aging is accompanied by a subfield-specific reduction of serotonergic fibers in the tree shrew...

Aging is accompanied by a subfield-specific reduction of

serotonergic fibers in the tree shrew hippocampal formation

Jeanine I.H. Keuker a,b,*, Jan N. Keijser b, Csaba Nyakas b,c,Paul G.M. Luiten b, Eberhard Fuchs a

a Clinical Neurobiology Laboratory, German Primate Center, Kellnerweg 4, 37077 Gottingen, Germanyb Department of Molecular Neurobiology, University of Groningen, The Netherlands

c Brain Physiology Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary

Received 22 March 2005; received in revised form 7 July 2005; accepted 15 August 2005

Available online 16 September 2005

Abstract

The hippocampal formation is a crucial structure for learning and memory, and serotonin together with other neurotransmitters is essential in

these processes. Although the effects of aging on various neurotransmitter systems in the hippocampus have been extensively investigated, it is not

entirely clear whether or how the hippocampal serotonergic innervation changes during aging. Rat studies, which have mostly focused on aging-

related changes in the dentate gyrus, have implied a loss of hippocampal serotonergic fibers. We used the tree shrew (Tupaia belangeri), an

intermediate between insectivores and primates, as a model of aging. We applied immunocytochemistry with an antibody against serotonin to

assess serotonergic fiber densities in the various hippocampal subfields of adult (0.9–1.3 years) and old (5–7 years) tree shrews. Our results have

revealed a reduction of serotonergic fiber densities in the stratum radiatum of CA1 and CA3, and in the stratum oriens of CA3. A partial depletion of

serotonin in the hippocampal formation, as can be expected from our current observations, will probably have an impact on the functioning of

hippocampal principal neurons. Our findings also indicate that the rat and the tree shrew hippocampal serotonergic innervation show some

variations that seem to be differentially affected during aging.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Aging; Hippocampus; Serotonin; 5-HT; Immunocytochemistry; Morphometry; Tree shrew

www.elsevier.com/locate/jchemneu

Journal of Chemical Neuroanatomy 30 (2005) 221–229

1. Introduction

During normal aging, various cognitive processes including

learning and memory are profoundly affected in some subjects.

These alterations probably underlie changes in the various

neurotransmitter systems in the brain as has been reported for

humans and experimental animals. The hippocampal formation

plays a crucial role in learning and memory and is known to be

affected during aging. The hippocampal formation is inner-

vated by cholinergic (Amaral and Kurz, 1985), serotonergic

(Azmitia and Segal, 1978), noradrenergic (Loy et al., 1980),

and dopaminergic (Gasbarri et al., 1994) fibers that, among

other systems like the GABAergic and glutamatergic systems,

are involved in various aspects of cognitive processing. A

study in rats, assessing spatial memory performance and

* Corresponding author. Tel.: +49 551 3851134; fax: +49 551 3851307.

E-mail addresses: Jeanine.Keuker@dpz.gwdg.de,

jkeuker@dpz.gwdg.de (Jeanine I.H. Keuker).

0891-0618/$ – see front matter # 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.jchemneu.2005.08.005

neurotransmitter concentrations in brain homogenates, indi-

cated that aging-related deficits in learning and memory might

involve concomitant alterations of various neurochemical

systems in several brain regions, including the hippocampus

(Stemmelin et al., 2000). Whereas the role of the cholinergic

system on hippocampal functioning and the aging-related

impairment of cholinergic afferents has been extensively

investigated (Fischer et al., 1992; Smith et al., 1999; Calhoun

et al., 2004), relatively few studies have examined the

hippocampal monoaminergic innervation during aging. In

the current study, we will focus on the serotonergic system.

Serotonin (5-hydroxytryptamine, 5-HT) modulates many

physiological and behavioral processes, such as sleep, circadian

rhythms, neuroendocrine function, and affective state (McEn-

tee and Crook, 1991), processes that are known to be affected

during aging. 5-HTon its own may have only a minor effect on

cognitive function; however, there is a potent interaction

between 5-HT and other neurotransmitters, for example

acetylcholine, in learning and memory (Steckler and Sahgal,

J.I.H. Keuker et al. / Journal of Chemical Neuroanatomy 30 (2005) 221–229222

1995; Vizi and Kiss, 1998), and this interaction is probably

disturbed during aging (Richter-Levin and Segal, 1996).

