aging is accompanied by a subfield-specific reduction of serotonergic fibers in the tree shrew...
TRANSCRIPT
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: [email protected],
[email protected] (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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