Laboratory of Physiology, Department of Basic Gerontology, National
Institute for Longevity Sciences, Obu 474-8522, Japan
 |
INTRODUCTION |
The locus coeruleus
(LC) is the densely packed cluster of noradrenergic neurons in the
brain stem. These neurons innervate widely to different target regions
such as the frontal cortex (FC) and hippocampus dentate gyrus (DG)
(Foote et al. 1983
; Swanson 1976
).
However, it is unclear at present how the multi-target innervations of
individual LC neurons change with age.
We recently observed that the percentage of LC neurons showing
multi-threshold antidromic responses, which suggests axonal branching
of individual LC neurons, increased critically between 15 and 17 mo of
age in the FC, whereas in the DG the branching of LC neurons steadily
increased up to 24 mo of age (Ishida et al. 2000
).
Although these findings suggest that the morphological plasticity
occurs with age in the axon terminals of LC neurons, the possibility of
changes in the electrophysiological properties of axon terminals still
remains (Morales et al. 1987
).
In the present study, we focused on the age-dependent changes in the
multi-target LC neurons that innervate both the FC and DG (FC-DG
neurons), using in vivo electrophysiological techniques (Nakamura 1977
).
 |
METHODS |
Male F344 rats (8 age groups: 7, 11, 15, 17, 19, 21, 24, and 27 mo of age, 6 animals for each group) were used. Animals were obtained
from the aging colony at the National Institute on Aging (Harlan). They
were housed with food and water available ad libitum on a 12-h
light/dark cycle. All animal procedures complied with the National
Institutes of Health guidelines and were approved by the Laboratory
Animal Research Facilities Committee of the National Institute for
Longevity Sciences.
Animals were anesthetized with urethan (1.2 g/kg ip). The anesthetic
was supplemented as necessary during the experiments. Lidocaine (4%
Xylocaine) was applied locally to all incisions. Rectal temperature was
maintained at 36.5°C. Electrocardiogram (ECG) and
electroencephalogram (EEG) were monitored throughout the experiment.
Stimulating electrodes of two insulated stainless steel wires (200 µm
diam) with an exposed tip of approximately 0.5 mm were implanted in the
right FC (AP 3.0 mm, L 1.5 mm, D 1.5 mm) and hippocampus DG (AP
4.0
mm, L 2.5 mm, D 3.5 mm), according to the atlas by Paxinos and
Watson (1986)
.
The electrical activity of LC neurons was recorded
extracellularly by means of a glass micropipette filled with 2 M NaCl, with resistance ranging from 10 to 18 M
. The location of the LC was
determined by the appearance of a short train of multiple units with
small amplitudes following electrical stimulation of FC
(Nakamura 1977
). Single-unit activity of LC neurons was
superimposed on the multi-unit response. The LC neurons were identified
according to the criteria (Aston-Jones et al. 1980
;
Nakamura 1977
). Briefly, the LC neurons revealed wide
spike duration (~2 ms), slow and tonic spontaneous firing (0.5-6
Hz), and excitation by tail pinches followed by a long-lasting
suppression of firing. In each animal, 60-66 LC neurons were recorded
from the right LC by moving a recording electrode within about 100 µm
rostrocaudally or mediolaterally to avoid sampling bias. Responses of
LC neurons were considered to be antidromic provided that the following
criteria were satisfied: 1) fixed latency, 2)
ability to follow high-frequency stimulation (>200 Hz), and
3) collision with spontaneous action potentials (Nakamura 1977
). The stimulation consisted of single
square pulses of 0.5 ms duration with currents ranging from 0.1 to 6.0 mA (in 0.01-mA steps). The cycle of stimulation was 1.5 s. The
data were means ± SE and were compared by one-way ANOVA.
 |
RESULTS |
Based on the threshold currents for antidromic activation, we
classified the responses of FC-DG neurons into two types:
"single-threshold" and "multi-threshold." In young rats, the
great majority of FC-DG neurons show single-threshold antidromic
responses (single-threshold). In contrast, in the aged animals, we
found that most of the FC-DG neurons show two or more discrete
antidromic responses (multi-threshold) (Nakamura et al. 1989
). An
example of multi-threshold responses in an FC-DG neuron to stimulation
of the DG is shown in Fig. 1.

