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Apparent absence of aging and gender effects on serotonin 1A receptors in human neocortex and hippocampus
Brain Rosearch 755 (1997) 26-32
Researchreport
Apparent absenceof aging ancl gen human neocortex and hippocxn Lionella Palego a** , Donatella Marazziti ‘, Alessandra Rossi f Gino Antonio G. Naccxato ‘, Antonio Lucacchini b, Giovanni
Accepted 20 Novernher 1996
Abstract
The effects of gender, aging and gender x age on the binding of the S-HT,, receptor high-affinity agonist [’ H]8-hydroxy-2(di-N-propylamino)tetralin ([ “H]8-OH-DPAT), were evaluatedand compared in tissues of human prefrontal, temporal, parietal, occipital cortex and hippocampusobtained from 21 autopsy subjects. The results revealed no variation with age or gender in either the [ 3 maximum binding capacity (B,,,, 1 or dissociation constant (K,) values. On the other hand, when separatecorrelations to subject ages were performed for men and women, aging effects on [ 3H]8-OH-DPAT B,,,, and K, were detected: in men, a significant age-dependent decreasein K, values was observedin the occipital cortex; in women, the R,,,ilXsignificantly decreasedwith aging in the parietal cortex and hippocampus,while increasing in occipito-cortical membranes.Overall, the present study reveals that. although neither gender nor aging ‘per se’seem to modify the S-NT,,, receptor binding, gender may reveal region-specific aging effects. i.e. on receptor affinity in men and receptor density in women. Such findings should stimulate further investigation on the hypothesizedexistence of gender x agerelated cross-connectionsbetween serotonergic system and hypothalamus-pituitary-gonudulcircuits.
Over the lust decade,rr c\chevidencehas surfacedto supportthe notion that raphe-nucleiserotonergicneurons may regulatea wide varietyof behavioursand functionsin the centralnervoussystem(CNS), especiallythoseinvolving neocartical,limbic and hypothalamus-pituitary circuits: serotonin6HT) seemsto exert an importantmodulatory role on memory,arousaland sleep,pain perception,food intake, thermoregulation,libido, impulsivity, aggressivenessand mood (for reviews:[2 1.29.361).All these functions may decline during the aging process:in fact, incidenceof cognitive,neuroendocrine and mooddistu+mces has n found increasedin elderly subjects[5,13]. Another physiologicalparameterwhich may influence CNS serotonergicfunctions is gender: neuropsychiatric disorders,such as major depresGon,where serotonergic
l
Comspondinp
author.Fax: + 39 (SO)2 I
0006-8SP3/97/$17.00 Copyright Plf SOOO6-8993(96)01415-l
58 I.
abnormalitieshave been reported [27], show a different male-femaledistribution [37]. Furthermore,male-female anatomicaland histologicaldifferenceshave beenfound in 5-HT-relatedregions,suchas humancortex and hypothalamus [ 11,and brain neurochemicaldimorphism has been postulatedas reflecting the aforementionedgender-related epidemiology of psychiatric disorders [ 18,191.Since at least seventeen5-HT receptorsubtypeshave been classiAed and localized in the CNS [ 17,341,assessmentof possiblegender-relatedand age-dependentdifferencesin 5-HT neurotransmissionseemswarrantedat the level of distinct 5-HT receptorsin discretebrain areas. In this regard,the 5-HT,, receptorsubtype has attracted much attentionas this receptorhas been extensivelycharacterizedin the brain of severalmammals,including humans[14,16,23,25,3 11.It furthermoreseemsto play a key role in the physiologyof the serotonergicsystemand has been postulatedas being involved in mood and anxiety disorders[8,35]. Moreover,relatively little data is availableregardingttie
Q 1997 Ekcvier ScienceB.V. All rights reserved.
