Devolx, B., Jaquet, P., Codaccioni, J.L. and Oliver, C., Immunoreactive somatostatin in hypophyseal portal blood, Endocrinology, 104 (1979) 1407-1410. 22 Graybiel, A.M., Neuropeptides in the basal ganglia. In J.B. Martin and J.D. Barchas (Eds.), Neuropeptides in Neurologic and Psychiatric Disease, Raven, New York (1986), pp. 135-161. 23 Heilig, M. and Murison, R., Intracerebroventricular neuropeptide Y suppresses open field and home cage activity in the rat, Regul. Pept., 19 (1987) 221-231. 24 Hendry, S.H.C., Jones, E.G. and Emson, P.C., Morphology, distribution, and synaptic relations of somatostatin- and neuropeptide Y-immunoreactive neurons in rat and monkey neocortex, J. Neurosci., 4 (1984) 2497-2517. 25 Kerkerian, L., Bosler, O., Pelletier, G. and Nieoullon, A., Striatal neuropeptide Y neurons are under influence of the nigrostriatal dopaminergic pathway: immunohistochemical evidence, Neurosci. Lett., 66 (1986) 106-112. 26 Kerkerian, L., Salin, P. and Nieoullon, A., Pharmacological characterization of dopaminergic influence on expression of neuropeptide Y immunoreactivity by rat striatal neurons, Neuroscience, 26 (1988) 809-817. 27 Khachaturian, H., Lewis, M.E., Schafer, M.G.H. and Watson, S.J., Anatomy of the CNS opioid systems, Trends Neurosci., 7
(1984) 111-119. 28 Lissitzky, J.C., Giraud, P., Conte-Devolx, B., Gillioz, P., Boudouresque, F., Eskay, R.L. and Oliver, C., fl-Endorphin is present in high concentration in the hypophysial portal vessels of rats, Neurosci. Lett., 19 (1980) 191-195. 29 Meister, B., H6kfelt, T., Brown, J., Joh, T. and Goldstein, M., Dopaminergic cells in the caudal A13 cell group express somatostatin-like immunoreactivity, Exp. Brain Res., 67 (1987) 441-444. 30 Mesco, E.R., Joseph, J.A., Blake, M.J. and Roth, G.S., Loss of D 2 receptors during aging is partially due to decreased levels of mRNA, Brain Res., 545 (1991) 355-357. 31 Missale, C., Govoni, S. and Croce, L., Changes of/~-endorphin and met-enkephalin content in hypothalamus-pituitary axis induced by aging, J. Neurochem., 40 (1984) 20-24. 32 Morelli, M., Mennini, T., Cagnotto, A., Toffano, G. and Di Chiara, G., Quantitative autoradiographical analysis of the agerelated modulation of central dopamine D~ and D 2 receptors, Neuroscience, 36 (1990) 403-410. 33 Pradham, S.N., Central neurotransmitters and aging, Life Sci., 26 (1980) 1643-1656. 34 Salin, P., Kerkerian-Le-Goff, L. and Nieoullon, A., Expression of neuropeptide Y immunoreactivity in the rat nucleus accumbens is under the influence of the dopaminergic mesencephalic pathway, Exp. Brain Res., 81 (1990) 363-371. 35 Simpkins, J.M. and Millard, J., Influence of age on neurotransmitter function, Endocrinol. Metab. Clin., 16 (1987) 893-917. 36 Skofitsch, G. and Jacobowitz, D.M., Distribution of corticotropin releasing factor-like immunoreactivity in the rat brain by immunohistochemistry and radioimmunoassay: comparison and characterization of ovine and rat/human CRF antisera, Peptides, 6 (1985) 319-336. 37 Strong, R., Neurochemistry of aging: 1982-1984. In M. Rothstein (Eds). Review of Biological Research in Aging, Vol. 2, A.R. Liss, New York (1985) pp. 181-196. 38 Swanson, L.W., Sawchenko, P.E., Rivier, J. and Vale, W.W., Organization of ovine corticotropin-releasing factor immunoreactive cells and filters in the rat brain: an immunohistochemical study; Neuroendocrinology, 36 (1983) 166-186. 39 Vincent, S.R., Skirboll, L., H6kfelt, T., Johansson, O., Lundberg, J.M., Elde, R.P., Terenius, L. and Kimmel, J., Coexistence of somatostatin- and avian pancreatic polypeptide (APP)like immunoreactivity in some forebrain neurons, Neuroscience, 7 (1982) 439-446. 40 Vuillet, J., Kerkerian, L., Salin, P. and Nieoullon, A., Ultrastructural features of NPY-containing neurons in the rat striaturn. Relationships with the nigrostriatal dopaminergic pathway, Brain Res., 477 (1989) 241-251. 41 Wilkinson, C.W. and Dorsa, D.M., The effects of aging on molecular forms of beta- and gamma-endorphins in rat hypothalamus, Neuroendocrinology, 43 (1986) 124-131.