Whereas numerous studies provide detailed information

about 5-HT content or receptor binding characteristics in the

various subfields of the hippocampal formation, they do not

yield any information about the 5-HT innervation, such as the

fiber density. It is therefore important to study the histology and

morphology of the 5-HT fibers in the hippocampal formation.

In rats aged 24 and 28 months, a decreased 5-HT fiber density

was qualitatively observed in the hilus of the dentate gyrus and

in the strata oriens and lacunosum-moleculare of the

hippocampus (Davidoff and Lolova, 1991; Van Luijtelaar

et al., 1992). A quantitative investigation in rats determined 5-

HT fiber densities in the dentate gyrus molecular layer and

hilus, in which an aging-related reduction was found

(Nishimura et al., 1995).

Thus, these studies point to a decreased serotonergic

innervation of the hippocampal formation during aging, and

these findings should be confirmed in other species. The aim of

neurobiological aging research is to understand aging processes

in humans. The tree shrew is an interesting species to study the

effects such processes. Originally regarded as primitive

primates (Le Gross-Clark, 1956), tree shrews are nowadays

considered as an intermediate between insectivores and

primates and are placed in the separate order Scandentia

(Starck, 1978; Martin, 1990). In many characteristics, tree

shrews are closer to primates than are rodents. The high degree

of genetic homology between tree shrews and primates found

for several receptor proteins of neuromodulators and the

amyloid-b precursor protein (reviewed by Fuchs and Flugge

(2002)), and the three to four times longer life span of tree

shrews than rodents, raise the possibility for tree shrews to

become an alternative model for studying aging-related brain

changes in socially homogenous and stable cohorts (Michaelis

et al., 2001).

To our knowledge, aging-associated alterations of the

hippocampal 5-HT innervation in the tree shrew have not

been previously studied. We used immunocytochemistry for 5-

HT, and investigated the 5-HT innervation pattern throughout

the entire hippocampal formation in the various subfields of

adult (0.9–1.3 years) and old (5–7 years) tree shrews with

semiquantitative morphometry.

2. Materials and methods

2.1. Experimental animals

Five adult (0.9–1.3 years) and five old (5–7 years) male tree shrews (Keuker

et al., 2004) from the breeding colony at the German Primate Center (Gottingen,

Germany; for housing details see Fuchs (1999)) were used. The animals used in

the present study had been tested earlier in a spatial memory task, in which

reference and working memory can be differentiated (Keuker et al., 2004).

Because male tree shrews become fertile between 4 and 5 months, we

consider 1-year-old animals as adults. Animal records of 62 tree shrews from

our breeding colony showed that between the age of 5 and 7 years approxi-

mately 70% of the tree shrews die spontaneously, so we therefore regard tree

shrews in that age bracket as old.

The animal experimentation was conducted in accordance with ‘‘Principles

of laboratory animal care’’ (NIH publication No. 86-23, revised 1985) and the

European Communities Council Directive of November 24th, 1986 (86/EEC),

and was approved by the government of Lower Saxony, Germany.

2.2. Perfusion and tissue preparation

The animals were terminally anesthetized by intraperitoneal administration

of an overdose of ketamine (Ketavet1, Pharmacia & Upjohn, Erlangen,

Germany), xylazine (Rompun1, Bayer, Leverkusen, Germany) and atropine

(WDT, Hannover, Germany) 5:1:0.01. The descending aorta was clamped and

the animals were perfused transcardially with cold 0.9% NaCl for 3 min,

followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH

7.2 for 12 min. To prevent the development of postperfusion artifacts the heads

were postfixed in fresh fixative at 4 8C (Cammermeyer, 1978). On the following

day, the brains were gently removed and stored in 0.1 M PB at 4 8C for

approximately 1 month. For cryoprotection the brains were immersed in 2%

DMSO and 20% glycerol in 0.125 M phosphate-buffered saline (PBS) at 4 8Cfor five nights. The right hemispheres were then dissected into blocks that

contained the entire hippocampal formation, frozen on dry ice and stored at

�80 8C for 2 weeks before sectioning at a thickness of 50 mm on a Leica

cryostat. A stereotaxic brain atlas of the tree shrew (Tigges and Shantha, 1969)

was used for reference during the dissecting and cryosectioning procedures.

Because the orientation of the hippocampal formation is almost vertical, we

chose to cut horizontal sections, which provided the best possibilities for

hippocampal subfield delineation (Keuker et al., 2003).