.c~r(9~%~t
ca9ruep;
en
I’(Ph4I.I) P W0mt31
dPMD) P
gender, aging
and gender x age cffecls i~~i.~urn~s~tu~ti(~n bi~~d~l~~ parameters of T, i.e. the maximum binding capacity (
40+ to 0. I
19 t IO - 0. I4 0.6H 4Of IO 0. I7 0.64
4 1~3 t O.F7 1,.25 0.46 4.58 It: 0.55 - 0.20 0.57
34kY - 0. IS 0.65 37fK 0.04 0.0 I
4. I6 IO.79 - 0. I5 0.65 4. I6 &(I.63 0.23 0.53
04f I’) - 0.26 0.44 86rt IX -’0.07 O.riS
4.06 -t 0.63 - 0.5 I 0.07 3.65 f 0.74 0.01 0.95
Ocdpilul cortex
Hippocampus Men dPMD1 P Women 4J’MD) P
the
dissociation constant ( K,, ), in the human prefr itaI cortices and l~i~~~~~~~~~~~usfrom a w99ple o!’q:
0.77 35 fY 0. t 2 0.7-l
Men APMD) P Women I’U’MD) P
on
VillUes are mean f S.D. of specimens from uutophy subjects (1lle11: II = 1 I ; women: II = IO). r(PMD): Pearson and Spearman coefficient of correlation between U,,,,, and K,,. rehpectively and postmortem delay. and P: its level of significance.
1”HJ&OH-DPAT (specific activity: 147. was purchasedfrom New England Nuclear CN MA, USA); S-HT was obtained from Sigma (St. Loui% USA); all other reagents used were of analytical grade. 2.2. Hmart brain tissues
Human brain tissues were obtained at the University of Piss Pathology Institute by autopsy on 21 subjects. 11 men and 10 women, who died from causes not invcllving primarily or secondarily the CNS and whose histories cxeluded evidence of psychiatric illness or treatment with psychotropic drugs before demise. Male ages and postmortem delay ranged from 32 to 83 years (mean It SD. = 64.45 f IS. 18) and from 18 to 42 h (mean + S.D. = 29.4 k 7.1) respectively; female ages and postmortem delay rangedfrom 30 to 78 years (mean k S.D. = 58.70 f 15.49) and from I8 to 43 h (mean i S.D. = 30.6 f 10.5) respectively. Male causes of death were myocardial infarction
E, &“trlqct et (11,/ l~~~l~tll?w’clrl’ll
28
41, respirataryf;rilure (11= 3, gastr~intestinulcancer I), lung cancer(12= 2). liver cnncerbt = 11;female causesof deathwere myocardialinfarction (tl = 4k respi-
ratory failure tn = 3,
Prefrontal cortex
AGE (Yesrr)
AGE (Years)
Temporal cortex 60
6,s
55
6
50
5,s
43
5 4,s 4 0
385 3 55 3
20
30
40
50
60
70
80
90
-iO
30
40
50
60
90
80
90
Parietal cortex 6ob.
.
-
.
-
.
.
.
_
.
_
.
0 0
0 0
I
mm (r 0 4.41; P - 0.2) women (r I -0.09; P = 079)
20
30
40
50
60
70
80
AGE (Vour)
90
20
30
40
50
I
60
70
80
90
AGE (Veur)
Fig. I. Gender x ape correlations of [ 7~]8-~~-~~~~ binding capacity ( B map) and affinity ( K,) in the prefrontal. temporal and parktol cortices. .o represent Bm,, ~LSfmol/mg protein. and K,, IIS nM. of men (n = I I ) and women (N = IO), respectively. Straight and broken lines iIre the best tit obtained from linear repression analysis in men and women rqxctively. Inset: r = Pearson’s (for B,,,,, anrrlysis) and Spearman’s (ior AT;,,analya~s) cwfftcicnt of condiition and its level of significunce. Significimt coefficient of correlation ( P < 0.05). l
l
55 so 45 40 35 30 25 20 -
..m
-.s
v,
3
B
10 2
2,s
2
50 20
30
40
50
AGE (Years)
60
70
60
90
20
30
40
50
60
70
80
90
AGE (Years)
* .cd rcprcWt Fig. 2. Gcndcr X ;~gc corrclutions of [ ‘l-i]%Oi-I-DPA’I’ binding capitcity f Ii,,,,,, ) illlll affinity ( K,, ) iI1 the occipiLd Wrtex od Ili[~pOCiLlllpU\. ( II = IO). respccrively. Straight and broken lrneh art’ the hesl fil ohtai~ul frm B ,,,,,%, ih flllOl/lllg prcttcin. ilnd K,,. as nM. 01’ 1lw11 (II = I I) id wo11w1 01’ illld Spm-man’s (fin k’,, analysis) codficicnl linear rqrcssioa analysis in il~t211 aid women respcctivoly. Iiiael: r = Pearson‘s (for fI,,,S,, illlillySiS) l Significanl cotflicient d correlrrlion ( P < 0.05). ccmelation alld its IWel Of SigllifilXlllC~.
between age and B,,,,, was obser (I.== --0.81; P < 0.00s~ ( (F= -0.80; P < 0.005) ( 2.4.