Brain Research, 582 (1992) 38-46 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17774
Age-related changes in cortico-releasing factor, somatostatin, neuropeptide Y, methionine enkephalin and fl-endorphin in specific rat brain areas C. Kowalski a, J. Micheau b, R. Corder c, R.
Gaillard c and
B.
Conte-Devolx
a
aLaboratoire de Neuroendrocinologie Expdrimentale, 1NSERM U 297, Facultd de Mddecine Nord, Marseille (France), bCentre de Recherche Delalande, Rueil-Malmaison, (France) and CDepartment of Medicine, University Hospital Micheli-Du-Crest, Geneva (Switzerland) (Accepted 21 January 1992) Key words: Cortico-releasing factor; Somatostatin, Neuropeptide Y; Methionine enkephalin; fl-Endorphin, Aging
We investigated the age-related changes in the tissular protein, cortico-releasing factor (CRF), somatostatin (SOM), neuropeptide Y(NPY), methionine enkephalin (M-ENK) and fl-endorphin (r-END) levels in frontal cortex, hippocampus, striatum and hypothalamus of young (4month-old), mature (18-month-old) and senescent (26-month-old) Wistar male rats, bred in a specific pathogen free environment. Between the age of 4 and 18 months, the tissular protein levels increased in all 4 structures studied. The CRF and SOM levels increased in the hippocampus, while the NPY levels decreased. During this time, the NPY content increased in the striatum, whereas the SOM and M-Enk striatal levels decreased. Concomitantly, the NPY and r-End levels decreased in the hypothalamus. Interestingly, no significant variations were found to occur in the frontal cortex whatever the neuropeptide studied. Between the age of 18 and 26 months, no significant changes in the tissular protein levels were detected, except in the hippocampus. The changes in the neuropeptide concentrations observed during this period depended on the neuropeptide and the brain structure studied. The CRF and r-End levels decreased in the frontal cortex and the hypothalamus, respectively. The NPY peptidergic systems seem to be preferentially affected by aging processes since 3 out of the 4 structures studied -- the frontal cortex, the striatum and the hypothalamus -- showed a decrease in their tissular NPY content. During the same period, none of the 5 neuropeptides studied were affected in the hippocampus. INTRODUCTION The alterations observed in the aging brain have been described as a progressive decline in the activities of the neuronal system, sometimes accompanied by neuronal loss 33. For example, age-related deficits in cognitive functions have been observed along with a decrease in the cholinergic transmission in the neocortex ~7, while deficits in extrapyramidal motor function have been associated with an impairment of the striatal dopaminergic transmission37. The cholinergic and monoaminergic systems have therefore been described up to now as the main central systems to be altered with aging. In view of the close relationships between these systems and the central neuropeptidergic systems, which are sometimes expressed in terms of intraneuronal colocalization 13'22'29, the age-related changes in these n e u r o m o d u l a t o r y peptides within the brain may also been decisive, however. Despite the increasing a m o u n t of information being published at present about the distribution, regulation and putative functions of these neuropeptides, few data are available about the age-related changes in the central
