Neuroscience Letters, 133 (1991) 279-283 279 © 1991 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/91/$ 03.50

NSL 08245

Regionally specific age-dependent decline in 2-adrenoceptors: an autoradiographic study in human brain

Jul io Pascuap '2 , C a r m e n del A r c o 1, A n t o n i o M. Gonzf i lez 1, A l v a r o D iaz 1, E lena del O l m o 1

a n d Ange l Pazos 1

1Department of Physiology and Pharmacology, Unit of Pharmacology and ~ Service of Neurology, Universitary Hospital 'Marqubs de Valdecilla', University of Cantabria, Santander (Spain)

(Received 10 June 1991; Revised version received 11 September 1991; Accepted 13 September 1991)

Key words: Aging; ct2-Adrenoceptor; Autoradiography; Human brain; [3H]UK-14304

The effect of sex, postmortem delay and aging on ~t2-adrenoceptor binding was studied in tissue sections from several representative regions of the human brain from 21 subjects using pH]UK-14304 as a ligand. Sex and postmortem delay did not influence the density of ct2-receptors. Aging resulted in clear decreases in most forebrain areas examined (n. basalis > basal ganglia > hypothalamus > fronto-temporal cortex > hippocampus > visual cortex), whereas ~2-receptors did not significantly change with age in the amygdala and several infratentorial areas. We conclude that age-related, regionally specific decreases in the density of ct2-receptors occur in the human brain. The implications of these findings for age-dependent noradrenergic degeneration are discussed.

Both in aging and in the neurodegenerative disorders related to aging, that is Parkinson's disease (PD) and, mainly, Alzheimer's disease (AD), noradrenaline (NA) levels decrease in several human brain areas [1, 2, 13]. This decrease is thought to be secondary to the degener- ation of the locus ceruleus [14], known to occur with aging [22], AD [4, 5, 11] and PD [2], and has been tenta- tively related to many of the symptoms and signs seen in these conditions [2, 4]. Functional noradrenergic neuro- transmission depends not only on the amount of NA but also on the state of the 4 types of adrenergic receptors. Brain ~2-adrenoceptors are of special theoretical interest in the aging process. First, the majority of ct2-receptors appear to be presynaptic autoreceptors, thus located on the NA terminals arising from the locus ceruleus; and second, they play a key role in the regulation of N A re- lease [12].

The pharmacological profile and regional distribution of this noradrenergic receptor subtype in the human brain have been studied in detail in membrane homoge- nates by means of radioligand binding methods [16]. Whilst there is no reliable information about the behav- iour of ~2-receptors with human aging, scarce data, owing to the lack of anatomical resolution of binding

Correspondence: J. Pascual, Department of Physiology and Pharmaco- logy, Unit of Pharmacology, Faculty of Medicine, c/Cardenal Herrera Oria s/n, 39011 Santander, Spain.

methods and the different radioligands employed, have been reported on the state of these receptors in mem- branes of several brain areas in AD [8, 21] and PD [7]. Recently, it has become possible to study these receptors in human tissue sections by quantitative autoradio- graphy, using the selective agonists [3H]p-aminoclon- idine [20] and [3H]UK-14304 [18] as radioligands. This allows the detailed study of the distribution of the ct2- adrenoceptor in discrete human brain areas.

The purpose of the present study was to assess the ef- fect of aging, sex and postmortem delay on the density of human ct2-receptors in those areas where the number of such receptors is relevant using as radioligand the se- lective • 2-agonist [3H]UK-14304.

Postmortem tissues from 21 patients, 12 males and 9 females, who died in our hospital without a clinical his-

tory of neuropsychiatric disorder were obtained at au- topsy. To our knowledge these patients had taken no psychotropic medications before death. One hemibrain was immediately dissected and frozen in dry ice and then stored at - 7 0 ° C until use. The neuropathological exam performed in the remaining hemibrain was normal. To better assess the possible changes due to the postmortem delay and aging we chose brains with a wide range of both postmortem period, which ranged from 3 to 42 h (mean _+ S.D. = 16 _ 10), and age, which ranged from 19 to 84 years (mean + S.D. = 58 _ 17). Ten pm sec- tions from several brain regions were mounted onto