Data
ilnaly.~is
trr1d statistks
Each experiment was performed in triplicate and the equilibrium-saturationbinding parameters(i.e. maximum binding capacity, Bmua,in fmol/mg protein, and dissociation constant, K,, in nM) were analysedwith the iterative curve fitting computerprogramsEBDA and LIGAND 1221. [ 3H&OH-DPAT binding parameterdifferences between correspondingbrain regions from men and women were evaluatedusing the Student’s t-test for independentsamples. Correlation studies between Bmilx,subject age and postmortemdelay were performed through the Pearson’s method.As K, values are not normally distributed, correlation studies between K,, subject age and postmortem delay were carried out through the non parametric Spearman’s method. All test of significance were two-tailed, with a preset cy level of P < 0.05. 3, Results
Under our experimental conditions, the [JH]8-QHDPAT specific binding ranged approximately from 58 to 75% of the total binding. Scatchardanalysis of binding data yielded a better fit to a single-site rather than a two-site model (Hill number close to unity) (data not shota,n).For the sake of comparison,Table 1 presentsthe BI’i,,,x tnd K, values obtained in the cortIca1 areas and hipprwampusof men and women, Student’s t-test rsvcnlcd data obtained in men wcrc undistinguishable from those in women, No significant correlation was found in any region between f$,,, or K, and postmortem delay in males or females (Table 1). Pearson analysis revealed no significant correlation between [ 3H]8-OHDPAT binding parametersand age in the full group of autopsy subjects,though the coefficient of correlation for BII\i4x~ahcs in the parictul cortex and hippocampus approached significance (r = -0.42; P = 0.058 and r = -0.43; P = 0.0%) (Table 21, as well as no effect of postmortemdelay (data not shown).
As shown in Figs. 1 and 2, aging effects were deteetablewhen men and women were analysedseparately:a significant negative correlation coefficient between age and & was found in men at the level of the occipital cortex (r = -0.67: P < 0.05) (Fig. 2); moreover, in women. a significant negative coefficient of correlation
Firstly, it emergedthat in no region does gender seem to alter the 5-HT,, receptor binding, ment with both autoradiographic an saturationbinding studies [9]. Other a have previously found that a 5-HT,, receptor bin Here we reconfirm B,,, values with age was nearly si cortex and hippocampus.However, effects of aging on 5-HT1, receptor binding are detectable when men and women are analysedseparately.In fact, in males, K, valuesshowed a significant nc ’ correlation -DPAT K, with age in the occipital cortex. As [“H values have never been found to vary with age in hu~~an brain, this representsa novel finding and indicates age-related changes in 5-HT,,1 receptor affinity. Interestingly, aging seems to decrease5-HT2A receptor affinity in the human cortex [4,12,24], thus revealing oppo age on different S-H? receptor subtypes. significant age-dependentdecrease in 5-HT,, receptor density ( Blllilr() was found in the parietal cortex and hippocampus. Such a result seems in conflict with that obtained by Dillon et al. [9 who revealedan agi effect on ~9calnpus ill Y values from I‘ronto arietal cwices and h in men only. Howcvcr, it should bc consideredthat these authors analysed autopsy subjects of al~proximatcmean age of 40 years, whereas the present work dealt with (111 older group of subjects, As aging effects on S-HTergic system seem to depend on age group [2,3], it can be also speculated that gender X age effects on 5-HT receptors may be affected by correspondingly different ranges of age, even in the same cerebral region. If true, Dillon’s findings, together with our results, might indicate that the decreasein 5-HT,, receptor density occurring in the parieta1 cortex and hippocampus is more pronounced in younger men and in older women. A further novel finding presentedhere is the significant increasewith age of the 5-HT,, receptordensity in occipito-cortical membranesin women. Differences in aging effects between the sexes have been also previously observedin dopamineD, receptorsin the living human brain [39]. Therefore, as already assumed for dopaminergicsystem, the finding that gender strongly intluences age effects on cortical and hippocampal5-HT,, receptors suggests the existence of a gender-dependent control of brain aging mechanismsexerted through alterations of 5-HT-related cross-connectionsbetween cortical,