2.3. Immunocytochemical staining for serotonin

For the immunocytochemical procedure, a 1-in-10 series was randomly

selected, yielding 13–16 sections per animal. The free-floating sections were

washed in 0.1 M PBS and then treated with 0.5% H2O2 for 30 min. After

washing, nonspecific binding of antibodies was blocked by incubating the

sections for 1 h with 5% normal goat serum (NGS; DAKO, Glostrup, Den-

mark) in 0.1 M PBS containing 0.5% Triton X-100. The sections were

subsequently incubated for 3 h at room temperature and 3 days at 4 8C with

rabbit anti-5-HT antibody (Zymed, San Francisco, CA, USA) at a dilution of

1:200 in PBS containing 0.5% Triton X-100 and 1% NGS. After incubation

with the primary antibody, the sections were thoroughly washed in PBS with

0.5% Triton X-100, and incubated with biotinylated goat anti-rabbit antibody

(DAKO) 1:400 in 0.1 M PBS with 1% NGS and 0.5% Triton X-100 for 4 h,

followed by washing in PBS with 0.5% Triton X-100. Then, the sections were

incubated with 1:200 horseradish peroxidase-conjugated streptavidin

(DAKO) in PBS with 1% NGS and 0.5% Triton X-100 for 2 h. After washing

with PBS and 0.05 M Tris–HCl (pH 7.6), the sections were stained with a

DAB kit (Vector Laboratories, Burlingame, CA, USA), which employs 3,30-diaminobenzidine (DAB) as a chromogen, and NiCl2 to enhance the DAB

staining. The concentrations of DAB, NiCl2, and H2O2 were 0.025%, 0.08%

and 0.01%, respectively. The exposure to NiCl2-DAB was 16 min for all

sections. The reaction was stopped by washing the sections in 0.05 M Tris–

HCl. The sections were mounted on glass slides in a 0.1% gelatin solution and

dried overnight at 37 8C, after which they were cleared in xylene for 30 min

and finally coverslipped with Eukitt (O. Kindler, Freiburg, Germany). For

comparison with the immunocytochemical images, series of adjacent hor-

izontal sections were mounted on glass slides, dried overnight, and stained

with cresyl violet. The sections were then dehydrated in a graded series of

ethanol, cleared in xylene and coverslipped. Careful examination revealed

that the sections were stained throughout the complete section thickness.

Labeling was absent when the primary antibody was omitted from immu-

nocytochemical incubations.

2.4. Semiquantitative image analysis of serotonergic fibers

The term ‘hippocampal formation’, as used in the current study, includes the

subiculum, the hippocampus (Bontempi et al., 2001), and the dentate gyrus

(Rosene and Van Hoesen, 1987). Seven areas in the hippocampal formation

were studied (see Fig. 1D) throughout the dorsoventral axis: (1) the pyramidal

layer of the subiculum, (2) CA1 stratum oriens, (3) CA1 stratum radiatum, (4)

CA3 stratum oriens, (5) CA3 stratum radiatum, (6) inner one-third, and (7) outer

J.I.H. Keuker et al. / Journal of Chemical Neuroanatomy 30 (2005) 221–229 223

Fig. 1. Gross distribution of 5-HT positive fibers in the hippocampal formation of an adult (A) and old (B) tree shrew. The frames in (A) and (B) show the position of

the photomicrographs in Fig. 2 (A) and (B), respectively. Nissl staining showing the principal cell layers of the hippocampal formation (C): the pyramidal cell layer of

the subiculum and hippocampal fields CA1–3, and the granule cell layer of the dentate gyrus (Rosene and Van Hoesen, 1987). Outline of the delineated areas (gray)

(D), in which the measurements were performed: (1) pyramidal layer of the subiculum; (2) CA1 stratum oriens; (3) CA1 stratum radiatum; (4) CA3 stratum oriens; (5)

CA3 stratum radiatum; (6) inner one-third; (7) outer two-thirds of the DG molecular layer. Scale bar = 1 mm (D, also applies to A–C).

two-thirds of the molecular layer of the dentate gyrus (Rosene and Van Hoesen,

1987). The animal numbers were coded prior to the quantification, and the code

was only revealed when all data had been collected.