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ge la t in-coated slides and s tored at - 2 0 ° C until used. I f

necessary, consecutive, 20 /zm sections, were avai lable

for rout ine neu ropa tho log ica l staining. Af te r a 15 min

pre incuba t ion at r o o m tempera tu re in Tris-HC1 buffer

(50 m M , p H 7.7) conta in ing 0.1 m M MnC12, tissue sec-

t ions were incuba ted with 6 n M [3H]UK-14304 (82.7 Ci/

mmol , New England Nuclear ) for 90 min in the same

buffer, also at r oom tempera ture . Fo l lowing the incuba-

t ion, sections were washed for 5 min in ice-cold buffer

and then dr ied in a cold air s tream. Non-specif ic b inding

(5-15% of the to ta l b inding) was defined as tha t remain-

ing in the presence o f 10 -5 M phento lamine . Sections

were exposed at 4°C for 10 weeks to t r i t ium-sensi t ive

film (3H-Ultrofi lm), together with the a p p r o p r i a t e s tand-

ards, as previously descr ibed [23]. The a u t o r a d i o g r a m s

were analyzed and quant i f ied using a computer -ass i s ted

image analysis system (Mic rom-IP , Microm-Espaf ia ,

Barcelona, Spain). The results were analyzed by Stu-

dent ' s two- ta i led t-test (sex vs receptor densi ty) and by

regression analysis ( pos tmor t em per iod vs recep tor den-

sity; age vs receptor densi ty) using the Stat View pro-

g ram in a Mac in tosh SE 1/20 computer . The significance

level was preset a t P = 0.05.

[~H]UK-14304 specific b inding to h u m a n bra in sec-

t ions f rom a large number o f subjects did not signifi-

cant ly corre la te ei ther with sex or with p o s t m o r t e m delay

(da ta not shown). By contras t , aging resulted in clear

changes in a number o f the regions examined (see Table

I). The changes observed in the densi ty o f ~ r r e c e p t o r s

var ied depending on the h u m a n bra in area tested. In

general , all the h u m a n forebra in regions examined

showed an age-re la ted decline in the densi ty o f e2-recep-

tors. However , the degree o f receptor loss in h u m a n fore-

b ra in was not uniform. In fact, the decline per decade

ranged f rom 13.8% to 0%. The nucleus basal is o f Mey-

nert showed the highest loss o f this recep tor with age,

with a decade-decl ine o f 13.8% and a 69% receptor loss

f rom 20 to 70 years o f age (Figs. 1 and 2A). The basal

gangl ia (caudate = pu tamen = c laus t rum) (Fig. 1) and the

neocor tex ( t empora l cortex, layer I > f ronta l cortex,

layer I > p r imary visual cortex, layer III) (Fig. 1) fol-

lowed the nucleus basal is in the degree o f recep tor loss

with a significant decline per decade ranging f rom 6.2%

to 10.6%. Final ly , o ther forebra in areas examined

showed lesser, non significant, or no decline at all in the

number o f these receptors . Whi ls t the hypo tha lamus , nu-

cleus vent romedia l i s , and the CA1 area o f the h ippocam-

pus, s t r a tum lacunosum-molecu la re , exhibi ted a clear

but non significant decline in receptor loss per decade,

the amygda la was the only forebra in area keeping the

number o f ~2-receptors with age. In con t ras t to the re-

sults observed in most forebra in areas s tudied, the densi-

ty o f ct2-receptors did not significantly decrease in several

inf ra tentor ia l regions examined (see Table I, Fig. 2B). In

fact, areas with relevant densit ies o f e2-receptors, such as

the lateral midbra in per iaqueducta l area, the t r igeminal

mesencephal ic nucleus and the nucleus in te rpeduncular i s

showed a non significant receptor loss, with a decline per

decade o f less than 5.7%. The cerebel lar cortex, s t ra tum

granulare , an inf ra tentor ia l a rea with relevant densit ies

o f ~2-receptors, showed a very slight, non-signif icant in-

crease in a u t o r a d i o g r a p h i c grains with aging (Fig. 2B).