In the present work we also found that gcnder-dependent cffccls of aging on 54IT,, receptors in the human brain are region-specific: they involve the parietal, Ehe occipital cortex and the hippocampus while excluding prefronto- and temporo-cortical tissues. The mechanismof such regional&y still remain obscure, though it has already been observed in previous studies on the effects of aging, alcohol use and suicide on S-HTI receptors and other 5-MT markers [7,9, I 1,121. In conclusion, a distinction in S-HT,, binding parameters has been revealed between men and women which deserves further investigation: in women, region-specific [ ‘H]X-OH-DPAT binding alterations with age are related to receptor density, while in men they are related to receptor affinity. Such tlndings underscorethe need for future studies on the physiological variables, such as sex and age which may influence the number and the affinity state of S-HT receptors in the human brain and indicate simultaneous analysis of 5-HT receptor subtypes in severa! age and sex-matched
32 from patients with chronic sohir,ophreni:i, Life Sci. 48 (1991) 35% 363. [ Ihj D. Moyer, A. Puzos, A. Prukst. J.M. Pulitcios, Somtwin rccepton in the humtrtt bruin. I. Ch,rntc:erizution and uutoradiographic localization of 5-HTIA recognition sites. Appitrent ilbb\etlCe Of S-HT,B recognition sites, Bmin Res. 376 ( 1986) 85-96. [17] D. Hoyer. D.E. Clarke, J.R. Fozard, P.R. Hartig. G.R. Martin. E.J. Mylechanne, P.R. Saxena. P.A. Humphrey, International Union of Pharmacology Classification of receptors for S-hydroxytryptamine (serotonin). Phttrmucol, Rev. 46 (1994) 157-203. [IS] B. Kirkpatrick, N.L. Bryant, Sexual dimorphism in the brain: It’s worse than you thought, Biol. Psychiatry 38 (1995) 347-348. [ 191 P. Lepage and M. Steiner, Gender tend serotonergic dysregulation: Implications for late luteal phase dysphoric disorders, in: G.B. Cassano and H.S. Akiskal (Eds.). Serotonin-Related Psychiatric Syndromes: Clinical and Therttpeuticttl Links, Royal Society of Medicine Services Ltd., London/New York, 1991. pp. 131- 143. [2O] K.P. Lesch, S. Mayer. J. Disselkamp-Tietze, A. Hoh. C. Schoellnhammer. H.M. Shultze. Subsensitivity of the 5-hydroxytryptantine I A (S-HT,, 1 receptor-mediated hypothermic response to ipsapirone in unipolttr depression, Life Sci. 46 ( 1990) I27 I - 1277. [?I] 1. Lucki. S. Wieland, 5-H y d roxytryptamine receptors and behavioral responses, Neuropsychophartnricology 3 ( 1990) 4%I-493. (22) McPherson, G.A.. KINETIC, EBDA, LIGAND. LOWRY: A collection of rttdioligand binding analysis programs. Biosaft. Cnmbtidge. UK, 1985. [23] D. Marazziti, S. Manacci, L. Palego. A. Rotondo. C. Mozzanti. I. Nttrdi, H. Ladinsky. E. Girttldo. F. Borsini, G.B. Cassnno, Locitlization and gene expression of serotonin 1A (5-HT,*) receptors in human brain postmortem, Brain Res. 658 (!994) 55-59. (241 J. Marcusson. L. Oreland, B. Win&d. Effect of age on serotonin (S-l) binding sites, J. Neurochem. 43 (1984) 1699-1705. [ZS] D.N. Middlemiss, A.N. Palmer. N. Edel. D.W. Bowen, Binding of the novel scrotonin agonist g-hydroxy-2-(di-N-propylnmino)tetrulin in normal und Alzhcimer brain, J. Neurochcm. 46 ( 1986) 993-996. [%I D.G. Morgan. The dopaminc rtnd scrotonin system\ during aging in lttmltttt and rodent bruin, A brief review, Prog. Nettropsychophitrntitcol. Biol. Psychiutr. 1 I ( 1987) l53- 157. (271 D.L. Murphy, Neuropsychiatric disorder\ itnd the mttltiplc l~tttnutt twin WiT receptor subtypes and +&5ystctns. Nettrol~ltitrttta~~~l~~~~ 3 ( Iwo) 457 -47 I,