peptidergic activities. The present study was designed to investigate the issue of cortico-releasing factor (CRF, fl-endorphin ( r - E N D ) , methionine enkephalin (MENK), neuropeptide Y (NPY) and somatostatin (SOM) tissular levels in the aging brain, these 5 neuropeptides being present in large amounts throughout the adult brain 1'11'16'27'36. Putative changes in neuropeptide concentrations have been thought to parallel changes in tissular protein content possibly occurring with age in the brain. Age-related changes in protein and neuropeptide tissular concentrations were therefore simultaneously measured at hypothalamic, striatal, hippocampal and frontal cortex levels in young (4-month-old), mature (18month-old) and senescent (26-month-old) male Wistar rats so as to build up an overall picture of the changes liable to occur with aging. MATERIAL AND METHODS Tissue preparation Male Wistar rats 4 (n = 20), 18 (n = 18) and 26 (n = 16) months of age were purchased together from IFFA-CREDO, where they were bred in specific pathogen free (SPF) facilities. The animals were housed in animal care facilities under a 12-h light-dark cycle
Correspondence: C. Kowalski, Laboratoire de Neuroendocrinologie Exp6rimentale, INSERM U 297, Facult6 de M6decine Nord, Bd. P. Dramard, 13326 Marseille Cedex 15, France.
39 at 24 + 2°C and provided with water and food at libitum for one week before the experiment was carried out. The animals were killed by decapitation and the brains rapidly dissected out from the skull before being rinsed in a phosphate buffered saline solution (PBS: 100 Mm, pH 7.4). The hypothalami, hippocampi, striata and frontal cortex were then quickly removed and dropped into cryogenic tubes filled with liquid nitrogen. The brain structures were kept at -80°C until neuropeptide extraction.
Neuropeptide extraction Neuropeptide extractions were performed at the same time on each structure of interest in all 3 groups of rats. Brain structures were sonicated in HC1 0.1 N, and centrifuged at 6,000 rpm. The pellets were kept aside and their protein content measured using the BIORAD assay. The supernatants were collected, and protease denaturation was performed by heating for 10 min at 80°C followed by cooling for 15 rain at 4°C. Supernatants were then divided into 5 aliquots (one for each type of neuropeptide assay) and lyophilised. The lyophilates were kept at -80°C until the specific neuropeptide measurements were performed. Using this methodology, the extraction recovery rate ranged from 72 to 85% with all 5 neuropeptides, as determined by spiking some separate samples with iodinated or tritiated tracer peptides. Measurement of tissular neuropeptide levels The concentration of each neuropeptide was determined in each of the 4 brain structures in all 3 groups of rats at a single experimental session. Aliquoted samples were dissolved in assay buffer at an appropriate dilution for obtaining 20-80% displacement of radioactive tracer by the sample. The CRF 7, SOM21, d-END 2s and M-ENK5"9 tissular contents were measured at our laboratory by specific radio-immunoassay (RIA) as previously described. One series of samples was sent on dry ice to R. Corder in Geneva (Switzerland) to have the NPY concentration measured by immunoradiometric assays. Results Protein and neuropeptide tissular levels were expressed in mg and ng per structure, respectively (paired structures were pooled). Significance among groups of data was evaluated by performing one way ANOVA and post hoc Scheffe F-test. RESULTS