TABLE I

AGING AND DENSITY OF ct2-RECEPTORS IN SEVERAL HUMAN BRAIN AREAS

The densities correspond to 6 nM [3H]UK-14304 in fmol/mg protein. *% change from 40-70 years. N.S. = statistically non-significant.

Brain area n Age range Density Density Mean % change P (years) (mean ___ range change per from 2(~70

S.D.) decade (%) years

Frontal cortex, layer I 15 19-84 663+262 307-1163 - 9.0% -45% Temporal cortex, layer I 13 39-84 681 + 175 314-998 - 10.1% -35%* Primary visual cortex, layer III 9 19-81 1035+301 628 1369 - 6.2% -31% Hippocampus, CA~ 11 19 84 987+376 463-1437 - 7.4% -37% Amygdala, nucleus medialis 9 19-81 186+ 106 98 344 + 1.4% + 7% Amygdala, n. basalis accesorius 7 19 81 189_+ 103 98-366 + 2.2% + 11% Nucleus basalis of Meynert 11 19-84 171 _+ 118 19-352 - 13.8% -69% Caudate, head-body 14 19-84 102 _+ 37 3(~171 - 10.5% - 52% Putamen 18 19-84 116 + 60 34-300 - 10.0% - 50% Claustrum 13 19-84 243+125 115~175 -10.6% -53% Hypothalamus, n. ventromedialis 8 39 84 331 _ 168 83-574 - 10.3% -31%* Lateral periaqueductal area 9 19-74 367__+ 181 224 804 0% 0% Trigeminal nucleus, midbrain 7 19-74 267-+81 137 375 - 5.7% -28% Nucleusinterpeduncularis 10 19 81 207-+61 118 298 - 2.6% -13% Cerebellum, stratum granulare 10 19 84 668_+ 202 368--934 + 1.5% + 8%

< 0.025 <0.05 < 0.025 <0.1 (N.S.) >0.25 (N.S.) > 0.25 (N.S.) < 0.005 < 0.005 < 0.005 < 0.005 <0.25 (N.S.) > 0.25 (N.S.) <0.25 (N.S.) >0.25 (N.S.) >0.25 (N.S.)

Fig. I. Photomicrographs of autoradiographic images of pH]UK- 14304 binding to posterior striatum (10/tm sections) of a young subject (A) and an elderly subject (B). Note the clear decrease occurring with age for neocortex (Cx), putamen (P) and nucleus basalis of Meynert

(NB). Bar = 2 mm.

281

FMOL/MG PROTEIN Nucleus basalis 400' (A)

3 5 0 '

300'

250'

200'

150' •

100'

50'

0 • , • , • , • , • , - , - , - •

10 20 30 40 50 60 70 80 90 AGE, YEARS

FMOL/MG PROTEIN 1 0 0 0 (B)

900

800

70o

600

5 0 0 •

4OO

3O0 0

Cerebellar cortex

• • • , . . , . , . .

• , , , . 0 . , . , , , . , . ,

2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

AGE, YEARS

Fig. 2. Correlation pattern of specific [3H]UK-14304 binding in fmol/ mg protein to 10/am human brain sections against age in the nucleus basalis (A) and in the cerebellar cortex (B). Note the striking age-de- pendent decline in the density of =2-receptors in the nucleus basalis (P < 0.005) and the absence of significant changes in the cerebellar cor-

tex (P> 0.25).

In addition, other brainstem areas with relevant densities of ~2-receptors, such as locus ceruleus, nucleus tractus solitarius, dorsal nucleus of X or hypoglossal nucleus, exhibited no decline in these receptors from 61 to 84 years of age (data not shown).

The most important finding of this work is the demostration of age-related, regionally specific decreases in the density of ~a-receptors in the human brain. Although one potential explanation for this loss of ~2- receptors could be the existence of regionally-specific, age-related changes in the state of affinity of these recep- tors, Ka values for [3HUK-14304 specific binding in membrane homogenates of several forebrain areas do not change with age (Garcia-Sevilla, J. submitted, per- sonal communication). It is well-known that the adre- nergic innervation of the whole human brain arises in the brainstem, specifically from the locus ceruleus and adya- cent nuclei [14]. Owing to the proposed presynaptic loca- lization of these receptors, age-related ~2-receptor losses could theoretically be explained by the loss of presynap-

tic noradrenergic terminals arising from the locus ceru- leus neurons which are known to degenerate with aging [4, 22].