[28] C. O’Ncilt. R.F. Cowbun, B. Wiehitgcr. I. Rlitftt~~~P’t;B. Wittdblitttd. C.J. Fowler. Preservation of 5-hydroxvtryl~titttttttc- I A receptor-G protein iuteritctions in the ccrcbml torte\ 01’pittictlt\ ‘1~ith Al/hcimer’s diseusc. Ncurosci. Lctt. I23 (1991 P 15-19. [29] N.N. Ohborne and M. Hamon. Neuronat Srrotonin. Wile?. Chichester. 1988. [30] L. Palego. D. Marazriti. A. Rotondo. A. Batistini. A. Imxcchi~ti. A.G. Naccaritto. 0. Bcvilaccluit. F. Borsini. H. Ladin&) unJ G.B. Cassano. Further characteriatton of [ ‘H]Xpylaminohetralin binding sites in hutnan bran postmortem, Ncurochem. Int. (in press). [31] S.J. Peroutka, Pharmacological differentiation and chatcterizarion of 5-HT,,, 5-HT,, and 5-HT,,. binding sites in ral frontal cortex. J. Neurochem. 47 ( 1986) 529-540. [32] G.L. Peterson. A simplification of the pro&in assay method which is more generally applicable, Anal. Biochctn. 83 ( 1087) 356-366. [33] S.N. Pradhan. Central nettrc~tr;rn~mittcrs itttd aging. Lit’e Sci. 26 (1980) 1643- 1656. [34] F. Saudou. R. Hen. 5-tlytlroxyt~~pt;tttlitl~ rcccptc~r sub~ypc~ in vcr~cbrutes and invcrtchra\. NW-~K~KYIL PIII. 3 (lW-1) 503- 5.19. (351 R. Schreibcr. J. De Vry, 5-HT,, rcccptor ligiitl& in animal 111oJci\ of anxiety. itnpul\iviry and depression: Muttiplc ltt~ChilltiSIll\. Pn~g. Neun)psychophann~t~ot. Biol. P\qchiatry I7 ( I ) ( 1093 1 R7- 10-l. [36] L.D. Van der Kar. Neurocndocrinc phnrnlacolog~ of scrotonergic (5HT) neurons. Annu. Rev. Phitrttl;t~o!. Toxicol. Z I ( I99 i )3X9-3’?!!. [.i7] M.M. Weissmann. Aft’cctivc disorder\. in: L.N. Robin\ and D.A. Regier (Eds.). Psychiatric Disorders in America. The Epidemiologic Cntchment Area Study, Free Press, New York. 1991. pp. 53-80. 1381 S. Weiss, M. Sebben. D.E. Kemp, J. Bockurn. Serotonin 5-HT, rcccptors mediate inhibition of cyclic AMP production in neurons. Eur. J. Ph,~rmitcol. 120 ( 1986) 217-330. [39] D.F. Wang. H.N. Wagner Jr.. R.F. Dannats. J.M. Liuks. J.J. Frost. H.T. Ritvert. A.A. Wilson. A.E. Rosenhaum, A. DOU~+IQ, J.D. Petron; ,. M.F. Folstcin. J.K.T. Touttg. 0’ M.J. Kuhiil. Effects ot’ age ott dopaminc and \crotc)ttitt I’LXXB~I~~~~ ttteasurcd by po4ttron tOtll0gKlphy in lhc living tllltltiltl brain. Scicncc 22G ( 1984) I3c)3- I3Y6.
Researchreport
Apparent absenceof aging ancl gen human neocortex and hippocxn Lionella Palego a** , Donatella Marazziti ‘, Alessandra Rossi f Gino Antonio G. Naccxato ‘, Antonio Lucacchini b, Giovanni
Accepted 20 Novernher 1996
Abstract
The effects of gender, aging and gender x age on the binding of the S-HT,, receptor high-affinity agonist [’ H]8-hydroxy-2(di-N-propylamino)tetralin ([ “H]8-OH-DPAT), were evaluatedand compared in tissues of human prefrontal, temporal, parietal, occipital cortex and hippocampusobtained from 21 autopsy subjects. The results revealed no variation with age or gender in either the [ 3 maximum binding capacity (B,,,, 1 or dissociation constant (K,) values. On the other hand, when separatecorrelations to subject ages were performed for men and women, aging effects on [ 3H]8-OH-DPAT B,,,, and K, were detected: in men, a significant age-dependent decreasein K, values was observedin the occipital cortex; in women, the R,,,ilXsignificantly decreasedwith aging in the parietal cortex and hippocampus,while increasing in occipito-cortical membranes.Overall, the present study reveals that. although neither gender nor aging ‘per se’seem to modify the S-NT,,, receptor binding, gender may reveal region-specific aging effects. i.e. on receptor affinity in men and receptor density in women. Such findings should stimulate further investigation on the hypothesizedexistence of gender x agerelated cross-connectionsbetween serotonergic system and hypothalamus-pituitary-gonudulcircuits.