Frontal cortex The tissular p r o t e i n and n e u r o p e p t i d e concentrations m e a s u r e d in the frontal cortex of 4-, 18- and 26-monthold rats are given in Fig. 1. B e t w e e n 4 and 18 months, the protein levels increased by 26% ( P < 0.01) from 0.76 + 0.03 mg to 0.96 + 0.04 mg. N o significant differences were o b s e r v e d in the protein concentration b e t w e e n 18and 26-month-old rat frontal cortices. N o r was any significant variation found to have occurred b e t w e e n 4- and 18-month-old rats whatever the n e u r o p e p t i d e studied. B e t w e e n the age of 18 and 26 months, the S O M , d - E N D and M - E N K levels did not vary significantly. O n the contrary, during this time, the C R F and N P Y levels decreased ( P < 0.01) from 0.29 ___ 0.02 ng to 0.2 + 0.01 ng (-35.5%) and from 0.83 + 0.06 to 0.59 + 0.05 ng (-36%), respectively. Hippocampus The protein, CRF, S O M , NPY, d - E N D and M - E N K
h i p p o c a m p a l levels are given in Fig. 2. The hippocampal protein levels showed a biphasic profile d e p e n d i n g on the animals' age. B e t w e e n 4 and 18 months, the protein levels increased by 30% ( P < 0.01), but between 18 and 26 months they decreased to a value similar to that obtained at 4 months ( P < 0.01). The d - E N D and M - E N K levels were not found to change significantly with age (Fig. 2). Interestingly, the C R F and S O M levels increased by 94% and 70% ( P < 0.01) b e t w e e n 4 and 18 months, from 0.17 + 0.01 ng to 0.33 + 0.03 ng in the case of C R F and from 2.73 + 0.13 ng to 4.63 + 0.5 ng in the case of SOM. During this time, the N P Y decreased by 34% ( P < 0.01) from 2.26 + 0.15 ng to 1.49 _+ 0.16 ng. No significant variations were detected in any of these n e u r o p e p t i d e levels b e t w e e n 18- and 26-monthold rat hippocampi.
Striatum Fig. 3 shows the striatal protein and n e u r o p e p t i d e contents in the 3 groups of rats. A s already observed in the frontal cortex and the hippocampi, a slight (18,5%) but significant ( P < 0.01) increase in the striatal protein content occurred between the age of 4 and 18 months. No significant changes in the striatal protein levels were m e a s u r e d after the age of 18 months. N o further significant variations in the C R F or d - E N D striatal levels were observed whatever the group studied (Fig. 3). B e t w e e n the age of 4 and 18 months, the striatal M - E N K and S O M decreased by 43% and 48%, from 8.28 + 0.54 ng to 4.76 + 0.32 ng in the case of M - E N K ; from 11.23 + 0.74 ng to 5.88 + 0.61 ng in the case of S O M ( P < 0.01). The most noteworthy finding was that during this time, the N P Y levels m a r k e d l y increased (566%) from 2.5 + 0.17 ng to 14.16 + 0.79 ng ( P < 0.01). A t the age of 26 months, the N P Y striatal levels were still significantly ( P < 0.01) higher than the striatal N P Y content at 4 months, but lower ( P < 0.01) than the 18-month levels (Fig. 3). No changes in the striatal tissular M - E N K or S O M contents were observed between the age of 18 and 26 months. Hypothalamus Protein, CRF, S O M , NPY, d - E N D and M - E N K hypothalamic tissular contents versus age are given in Fig. 4. The hypothalamic protein levels increased by 32% between the age of 4 and 18 months ( P < 0.01). N o difference was then d e t e c t e d b e t w e e n the age of 18 and 26 months. N o r were any significant differences observed between the respective concentrations of CRF, S O M and M - E N K in the 3 groups of rats. The N P Y and d - E N D hypothalamic contents gradually decreased, however, with time (Fig. 4). The N P Y content first decreased by 28% (from 21.85 + 1.63 ng to 15.75 + 0.6 ng, P < 0.01)
40
PROTEIN
CRF 0,5.
1,5
I
0,4.
~1,O.
~
0,3.
~
0,2.
~
0,1.
000~.
o,O.
0,o. 4
18
26
4
AGE
18
26
AGE
SOM
NPY
2,O.
2,0_ "~v 1,5
1,5.
,~ 1,O.
t13
b,O 0,s.
01) 0,5.
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0,10 "1
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,~ 0,06.
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m 0,04.
0,04.
0,02.