All measurements were carried out with a Quantimet Q-600HR digital

image analysis system (Leica). The procedures have been previously described

in detail (Harkany et al., 2000; Horvath et al., 2004). All data were obtained

with a 20� objective lens and a 469.4 nm emission filter. A shading correction

(assessed with the microscopic slides, just aside of the tissue section) was

performed for each animal to eliminate any major irregularities in the

illumination of the microscopic field. After this correction, the gray-scale

threshold was determined and kept constant throughout the quantification

procedure. A microscopic field of 0.565 mm � 0.565 mm was shown on a

monitor, on which the areas of interest were manually delineated. The

delineated areas were approximately 0.2–0.4 mm2. Care was taken to keep

the positions and areas of the delineations for all the sections highly compar-

able for all animals. The computer used was programmed to recognize fibers

and varicosities, yielding an overlay with a digitized skeleton of the 5-HT-

immunoreactive (IR) fibers. Fig. 2 shows, as an example for the quantitative

analyses in the current experiment, digitized, skeletonized 5-HT-IR fibers in

the CA3 region for a representative adult and old tree shrew. Only structures in

the focal plane of the middle of the sections were converted into an overlay.

The depth of focus of a 20� objective lens is approximately 6 mm, and after the

immunocytochemical staining, the sections were approximately 10 mm thick.

Thus, the extent of the fibers in the overlay was largely proportional to the

amount of fibers in the sections (see Fig. 2C and D). Varicosities are thicker

than fibers, and they were identified by the computer throughout the section

depth. Because a random series of sections was selected, the plane of section

was not the same in all brains. However, measuring fiber densities in evenly

spaced sections perpendicular to the hippocampal formation will yield a

reliable mean value.

The percentual area of skeletonized 5-HT-IR structures was calculated for

the delineated areas using the image analysis software. Because staining

intensity varied strongly between certain hippocampal areas, two different

settings were used (lamp intensity and gray-scale threshold determination): one

setting for the strata oriens and radiatum of CA1 and CA3, and for the inner one-

third of the DG molecular layer, and another setting for the subiculum and the

outer two-thirds of the DG molecular layer. Fibers in the stratum lacunosum-

moleculare were not quantified, because the numerous fibers in this area would

visually overlay each other and would cause a substantial underestimation of the

fiber density. We did not measure in the hilus of the DG, because it was difficult

to delineate the area in a standardized and reproducible manner.

To determine the effect of aging on the 5-HT-IR fiber densities in the

evaluated hippocampal regions, we used a two-way analysis of variance

(ANOVA), with age group (adult and old) as the between-groups factor and

the sections along the hippocampal axis per animal as the within-subjects factor.

The significance level was set at P < 0.05.

J.I.H. Keuker et al. / Journal of Chemical Neuroanatomy 30 (2005) 221–229224

Fig. 2. Digital photomicrographs of 5-HT-immunoreactive fibers in the CA3 area in (A) an adult (0.9-year-old) and (B) an old (5-year-old) tree shrew. Computer

images showing the digitized, skeletonized fibers in the CA3 area of the adult (C) and old (D) tree shrews, exactly matching (A) and (B), respectively. The digitized

images demonstrate how the 5-HT-positive structures are recognized by the computer analysis system to produce a skeleton, of which in the delineated areas the fiber

area percentage is determined. Note the lower 5-HT fiber densities in the strata oriens and radiatum of the old animal. rad., stratum radiatum; luc., stratum lucidum;

pyr., stratum pyramidale; or., stratum oriens. Scale bar = 100 mm (A, also applies to B–D).

3. Results

3.1. Qualitative observations

The hippocampal subfields as they appear after Nissl

staining are shown in Fig. 1C (see also Keuker et al., 2003). The

immunocytochemical staining for 5-HT resulted in a strong

specific signal with low background (Fig. 1A and B). The

highest 5-HT-IR fiber density in the tree shrew hippocampal

formation was observed in the stratum lacunosum-moleculare

of subfields CA1, CA2 and CA3 (dark layer adjacent to the

dentate gyrus). High fiber densities were found in the

subiculum, especially towards the border with CA1 subfield,

and in the outer two-thirds of the DG molecular layer. Lower 5-

HT-IR fiber densities were observed in the strata oriens and

radiatum of fields CA1–3, the inner one-third of the DG

molecular layer, and in the hilus of the DG. The principal cell

layers of the hippocampus and DG, and the stratum lucidum of

subfield CA3 contained only few 5-HT-IR fibers. On a low

magnification, density differences in hippocampal 5-HT-IR

fibers between adult (Fig. 1A) and old (Fig. 1B) tree shrews can

barely be observed.