Over the lust decade,rr c\chevidencehas surfacedto supportthe notion that raphe-nucleiserotonergicneurons may regulatea wide varietyof behavioursand functionsin the centralnervoussystem(CNS), especiallythoseinvolving neocartical,limbic and hypothalamus-pituitary circuits: serotonin6HT) seemsto exert an importantmodulatory role on memory,arousaland sleep,pain perception,food intake, thermoregulation,libido, impulsivity, aggressivenessand mood (for reviews:[2 1.29.361).All these functions may decline during the aging process:in fact, incidenceof cognitive,neuroendocrine and mooddistu+mces has n found increasedin elderly subjects[5,13]. Another physiologicalparameterwhich may influence CNS serotonergicfunctions is gender: neuropsychiatric disorders,such as major depresGon,where serotonergic
l
Comspondinp
author.Fax: + 39 (SO)2 I
0006-8SP3/97/$17.00 Copyright Plf SOOO6-8993(96)01415-l
58 I.
abnormalitieshave been reported [27], show a different male-femaledistribution [37]. Furthermore,male-female anatomicaland histologicaldifferenceshave beenfound in 5-HT-relatedregions,suchas humancortex and hypothalamus [ 11,and brain neurochemicaldimorphism has been postulatedas reflecting the aforementionedgender-related epidemiology of psychiatric disorders [ 18,191.Since at least seventeen5-HT receptorsubtypeshave been classiAed and localized in the CNS [ 17,341,assessmentof possiblegender-relatedand age-dependentdifferencesin 5-HT neurotransmissionseemswarrantedat the level of distinct 5-HT receptorsin discretebrain areas. In this regard,the 5-HT,, receptorsubtype has attracted much attentionas this receptorhas been extensivelycharacterizedin the brain of severalmammals,including humans[14,16,23,25,3 11.It furthermoreseemsto play a key role in the physiologyof the serotonergicsystemand has been postulatedas being involved in mood and anxiety disorders[8,35]. Moreover,relatively little data is availableregardingttie
Q 1997 Ekcvier ScienceB.V. All rights reserved.
.c~r(9~%~t
ca9ruep;
en
I’(Ph4I.I) P W0mt31
dPMD) P
gender, aging
and gender x age cffecls i~~i.~urn~s~tu~ti(~n bi~~d~l~~ parameters of T, i.e. the maximum binding capacity (
40+ to 0. I
19 t IO - 0. I4 0.6H 4Of IO 0. I7 0.64
4 1~3 t O.F7 1,.25 0.46 4.58 It: 0.55 - 0.20 0.57
34kY - 0. IS 0.65 37fK 0.04 0.0 I
4. I6 IO.79 - 0. I5 0.65 4. I6 &(I.63 0.23 0.53
04f I’) - 0.26 0.44 86rt IX -’0.07 O.riS
4.06 -t 0.63 - 0.5 I 0.07 3.65 f 0.74 0.01 0.95
Ocdpilul cortex
Hippocampus Men dPMD1 P Women 4J’MD) P
the
dissociation constant ( K,, ), in the human prefr itaI cortices and l~i~~~~~~~~~~~usfrom a w99ple o!’q:
0.77 35 fY 0. t 2 0.7-l
Men APMD) P Women I’U’MD) P
on
VillUes are mean f S.D. of specimens from uutophy subjects (1lle11: II = 1 I ; women: II = IO). r(PMD): Pearson and Spearman coefficient of correlation between U,,,,, and K,,. rehpectively and postmortem delay. and P: its level of significance.