Data about the behaviour of human brain receptors in relation to aging are scarce [19]. Most studies on receptors and aging have been performed in rats or on gross human brain areas, usually in membrane homoge- nates, thus preventing any analysis of differential changes between discrete brain areas. Interestingly, the most dra- matic decrease in the density of ~2-adrenoceptors with age takes place in the nucleus basalis of Meynert, one of the brain regions mostly involved in the aging process up to date. It is tempting to relate this loss in ~2-adrenocep- tors in the nucleus basalis of Meynert and neocortical re- gions with the normal decline in some aspects of higher intellectual functions, such as attention or learning memory, occurring with age. Similarly, the relevant loss of ~2-adrenoceptors in the basal ganglia could partly explain the aging-related motor abnormalities. In fact, in parkinsonism, where locus ceruleus degeneration is

282

fairly cons tan t and a loss o f ~2-receptors in f ronta l cor tex

has been repor ted , cer tain symptoms , such as freezing

episodes, have been re la ted to the loss o f noradrenerg ic

innerva t ion [2].

One interest ing f inding in this s tudy is the absence o f

significant age-re la ted effects on the specific b inding

observed in several b ra in areas, main ly the amygda la

and inf ra ten tor ia l regions with re levant ct2-receptor b ind-

ing such as the cerebel lum. Our results for ~2-receptors

are very s imilar to those recent ly found for muscar in ic

receptors in h u m a n brain, bo th by means o f au to rad io -

graphic me thods in p o s t m o r t e m h u m a n bra in [3] and

with h igh-resolu t ion PET in the living h u m a n bra in [9].

In these studies a s t r iking decrease in muscar in ic b inding

was observed with age in neocor tex and corpus s t r ia tum,

whereas there were no a p p a r e n t age-re la ted effects on the

specific b inding observed in regions such as the thala-

mus, h i p p o c a m p a l fo rma t ion or cerebel lum [3, 9]. These

results suggest that aging is no t a non-specific process

t h roughou t the brain, but tha t it follows differential pat -

terns and degrees depending on the region o f the human

brain. Recent studies in A D bra ins have found that the

enzymat ic presynapt ic marke r s for bo th chol inergic and

noradrenerg ic presynapt ic nerve terminals precede the

loss o f neurons that synthesize these enzymes [6, 10].

These decreases are not general ized, only occurr ing in re-

gions with a previous loss o f pos t synap t i c neurons or o f

monoamine rg i c neuro t ransmi t te r s , such as h i p p o c a m p u s

or amygda la . In areas little affected by the disease, such

as the cerebel lum, presynapt ic enzymat ic marke r s

remained unaffected [6, 17]. Fu r the r works have shown

that the loss o f locus ceruleus noradrenerg ic somas in

A D is restr icted to those neurons tha t project to regions

o f the h u m a n bra in mos t affected in A D , whereas neu-

rons projec t ing to non- fo rebra in areas are relat ively

spared [15]. These findings suggest that a p r imary loss o f

pos t synap t ic neurons or o f p resynapt ic nerve terminals

may tr igger subsequent changes in the somas o f presy-

napt ic neurons leading to their secondary degenera t ion

[5, 15]. N e u r o t r o p h i c factors have been involved in this

p roposed re t rograde degenera t ion o f locus ceruleus in

A D [5, 10].

The noradrenerg ic deficit found in the h u m a n bra in

with aging seems to be very s imilar in mos t areas to tha t

found in A D , though o f lesser degree [4, 22]. Interest-

ingly, no cell loss in the h u m a n locus ceruleus can be

found under 65 years [22], while our da ta , showing a

s tr iking region-specific decrease in ct2-receptors s tar t ing

f rom the second decade o f life, suggest a c lear decrease

in noradrenerg ic terminals previous to this selective

senile locus ceruleus neurona l loss. These da t a seem to

lend weight to the p r o p o s e d re t rograde degenera t ion as

the mechanism o f locus ceruleus dea th in the aging proc-

ess.

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