~
O,OO.
o,o2. o,o0-
4
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26
AGE
4
18
26
AGE
Fig. 1. Age-related changes in frontal cortex tissular protein, corticotropin releasing factor (CRF), somatostatin (SOM), neuropeptide Y (NPY), fl-endorphin (B-END) and methionine enkephalin (M-ENK) levels in 4 (n = 20), 18 (n = 18) and 26 (n = 16) month-old rats. Brain structures were acid extracted, boiled, centrifuged and lyophilised before neuropeptides were measured by means of specific radioimmuno(CRF, SOM, B-END and M-ENK) or immunoradiometric- (NPY) assays (for details, see text). Scale bars represent means and S.E.M. Statistical analysis was carried out using one way ANOVA followed by Scheffe F-test comparisons. ** Significantly different from 4-monthold rat levels, P < 0.01. "'Significantly different from 18-month-old rat levels, P < 0.01.
between 4 and 18 months, and then decreased by 26% (15.75 + 0.6 ng to 11.67 + 1.26 ng, P < 0.01) between 18 and 26 months. The fl-END content decreased by 41% (from 18.7 + 1.94 ng to 11.08 ___ 0.75 ng, P < 0.01)
between 4 and 18 months, and then again by 30% (from 11.08 + 0.75 ng to 7.84 ___ 0.62 ng, P < 0.01) between 18 and 26 months (Fig. 4).
41
PROTEIN
CRF
lfl
0,5.
0~, ~
0,2. ~
0,1.
0,0. 4
18
~
4
AGE
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4
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-
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~ o~]
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,,I,,,*
26
5
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AGE
0,2.
0,2
0I) 0,1.
0,1
0,0
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18
26
AGE
Fig. 2. Age-related changes in hippocampal tissular protein, corticotropin releasing factor (CRF), somatostatin (SOM), neuropeptide Y (NPY), fl-endorphin (B-END) and methionine enkephalin (M-ENK) levels in 4- (n = 20), 18- (n = 18) and 26- (n = 16) month-old rats. Brain structures were acid extracted, boiled, centrifuged and lyophilised before neuropeptides were measured by means of specific radioimmuno- (CRF, SOM, B-END and M-ENK) or immunoradiometric- (NPY) assays (for details, see text). Scale bars represent means and S.E.M. Statistical analysis was carried out using one way ANOVA followed by Scheffe F-test comparisons. ** Significantly different from 4-monthold rat levels, P < 0.01. " Significantly different from 18-month-old rat levels, P < 0.01.
DISCUSSION A l t h o u g h measuring tissular n e u r o p e p t i d e concentrations does not provide any particular information a b o u t the biosynthesis, m a t u r a t i o n or release of a given neur o p e p t i d e , evaluating tissular n e u r o p e p t i d e concentrations, is actually a valuable tool for assessing the state
of a given n e u r o p e p t i d e r g i c system. O n the o t h e r hand, in o r d e r to minimize the effects of putative changes in aged brain weight (in particular with the shrinkage problem) or those of age-related changes in the brain protein concentration, we decided to express the tissular neur o p e p t i d e levels in mg p e r structure on the basis of a significant n u m b e r of rats (minimum 16 animals p e r age
42
PROTEIN
CRF
10
016. I .~-~
8
0,5. "~ 0,4. r~ ~ 0,2.
4 010
~ 0
0,1. 0,0.
18
26
4
AGE
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AGE
NPY
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II
20_
1210.
I
1-i lS. GI
8.
, • 6.
10-
2. 0.
O-
18
~
4
AGE
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AGE
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1,o
10.
°1
8.
6. 4.
0'4"t
2.
o,o.
o.
18
4
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4
AGE
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AGE
Fig. 3. Age-related changes in striatal tissular protein, corticotropin releasing factor (CRF), somatostatin (SOM), neuropeptide Y (NPY), /~-endorphin (B-END) and methionine enkephalin (M-ENK) levels in 4- (n = 20), 18- (n = 18) and 26- (n = 16) month-old rats. Brain structures were acid extracted, boiled, centrifuged and lyophilised before neuropeptides were measured by means of specific radioimmuno(CRF, SOM, B-END and M-ENK) or immunoradiometric- (NPY) assays (for details, see text). Scale bars represent means and S.E.M. Statistical analysis was carried out using one way ANOVA followed by Scheffe F-test comparisons. **Significantly different from 4-month-old rat levels, P < 0.01. "*Significantly different from 18-month-old rat levels, P < 0.01.
groups).