1”HJ&OH-DPAT (specific activity: 147. was purchasedfrom New England Nuclear CN MA, USA); S-HT was obtained from Sigma (St. Loui% USA); all other reagents used were of analytical grade. 2.2. Hmart brain tissues
Human brain tissues were obtained at the University of Piss Pathology Institute by autopsy on 21 subjects. 11 men and 10 women, who died from causes not invcllving primarily or secondarily the CNS and whose histories cxeluded evidence of psychiatric illness or treatment with psychotropic drugs before demise. Male ages and postmortem delay ranged from 32 to 83 years (mean It SD. = 64.45 f IS. 18) and from 18 to 42 h (mean + S.D. = 29.4 k 7.1) respectively; female ages and postmortem delay rangedfrom 30 to 78 years (mean k S.D. = 58.70 f 15.49) and from I8 to 43 h (mean i S.D. = 30.6 f 10.5) respectively. Male causes of death were myocardial infarction
E, &“trlqct et (11,/ l~~~l~tll?w’clrl’ll
28
41, respirataryf;rilure (11= 3, gastr~intestinulcancer I), lung cancer(12= 2). liver cnncerbt = 11;female causesof deathwere myocardialinfarction (tl = 4k respi-
ratory failure tn = 3,
Prefrontal cortex
AGE (Yesrr)
AGE (Years)
Temporal cortex 60
6,s
55
6
50
5,s
43
5 4,s 4 0
385 3 55 3
20
30
40
50
60
70
80
90
-iO
30
40
50
60
90
80
90
Parietal cortex 6ob.
.
-
.
-
.
.
.
_
.
_
.
0 0
0 0
I
mm (r 0 4.41; P - 0.2) women (r I -0.09; P = 079)
20
30
40
50
60
70
80
AGE (Vour)
90
20
30
40
50
I
60
70
80
90
AGE (Veur)
Fig. I. Gender x ape correlations of [ 7~]8-~~-~~~~ binding capacity ( B map) and affinity ( K,) in the prefrontal. temporal and parktol cortices. .o represent Bm,, ~LSfmol/mg protein. and K,, IIS nM. of men (n = I I ) and women (N = IO), respectively. Straight and broken lines iIre the best tit obtained from linear repression analysis in men and women rqxctively. Inset: r = Pearson’s (for B,,,,, anrrlysis) and Spearman’s (ior AT;,,analya~s) cwfftcicnt of condiition and its level of significunce. Significimt coefficient of correlation ( P < 0.05). l
l
55 so 45 40 35 30 25 20 -
..m
-.s
v,
3
B
10 2
2,s
2
50 20
30
40
50
AGE (Years)
60
70
60
90
20
30
40
50
60
70
80
90
AGE (Years)
* .cd rcprcWt Fig. 2. Gcndcr X ;~gc corrclutions of [ ‘l-i]%Oi-I-DPA’I’ binding capitcity f Ii,,,,,, ) illlll affinity ( K,, ) iI1 the occipiLd Wrtex od Ili[~pOCiLlllpU\. ( II = IO). respccrively. Straight and broken lrneh art’ the hesl fil ohtai~ul frm B ,,,,,%, ih flllOl/lllg prcttcin. ilnd K,,. as nM. 01’ 1lw11 (II = I I) id wo11w1 01’ illld Spm-man’s (fin k’,, analysis) codficicnl linear rqrcssioa analysis in il~t211 aid women respcctivoly. Iiiael: r = Pearson‘s (for fI,,,S,, illlillySiS) l Significanl cotflicient d correlrrlion ( P < 0.05). ccmelation alld its IWel Of SigllifilXlllC~.
between age and B,,,,, was obser (I.== --0.81; P < 0.00s~ ( (F= -0.80; P < 0.005) ( 2.4.