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Fig. 1.
An extracellular recording of multi-threshold responses in a frontal
cortex dentate gyrus (FC-DG) neuron to stimulation of the DG in a
21-mo-old rat. When the stimulus was adjusted to the minimum current
necessary to evoke antidromic responses on every trial (1.05 mA), an
antidromic response was evoked at a fixed, discrete latency (64 ms,
top). When the stimulus was increased to 1.87 mA, the
antidromic response occurred at a shorter latency (48 ms,
bottom). Further increases in stimulus currents (up to
6.0 mA) did not cause any latency change.
|
|
The total number of LC neurons, the number of FC-DG neurons, the mean
antidromic latency, and the number of latency jumps of FC-DG neurons
for each age group are shown in Table 1.
The mean antidromic latency is significantly longer in DG after 24 mo
of age. In FC the latency shows no significant difference between ages,
although it tends to be longer between 19 and 21 mo of age. The number
of latency jumps is highest at 19 mo of age in both targets, and it is
lowest at 15 mo of age. The proportion of multi-threshold FC-DG neurons
significantly increased with age, showing a different time course in FC
and DG (Fig. 2). The maximum rate of
increase was found between 15 and 17 mo in FC, against 19-21 mo in DG. The proportion of multi-threshold FC-DG neurons peaked at 24 mo in each
target. The increased proportion of multi-threshold FC-DG neurons
finally declined at 27 mo.
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Table 1.
The age-dependent changes in the number of LC neurons, FC-DG neurons,
mean antidromic latency, and the number of latency jumps of FC-DG
neurons
|
|

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Fig. 2.
Age-dependent changes in multi-threshold FC-DG neurons to stimulation
of FC and DG. Data, expressed as ratio of multi-threshold FC-DG
neurons, are the means ± SE of 6 animals.
* P < 0.05; ** P < 0.01;
*** P < 0.001 (ANOVA).
|
|
To clarify whether the axonal branching patterns of individual FC-DG
neurons are independently regulated by each target, we classified these
neurons into four types in terms of the appearance of the
multi-threshold responses to stimulation of each target as
follows
|
FC |
DG |
type
A |
single |
single |
type
B |
multi |
single |
type C |
multi |
multi |
type
D |
single |
multi |
As shown in Fig. 3, the type
of FC-DG neurons changes with age in a target-dependent manner. At 7 mo
of age, the ratio of type A (49%) and type B (28%) neurons is
significantly higher than the other types. Up to 15 mo of age, the
ratio of type A neurons significantly increases (66%), whereas the
ratio of type B neurons significantly decreases (3%). At the age of 19 mo, however, the ratios of type B (52%) and type C (28%) neurons are
significantly higher than the other types. At the age of 24 mo, the
ratio of type C neurons is significantly higher (72%) than the others. The type D neurons show no significant peak with age.

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Fig. 3.
Target-dependent changes in 4 types of FC-DG neurons. They are
classified by the appearance of multi-threshold responses to
stimulation of the FC and DG as follows
|
|
FC |
DG |
type A
(circles) |
single |
single |
type B
(triangles) |
multi |
single |
type C (squares) |
multi |
multi |
type D (downward triangles) |
single |
multi |
|
Data,
expressed as the ratio of each type, are the means ± SE of 6 animals.
* P < 0.05; ** P < 0.01; *** P < 0.001 (ANOVA).
|
|
 |
DISCUSSION |
Our results strongly suggest that in both frontal cortex and
hippocampus dentate gyrus there is an age-dependent increase in axonal
branching of FC-DG neurons up to 24 mo of age, and then a decrease
thereafter. In addition, the time course of the increase differs in the
two terminal fields, suggesting target dependency, i.e., the degree of
plasticity or age-dependent increase in axonal arborization can differ
across terminal fields.
Recently we reported age-dependent changes in the LC projection of FC
and DG that suggested a decrease in density between 7 and 15 mo of age,
giving rise to axonal branching following the loss of projections
(Ishida et al. 2000
). Since there is no direct
morphological data supporting the axonal branching of single LC
neurons, the possibility remains that the appearance of multi-threshold antidromic responses is due to changes in electrophysiological properties of axon terminals of LC neurons. It is likely that the
threshold for activation of remote axon terminals is lower in the aged
brain, since a decrease in rheobase is observed in the spinal cord
motoneurons in the aged cat (Morales et al. 1987
). Thus
these plastic changes in the axon terminals that occurs with age may
compensate for the loss of LC innervations in the aged brain.
We thank Dr. S. Nakamura for insightful comments on an earlier
version of the manuscript. We also thank Dr. H. Saito for advice on the
statistical analysis.
This work was supported by the Research Grants for Longevity Sciences
(10C-03) from the Ministry of Health and Welfare of Japan.
Address for reprint requests: T. Shirokawa, Dept. of Basic Gerontology,
National Institute for Longevity Sciences (NILS), Gengo 36-3, Morioka-cho, Obu 474-8522, Japan (E-mail:
shiro{at}nils.go.jp).
The costs of publication of this article were defrayed in
part by the payment of page charges. The article must
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in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.