We observed a few fine and numerous beaded fibers within

the hippocampal formation of the tree shrew (Fig. 3A–D) and

occasionally stem-axons in the fimbria and alveus (Fig. 3A).

From our material, it is difficult to judge how fine fibers are

affected by aging. However, we could detect clear signs of

degeneration in the beaded fibers. Fig. 3E–I demonstrates such

degenerative changes in a qualitative manner. In general, the

varicosities of beaded fibers seem fewer in number in old

animals, and certain segments of such fibers are thinner than

normal and practically devoid of varicosities. Varicosities

bordering such segments appear larger in old than in adult tree

shrews.

3.2. Results from semiquantitative image analysis

The semiquantitative results of 5-HT-IR fiber densities for

each evaluated subfield are presented as mean (�S.E.M.) per

group in Fig. 4. The two-way ANOVA revealed significant

reductions of 5-HT-IR fiber densities in the CA3 stratum oriens

(F1,8 = 19.94; P = 0.002) and in the stratum radiatum of CA1

(F1,8 = 13.43; P = 0.006) and CA3 (F1,8 = 27.39; P < 0.001),

and a trend towards a decrease in the CA1 stratum oriens

J.I.H. Keuker et al. / Journal of Chemical Neuroanatomy 30 (2005) 221–229 225

Fig. 3. Detailed digital photomicrographs of 5-HT fiber types in the hippocampal formation of adult (A–D) and old (E–I) tree shrews. A stem-axon in the alveus,

relatively thick without varicosities (A). Beaded fibers are thin with relatively large, spherical varicosities, which are regularly aligned (B). Fine fibers are thin with

fusiform or ovoid varicosities smaller and fewer than those of beaded fibers (C). The difference between fine and beaded fibers is easily recognized in (D).

Degenerative, beaded 5-HT fibers (E–I) clearly contain fewer varicosities than healthy beaded fibers. (G) Left to the branching point (arrow) of a beaded fiber runs a

fiber (upper) with fewer varicosities than normal and a fiber (lower) that is lacking varicosities. Varicosities that border a fiber segment with shrunken varicosities

sometimes appear larger than normal (E, F, and H). Thick and thin arrows in (B–D) point to varicosities of the thick and thin fibers, respectively. Segments of thick

fibers showing noticeable degenerative changes are marked between arrowheads (E–I). SA, stem-axon; BF, beaded fiber; FF, fine fiber. Scale bar = 10 mm (D, I, also

applies to A–C and E–H).

(F1,8 = 3.98; P = 0.081) of old tree shrews. The 5-HT-IR fiber

densities in subiculum (F1,8 = 1.32; P = 0.284), inner one-third

(F1,8 = 1.40; P = 0.270) and outer two-thirds (F1,8 = 0.28;

P = 0.611) of the DG molecular layer of old tree shrews were

not significantly different from those of adult animals.

Fig. 4. Average 5-HT-immunoreactive (IR) fiber densities (expressed as area

%) in evaluated hippocampal subfields of adult (n = 5, light gray) and old (n = 5,

dark gray) tree shrews. **P < 0.01; ***P < 0.001.

4. Discussion

4.1. Methodological considerations

A limitation of the current method is that perfusion–fixation

may cause awashout of the antigen (5-HT), resulting in reduced

concentrations of 5-HT in the fibers, or even in false negative

staining. To avoid this, the animals were perfused with NaCl for

only a relatively short time, and the prerinsing and fixation steps

(the latter with paraformaldehyde, a fast fixative) were kept

constant for all animals. Also during the immunocytochemical

procedure, the incubation and washing times were the same.

Furthermore, we used a NiCl2 enhanced DAB staining, which is

at least 7–10 times more sensitive (Coventry et al., 1995) than

DAB staining alone, yielding more positive staining and a

higher contrast over background. Moreover, we used strepta-

vidin instead of avidin, as it has a better tissue penetration and a

higher sensitivity than does avidin. Thus, by using the current

technique, we can be confident that a negative effect of a

J.I.H. Keuker et al. / Journal of Chemical Neuroanatomy 30 (2005) 221–229226

possible washout of the 5-HT antigen was kept to a minimum.