Data
ilnaly.~is
trr1d statistks
Each experiment was performed in triplicate and the equilibrium-saturationbinding parameters(i.e. maximum binding capacity, Bmua,in fmol/mg protein, and dissociation constant, K,, in nM) were analysedwith the iterative curve fitting computerprogramsEBDA and LIGAND 1221. [ 3H&OH-DPAT binding parameterdifferences between correspondingbrain regions from men and women were evaluatedusing the Student’s t-test for independentsamples. Correlation studies between Bmilx,subject age and postmortemdelay were performed through the Pearson’s method.As K, values are not normally distributed, correlation studies between K,, subject age and postmortem delay were carried out through the non parametric Spearman’s method. All test of significance were two-tailed, with a preset cy level of P < 0.05. 3, Results
Under our experimental conditions, the [JH]8-QHDPAT specific binding ranged approximately from 58 to 75% of the total binding. Scatchardanalysis of binding data yielded a better fit to a single-site rather than a two-site model (Hill number close to unity) (data not shota,n).For the sake of comparison,Table 1 presentsthe BI’i,,,x tnd K, values obtained in the cortIca1 areas and hipprwampusof men and women, Student’s t-test rsvcnlcd data obtained in men wcrc undistinguishable from those in women, No significant correlation was found in any region between f$,,, or K, and postmortem delay in males or females (Table 1). Pearson analysis revealed no significant correlation between [ 3H]8-OHDPAT binding parametersand age in the full group of autopsy subjects,though the coefficient of correlation for BII\i4x~ahcs in the parictul cortex and hippocampus approached significance (r = -0.42; P = 0.058 and r = -0.43; P = 0.0%) (Table 21, as well as no effect of postmortemdelay (data not shown).
As shown in Figs. 1 and 2, aging effects were deteetablewhen men and women were analysedseparately:a significant negative correlation coefficient between age and & was found in men at the level of the occipital cortex (r = -0.67: P < 0.05) (Fig. 2); moreover, in women. a significant negative coefficient of correlation
Firstly, it emergedthat in no region does gender seem to alter the 5-HT,, receptor binding, ment with both autoradiographic an saturationbinding studies [9]. Other a have previously found that a 5-HT,, receptor bin Here we reconfirm B,,, values with age was nearly si cortex and hippocampus.However, effects of aging on 5-HT1, receptor binding are detectable when men and women are analysedseparately.In fact, in males, K, valuesshowed a significant nc ’ correlation -DPAT K, with age in the occipital cortex. As [“H values have never been found to vary with age in hu~~an brain, this representsa novel finding and indicates age-related changes in 5-HT,,1 receptor affinity. Interestingly, aging seems to decrease5-HT2A receptor affinity in the human cortex [4,12,24], thus revealing oppo age on different S-H? receptor subtypes. significant age-dependentdecrease in 5-HT,, receptor density ( Blllilr() was found in the parietal cortex and hippocampus. Such a result seems in conflict with that obtained by Dillon et al. [9 who revealedan agi effect on ~9calnpus ill Y values from I‘ronto arietal cwices and h in men only. Howcvcr, it should bc consideredthat these authors analysed autopsy subjects of al~proximatcmean age of 40 years, whereas the present work dealt with (111 older group of subjects, As aging effects on S-HTergic system seem to depend on age group [2,3], it can be also speculated that gender X age effects on 5-HT receptors may be affected by correspondingly different ranges of age, even in the same cerebral region. If true, Dillon’s findings, together with our results, might indicate that the decreasein 5-HT,, receptor density occurring in the parieta1 cortex and hippocampus is more pronounced in younger men and in older women. A further novel finding presentedhere is the significant increasewith age of the 5-HT,, receptordensity in occipito-cortical membranesin women. Differences in aging effects between the sexes have been also previously observedin dopamineD, receptorsin the living human brain [39]. Therefore, as already assumed for dopaminergicsystem, the finding that gender strongly intluences age effects on cortical and hippocampal5-HT,, receptors suggests the existence of a gender-dependent control of brain aging mechanismsexerted through alterations of 5-HT-related cross-connectionsbetween cortical,
In the present work we also found that gcnder-dependent cffccls of aging on 54IT,, receptors in the human brain are region-specific: they involve the parietal, Ehe occipital cortex and the hippocampus while excluding prefronto- and temporo-cortical tissues. The mechanismof such regional&y still remain obscure, though it has already been observed in previous studies on the effects of aging, alcohol use and suicide on S-HTI receptors and other 5-MT markers [7,9, I 1,121. In conclusion, a distinction in S-HT,, binding parameters has been revealed between men and women which deserves further investigation: in women, region-specific [ ‘H]X-OH-DPAT binding alterations with age are related to receptor density, while in men they are related to receptor affinity. Such tlndings underscorethe need for future studies on the physiological variables, such as sex and age which may influence the number and the affinity state of S-HT receptors in the human brain and indicate simultaneous analysis of 5-HT receptor subtypes in severa! age and sex-matched
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