Under
present
experiment
these
conditions, revealed
the
results of the
complex
studied.
age-related
changes in the C R F , S O M , N P Y , E - E N D and M - E N K c o n c e n t r a t i o n s which d e p e n d e d o n the brain structure
Age-related changes in brain protein levels A g e n e r a l increase in the tissular p r o t e i n levels was
43
CRF
PROTEIN
I
r~
I
O~
4
18 AGE
18 AGE
26
26
NPY
SOM 3o]
1
II
t-i
•*., 21
2 00
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18 AGE
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B-END
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30
20
26
1P
t
I
II
I ,,t,,t
c/3
4
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26
4
18 AGE
26
Fig. 4. Age-related changes in hypothalamic tissular protein, corticotropin releasing factor (CRF), somatostatin (SOM), neuropeptide Y (NPY), fl-endorphin (B-END) and methionine enkephalin (M-ENK) levels in 4- (n = 20), 18- (n = 18) and 26- (n = 16) month-old rats. Brain structures were acid extracted, boiled, centrifuged and lyophilised before neuropeptides were measured by means of specific radioimmuno- (CRF, SOM, B-END and M-ENK) or immunoradiometric- (NPY) assays (for details, see text). Scale bars represent means and S.E.M. Statistical analysis was carried out using one way ANOVA followed by Scheffe F-test comparisons. ** Significantly different from 4-month-old rat levels, P < 0.01. "" Significantly different from 18-month-old rat levels, P < 0.01.
found to occur between 4- and 18-month-old rat groups whatever the brain structure studied. These findings can be compared with previous data on the rate of protein synthesis, which showed the occurrence of age-related
variations in the rate of protein synthesis between young and middle-aged rats 14. This increase in protein levels could be linked to the increasing weight of the brain in the young rat. A p a r t from the hippocampus, we did not
44 observe any significant differences in the protein content between 18- and 26-month-old rats which is in agreement with a previous report showing no significant differences in the rate of protein synthesis between old and middleaged rat brains TM. Since aging is frequently associated with cellular death, measuring protein levels throughout the brain helps to approximately quantify this putative cellular loss. The fact that we did not observe any significant decrease in the tissular protein levels in 3 out of the 4 structures studied argues against this idea, although the glial cell proliferation which occurs in response to neural loss may explain why the protein levels were preserved. Furthermore, despite the drop in the protein levels observed in the hippocampus, no significant differences in any of the neuropeptides studied were detected within the hippocampus, Since no direct relationship exists between protein decrease and neuropeptide levels, aging may therefore not involve neuronal loss of the peptidergic system studied here, at least in the hippocampus.
Age-related changes in the neuropeptide concentrations between 4- and 18-month-old rats Both increases and decreases in the tissular concentrations occurred in the rat brain between 4 and 18 months of life in the case of all the neuropeptides studied. The CRF and SOM contents increased in the hippocampus, while the NPY levels decreased in this structure. The NPY increased, on the contrary, in the striatum, whereas the SOM and M-ENK levels decreased during this time. On the other hand, as previously reported 4, we observed no changes in the SOM concentrations in the hypothalamus or the frontal cortices of young and mature rats. However, the above study reported a significant decrease in the rat striatal SOM levels between the age of 11 and 28 months, while we observed a decrease in the striatal SOM levels between 4 and 18 months but no significant changes between 18 and 26 months. The discrepancy between these two sets of data suggests that changes in the striatal SOM levels probably occurred between 11 and 18 months in the rat. Although the physiological significance of these variations cannot as yet be specified, these data taken together with the present observation of increased protein levels would lead to the conclusion that the variations measured between 4 and 18 months of age in the rat brain might be due to metabolic changes in the adult brain rather than being specifically associated with aging. The changes in neuropeptide concentrations observed during aging depend on the neuropeptide and the brain region investigated Interestingly, the changes in the neuropeptide concen-