The morphology of the fibers in Fig. 3 shows that there is in fact

an aging-related change in the fibers.

In a previous study, we estimated with stereology the total

volume of the hippocampal formation and its separate subfields

in the animals that were used in the current investigations, and

we did not find a difference between adult and old animals

(Keuker et al., 2004). Therefore, the present results probably do

not reflect an atrophic change in the hippocampal formation.

However, because we have not measured the volume of the

specific hippocampal strata, we could have overseen a potential

differential shrinkage, for example of the stratum lucidum,

which could indicate a redistribution of 5-HT fibers during old

age in the hippocampal formation.

4.2. Serotonergic fiber morphology

The anatomy of the 5-HT fiber system in the hippocampal

formation has been described extensively for the rat (Molliver,

1987; Swanson et al., 1987), rabbit (Bjarkam et al., 2003), cat

(Mulligan and Tork, 1988), nonhuman primate (Hornung and

Fritschy, 1990; Wilson and Molliver, 1991), and human (Tork,

1990). So-called fine fibers, thin with small fusiform or granular

boutons, originate from the dorsal raphe nucleus and primarily

innervate striatal and cortical areas. Beaded fibers are thin

axons with large spherical boutons, and primarily innervate

limbic areas and the septum. These two systems coexist in all

areas of the brain, but their ratio can vary considerably (Tork,

1990). The beaded fibers originate from thick, nonvaricose

stem-axons, representing the third fiber type, which arise from

the medial raphe nucleus. The appearance of the 5-HT fibers in

the tree shrew hippocampal formation was in agreement with

these descriptions.

Specific morphological alterations of 5-HT fibers have been

reported in the aging rat brain. Such aberrant 5-HT fibers have

swollen varicosities, are sometimes tortuous, and may appear in

clusters (Van Luijtelaar et al., 1988; Nishimura et al., 1998;

Nyakas et al., 2003). These alterations are supposed to be

indicative of fiber degeneration. In the aged rat hippocampus,

such degenerative 5-HT fibers were only infrequently observed

(Van Luijtelaar et al., 1992) and when present, appeared mainly

in the DG molecular layer and in the stratum lacunosum-

moleculare of the CA1 region (Venero et al., 1993). In the tree

shrew hippocampal subfields evaluated in the current study, we

observed almost no swollen varicosities so therefore concluded

that varicosities most probably did not affect the area

percentage measurements. It was proposed that, after several

stages of morphological aberrations, the disappearance of fibers

represents the final state of degeneration. However, not all 5-HT

fibers degenerate in the same manner. In some areas the

predominant form of degeneration is neurotransmitter accu-

mulation, resulting in swollen varicosities, while in the

hippocampus shrinkage of the 5-HT fibers appears to be

predominant (Van Luijtelaar et al., 1992). Accordingly, our

qualitative observations indicate that varicosities and fibers are

mainly shrinking, and that varicosities may eventually

disappear. Nevertheless, some varicosities that bordered

degenerative fiber segments with smaller or no varicosities

appeared larger than normal, and this could point to an

accumulation of 5-HT (Van Luijtelaar et al., 1992).

4.3. Species differences in the serotonergic fiber system

Although the 5-HT system generally shows a great degree of

homology between species, minor differences have been

reported. First of all, 5-HT neurons in the brain stem of the

New Zealand white rabbit, but not of the rat, undergo the same

kind of lateralization as seen in higher primates (Bjarkam et al.,

1997). Second, the 5-HT innervation pattern of the hippo-

campal formation shows species variations. The most apparent

difference between the tree shrew and the rat is that in the tree

shrew DG molecular layer, we observed a clear laminated

pattern with high densities in the outer two-thirds and a

relatively low density in the inner one-third, as has been

reported for the rabbit (Bjarkam et al., 2003), cat (Ihara et al.,

1988; Mulligan and Tork, 1988) and monkey dentate gyrus

(Hornung and Fritschy, 1990;Wilson andMolliver, 1991). Such

a specific laminated innervation is not observed in the rat DG

molecular layer (Molliver, 1987; Swanson et al., 1987; Ihara

et al., 1988). Furthermore, evidence exists that rat hippocampal

interneurons express not only 5-HT3 (Ropert and Guy, 1991),

but also 5-HT1A receptors (Levkovitz and Segal, 1997; Aznar

et al., 2003). It appears that such 5-HT1A receptor expressing

interneurons contain calbindin and parvalbumin (Aznar et al.,

2003). In contrast, a recent article has shown that calbindin- and

parvalbumin-containing interneurons in the tree shrew hippo-

campal formation do not express the 5-HT1A receptor

(Palchaudhuri and Flugge, 2005).

