Age-Dependent Changes in Axonal Branching of Single Locus Coeruleus Neurons Projecting to Two Different Terminal Fields

Tetsuya Shirokawa, Yoshiyuki Ishida, and Ken-Ichi Isobe

Laboratory of Physiology, Department of Basic Gerontology, National Institute for Longevity Sciences, Obu 474-8522, Japan


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ABSTRACT
INTRODUCTION
METHODS
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REFERENCES

Shirokawa, Tetsuya, Yoshiyuki Ishida, and Ken-Ichi Isobe. Age-Dependent Changes in Axonal Branching of Single Locus Coeruleus Neurons Projecting to Two Different Terminal Fields. J. Neurophysiol. 84: 1120-1122, 2000. Age-dependent changes in the axonal branching patterns of single locus coeruleus neurons, which innervate both the frontal cortex and hippocampus dentate gyrus, have been studied in male F344 rats. We used an electrophysiological approach involving antidromic activation to differentiate single from multi-threshold locus coeruleus neurons in each terminal field with age (7-27 mo of age). Most of these neurons have a single threshold in the young rats, whereas in the older brains, the neurons have multi-threshold responses. This implies an increased amount of axonal branching in the older brains. The time course of the increase differs in the two terminal fields, suggesting that the degree of plasticity or age-dependent increase in branching can differ across terminal fields.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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INTRODUCTION
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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 MOmega . 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
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INTRODUCTION
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DISCUSSION
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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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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 therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 November 1999; accepted in final form 17 April 2000.


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0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society




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