trations observed between the age of 18 and 26 months involved only a few of the neuropeptides studied. The tissular concentrations of 3 neuropeptides were selectively affected depending on the brain structure examined. The CRF levels decreased in the frontal cortex, the fl-END contents decreased specifically in the hypothalami, and the tissular NPY concentrations decreased in 3 out of the 4 structures studied. In the mammalian CNS, CRF is widely distributed and in rodents, CRF nerve cells have been immunocytochemically identified in cortical layers II and III and CRF terminal fields in laminae I and IV - areas with high densities of CRF receptors 12'38. The significance of the CRF cortical concentrations has not yet been elucidated, however. There exists, nevertheless, neuropharmacological evidence that complex interactions occur between the cholinergic system and CRF (for review see ref. 15). The decrease in the cortical CRF concentrations with age might be linked with the concomitant decreases in the activity of the cortical cholinergic neurons observed in aging animals. The authors of several reports in the literature have referred to the decrease in the hypothalamic fl-END levels t°'31. Furthermore not only is less fl-END to be found in the hypothalamus of aging rats, but a marked alteration in the post-translational processing of this peptide occurs during aging 41. Among the other pro-opiomelanocortin (POMC)-derived peptides, the concentrations of a-melanostimulating hormone (a-MSH), adreno-corticotropin hormone (ACTH) and fl-lipotropin have been found to decrease in the hypothalamus of aged male rats (for review see ref. 35). The decrease in the amounts of the 4 major POMC-derived peptides may be attributable to a decrease in POMC gene transcription, a reduced or inappropriate post-translational processing or an increase in the rate of release of these 4 peptides. CRF is known to be one of the two main hypothalamic stimulatory factors of the biosynthesis and the release of POMC-derived peptides (for review see ref. 3). Although, as mentioned above, measuring tissular n e u r o p e p t i d e concentrations does not provide any particular information about the peptide biosynthesis, maturation or release, it is worth noting that there is no change in hypothalamic CRF tissular concentrations occurring during aging which could explain the decrease observed in B-END hypothalamic level. Further studies will be necessary to solve the mechanisms at the origin of the decrease in hypothalamic POMC-derived peptides occurring during aging. As previously mentioned, we observed a decrease in NPY levels in 3 out of the 4 structures studied. Recently, Fuxe et al. demonstrated the vulnerability of the NPY peptidergic system to aging processes 19. In agreement
45 with our results, these authors have shown that a m a r k e d loss of N P Y immunoreactivity occurs in the nerve terminals of the paraventricular hypothalamic nuclei as well as a d r o p in the N P Y concentrations in many hypothalamic nuclei in the 24-month-old rat. F u r t h e r m o r e , in the same study, a loss of N P Y immunoreactive p e r i k a r y a (but not of the other p e p t i d e perikarya) was o b s e r v e d during aging within the striatum. It is worth noting that the N P Y and the S O M , known to be co-localized in the same neuronal population 6'24'39, were not affected in the same way during this time, since we did not observe any significant changes between 18 and 26 months in S O M levels whatever the brain region studied. These decreases in N P Y concentration in central brain structures are of particular interest in view of the recent evidence that central N P Y peptidergic systems are involved in the neur o m o d u l a t i o n of the sensori-motor axes 23 as well as in cognitive functions TM, these two neuronal systems being preferentially i m p a i r e d during aging. F u r t h e r m o r e , previous studies 25 have d e m o n s t r a t e d the close relationship as well as at the synaptological features 4° and the biochemical interaction levels 26'34 between the N P Y peptidergic and m o n o a m i n e r g i c systems - - in particular with the dopaminergic system. These studies r e p o r t e d that a decrease in N P Y m R N A expression, and in the n u m b e r of N P Y positive immunostaining p e r i k a r y a , occurs in the striatum of adult Wistar rats when the disruption of the dopaminergic nigrostriatal transmission is induced by a d o p a m i n e ( D A ) r e c e p t o r antagonist or a blocker of D A synthesis. Since it is known nowadays that the central dopaminergic systems are particularly strongly affected in the aged brain, as shown by the m a r k e d decrease in
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