4.4. Aging-related changes in serotonergic fiber densities

The rat studies on 5-HT innervation in the aging

hippocampal formation report decreased fiber densities in

the DG hilus and molecular layer, and in the strata oriens and

lacunosum-moleculare of the hippocampus (Davidoff and

Lolova, 1991; Van Luijtelaar et al., 1992; Nishimura et al.,

1995). These scarce data from aged rats are, mostly because of

methodological differences, difficult to compare with our

results in old tree shrews, but it seems that the 5-HT innervation

in the hippocampal formation in the rat and tree shrew

undergoes minor differential changes during aging. Our

semiquantitative results revealed a significant decrease in 5-

HT-IR fiber density in the stratum radiatum of CA1 and CA3

and in the CA3 stratum oriens of old tree shrews, but not in the

DG molecular layer.

4.5. 5-HT-producing neuron loss as a cause of fiber

degeneration?

Although the 5-HT system is a diffusely innervating system,

the 5-HT fibers in the tree shrew hippocampal formation

display a laminated pattern, which appears to be differentially

affected by the aging process. This effect may be related to the

differential innervation from the raphe nuclei.

J.I.H. Keuker et al. / Journal of Chemical Neuroanatomy 30 (2005) 221–229 227

The rat hippocampal formation is mainly innervated by the

dorsal raphe nucleus (DRN; with about 60% and 10% via the

supracallosal and ventral pathway, respectively), and to a lesser

extent from the medial raphe nucleus (MRN; 30% of

serotonergic innervation via the fimbria/dorsal fornix) (Swan-

son et al., 1987). Fibers from the DRN are fine with small

varicosities, whereas the MRN is the origin of nonvaricose

stem-axons that branch out to beaded fibers that are ramifying

to the hippocampal formation (Tork, 1990). The distributional

pattern of this dual fiber system within the hippocampal

formation is highly comparable between different mammalian

species (Ihara et al., 1988). Fine fibers from the supracallosal

pathway mainly innervate the CA1 stratum oriens, and those

from the central pathway primarily spread into the CA1 strata

radiatum and lacunosum-moleculare (Ihara et al., 1988). The

CA3 stratum oriens is, predominantly, innervated by beaded

fibers via the fimbria/fornix pathway, but the CA3 strata

radiatum and lacunosum-moleculare mostly receive fibers

coming from the DRN via the central pathway (Ihara et al.,

1988). The hilus of the dentate gyrus is innervated both from the

fimbria/fornix pathway (beaded fibers) and from the ventral

pathway of DRN (fine fibers), whereas the molecular layer of

the dentate gyrus receives input from the supracallosal pathway

(Ihara et al., 1988).

Having these 5-HT innervation patterns in mind, and having

found a significant decrease in 5-HT-IR fibers in the CA1

stratum radiatum and CA3 strata oriens and radiatum of old tree

shrews (current study), we speculate that predominantly fibers

from the MRN, via the fimbria/fornix and ventral pathways, are

deteriorated, whereas fibers coming from the DRN via the

supracallosal pathway are spared by the aging process in tree

shrews. Studies in rhesus monkeys and humans demonstrated

that neurons in the nucleus centralis superior and MRN,

respectively, are lost during aging, but that the percentage of 5-

HT-producing neurons in these nuclei is unaltered (Kemper

et al., 1997; Kloppel et al., 2001). Thus, a reduction of 5-HT-IR

fiber densities in the hippocampal formation could partly be due

to a loss of 5-HT-producing neurons. However, the fine and

beaded fibers are distinguished purely on a morphological

basis, and many fibers show an intermediate morphology

(Bjarkam et al., 2003). Therefore, we cannot prove the nuclear

origin of the fibers in the tree shrew hippocampal formation, nor

can we be sure whether a loss of 5-HT-producing neurons is the

cause of the aging-related fiber degeneration. A reduced

activity of the rate-limiting enzyme for 5-HT synthesis,

tryptophan hydroxylase, as demonstrated in 24-month-old rats

(Hussain and Mitra, 2000), could also cause a loss of 5-HT

phenotype fibers coming from the raphe nuclei.

4.6. Functional implications

5-HT can influence the principal cells in the hippocampal

region directly by 5-HT released from single fibers traversing

the hippocampal layers (mostly nonsynaptic transmission),

acting, for example, on 5-HT1A and 5-HT4 receptors situated on

the dendrites of the principal cells. However, the majority of the

5-HT fibers in the hippocampal formation are beaded fibers,

which form characteristic pericellular arrays (primarily

synaptic transmission) in the DG and hippocampus, often in

relation to calbindin-positive (Freund et al., 1990; Bjarkam

et al., 2003) or calretinin-positive (Acsady et al., 1993)

GABAergic interneurons. Thus, 5-HT can modulate the

calbindin- and calretinin-positive subsets of hippocampal

GABAergic interneurons. Direct excitation of GABAergic

interneurons via the 5-HT3 receptor subtype increases

inhibition of CA1 pyramidal cells (Ropert and Guy, 1991).

A partial depletion of 5-HT in the hippocampal formation, as

can be expected from our current observations, could, via the

action of 5-HT3 receptors on interneurons, result in a reduced

inhibition of principal neurons. Furthermore, the 5-HT1A

receptor-mediated hyperpolarization induced inhibition of

pyramidal cells could also be impaired in aging (Joels et al.,

1991). However, we have no data on changes in, for example, 5-

HT1A and 5-HT3 receptors in old tree shrews, and we thus have

no direct evidence of the relationship between 5-HT receptors

and 5-HT fiber depletion in the hippocampal formation during

aging.

A spatial learning task conducted earlier with the animals in

the current study revealed that the reference memory of old tree

shrews was as good as that of adult animals but that working

memory was impaired in old subjects (Keuker et al., 2004). In

the current study we found decreased 5-HT fiber densities in the

CA1 and CA3 areas, which could be due to a degeneration of

mostly the beaded fiber type coming from the MRN. A

speculative, yet interesting thought is that, if indeed the beaded

fibers are more susceptible to degeneration than are fine fibers,

the activity of the pyramidal neurons would be attenuated via

the reduced action of 5-HT on interneurons. Consistent with

this hypothesis, we found significant negative correlations

between working memory errors and 5-HT fiber densities in

stratum oriens of CA1 and CA3 (data not shown). This may

indicate that 5-HT could be at least partly responsible for the

working memory deficits we found in the old tree shrews.

5. Conclusion

The present study is, to our knowledge, the first to provide a

detailed investigation of the hippocampal 5-HT innervation

during aging in tree shrews. We demonstrate that hippocampal

5-HT fibers in old tree shrews are affected in a subfield-specific

manner. 5-HT should not be regarded as participating in any

specific behavior per se, but rather, in combination with other

neurotransmitter systems, as setting the computational mode of

central networks (Hille, 1994). The 5-HT system is known to

interact strongly with, for example, the cholinergic system

(Steckler and Sahgal, 1995; Vizi and Kiss, 1998; Stancampiano

et al., 1999), which is also impaired during aging (Fischer et al.,

1992; Smith et al., 1999; Calhoun et al., 2004). The

hippocampal formation, rather than cortical regions, is

supposed to be the region where 5-HT and acetylcholine

interact in working memory processes (Steckler and Sahgal,

1995). Thus, the subfield-specific reductions of 5-HT-IR fiber

densities, as demonstrated in the present study, probably cause a

neurochemical imbalance and impair the ability of the

J.I.H. Keuker et al. / Journal of Chemical Neuroanatomy 30 (2005) 221–229228

hippocampus to function appropriately (Stancampiano et al.,

1999), and might therefore account for the working memory

deficits that we observed earlier in old tree shrews (Keuker

et al., 2004). In order to understand how the imbalance between

various neurotransmitter systems exerts its effect on hippo-

campal functioning during aging, various aspects should be

considered in addition to innervation patterns, such as changes

in receptor numbers, binding capacity and affinity, and 5-HT-

producing neurons in the raphe nuclei.

Acknowledgements

The authors are grateful to Dr. Katalin Horvath for sharing

her excellent technical experience in the immunocytochemical

staining. We thank PD Dr. Gabriele Flugge for fruitful

discussion. Supported by the Graduate School ‘‘Perspectives

of Primatology’’ of the German Science Foundation.

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