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Electrophysiological evidence for terminal sprouting of locus coeruleus neurons following repeated mild stress
Neuroscience Letters, 100 (1989) 147 152 Elsevier ScientificPublishers Ireland Ltd.
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NSL 06060
Electrophysiological evidence for terminal sprouting of locus coeruleus neurons following repeated mild stress S. Nakamura, T. Sakaguchi and F. Aoki Department of Physiology, Faculty of Medicine, Kanazawa University, Kanazawa (Japan) (Received 5 December 1988; Revisedversion received 23 January 1989; Accepted 23 January 1989)
Key words." Locus ceruleus; Stress; Terminal sprouting; Cerebral cortex; Noradrenergic neuron; Antidromic response To see if repeated mild stress causes plastic changes in central noradrenergic terminal axons, the density of terminal axons arising in locus coeruleus (LC) neurons of rats was quantified by antidromic stimulation technique. After the termination of stress treatments (immersion in warm water for 10 min daily) for 1 or 2 weeks, electrophysiologicalexperiments were performed under urethane anesthesia. The frequency of LC neurons activated antidromically from the cerebral cortex increased in rats stressed for 2 weeks but not for 1 week. Since the increased frequency of antidromic responses was not due to a change in terminal excitability, the change observed here is considered to be morphological (terminal sprouting) rather than a physiologicalconsequence. The results suggest that LC neurons dynamically alter their terminal morphology in response to environmental stimuli.
Repeated stress has been k n o w n to cause a variety o f neurochemical changes in the central n o r a d r e n e r g i c system [1, 2, 4, 5, 14, 21, 22]. C h a n g e s such as subsensitivity o f f l - a d r e n o c e p t o r s a n d e n h a n c e d n o r a d r e n a l i n e synthesis become evident after a b o u t 2 weeks from the onset o f stress t r e a t m e n t s [I 9, 21]. This suggests that repeated stress results in a delayed n e u r o c h e m i c a l response. Here we report physiological evidence that, in a n i m a l s who repeatedly received mild stress, the n u m b e r of n o r a d r e n e r g i c a x o n terminals arising in the locus coeruleus (LC) increased in the cerebral cortex. Male S p r a g u e - D a w l e y rats (8-14 weeks o f age) ( n = 2 4 ) were used. The a n i m a l s were housed 2 or 3 per cage at 2 3 + 2 ° C u n d e r a 12 h l i g h t , l a r k cycle (light on at 8.00 h). F o o d a n d water were freely available. The a n i m a l s were divided into two groups, control (n = 8) a n d stressed (n = 16). In the stress group, a n i m a l s restrained in a small cage were immersed in w a r m water (36-37°C) up to the neck for 10 m i n daily. The stress g r o u p received stress t r e a t m e n t for 1 or 2 weeks. Electrophysiological experiments were m a d e on the day following the t e r m i n a t i o n of stress treatments.
Correspondence: S. Nakamura, Department of Physiology, Faculty of Medicine, Kanazawa University, Kanazawa 920, Japan. 0304-3940/89/$ 03.50 © 1989 Elsevier ScientificPublishers Ireland Ltd.
148
The density of noradrenergic terminal axons arising in the LC was quantified in two ways using antidromic stimulation techniques: (I) the percentage of LC neurons activated antidromically from a given stimulus site (projection (P)-index). (2) The proportion of LC neurons showing two or more discrete antidromic latencies (multiple antidromic latency) from a single stimulus site (Fig. 2A). The availability of the second physiological measure is based on the view that the multiple antidromic latency results from activation of two or more different axonal branches [7, 17]. Therefore, multiple antidromic latency can provide a physiological index of" axonal branching of an individual neuron around a single stimulus locus. For electrophysiological experiments, animals were anesthetized with urethane (1.3 g/kg, i.p.). The anesthetic was supplemented as necessary during experiments. Body temperature was maintained at 37_+ I C by a heating pad. For electrical stimulation of brain sites, stimulating electrodes consisting of two insulated stainless-steel wires (approx. diameter, 200 /~m) with an exposed tip of approximately 0.5 mm were implanted in each stimulating site. The frontal and occipital cortex (FC and OC) were subjected to electrical stimulation consisting of single square pulses of I ms duration with currents ranging from 0.1 to 5 mA. The rate of stimulation was 1 Hz in all experiments. The coordinates for the FC and OC were as follows: FC, 2.0 m m anterior to bregma, 1.5 m m lateral and 1.5 m m from the cortical surface; OC, 3.0 m m lateral to lambda and 1.0 mm from the cortical surface. The electrical activity of LC neurons was recorded extracellularly by means of a glass micropipette filled with 2 M NaCI. As described previously [10], the location of the LC was determined by appearance of field responses evoked by stimulation of the dorsal noradrenergic bundle arising in the LC. Single-unit activity of LC neurons was recorded superimposed upon the field response. When a single LC neuron was encountered, stimulation of each stimulating site was examined. Responses of LC neurons were considered to be antidromic in nature 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. In each animal, 40 70 LC neurons were recorded to assess the P-index, and 2-7 LC neurons evoked antidromically from the FC were examined to assess the presence of multiple antidromic latency. In all animals, stimulation and recording were made in the left hemisphere. The data on the P-indices were analyzed by analysis of variance and post hoc individual mean comparisons were made by Newman Keuls test. The P-index for each stimulating site in control rats varied from animal to animal (Fig. 1). The mean P-indices (+- S.E.M.) were not different between the control (FC, 38.8+3.3; OC, 23.5+2.9) and 1 week stress group (FC, 42.0+3.8; OC, 30.5+_1.9), whereas those for the FC and OC were significantly higher in the 2 week stress (FC, 50.8-+2.9; OC, 33.5 +_3.0) than the control group (Newman-Keuts test, P<0.05). The mean P-indices for the FC and OC in the 2 week stress group did not differ from those in the 1 week stress group. These results suggest that the apparent density of the projection from the LC to the cerebral cortex increases gradually, only reaching statistical significance after 2 weeks of stress. As reported previously [17], some LC neurons activated antidromically revealed
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Fig. 1. Physiological evidence for terminal sprouting of LC neurons in the cerebral cortex in response to 10 min restraint in warm water. The frequency of LC neurons activated antidromically from a given brain site was taken as a physiological index for the percentage of LC neurons projecting to that site (projection (P)-index). The mean P-indices for the FC and OC in 2 week stress groups were significantly higher than those in the control group (Newman-Keuls test, P<0.05). The P-indices for FC and OC did not differ between the control and 1 week stress group. Bar indicates the mean value of P-index.
two o r m o r e discrete a n t i d r o m i c latencies at different intensities o f stimulus currents (Fig. 2A). The m u l t i p l e a n t i d r o m i c latencies are c o n s i d e r e d to result f r o m a c t i v a t i o n o f different a x o n a l b r a n c h e s [7, 17]. Therefore, it is possible t h a t if i n d i v i d u a l L C neurons increase their b r a n c h i n g in response to r e p e a t e d mild stress, m o r e L C n e u r o n s m a y exhibit the m u l t i p l e a n t i d r o m i c latencies in stressed t h a n non-stressed rats. In the c o n t r o l rats, a total o f 47 L C n e u r o n s showing a n t i d r o m i c responses from F C s t i m u l a t i o n were e x a m i n e d , a n d 15 (31.9%) o f t h e m revealed the m u l t i p l e a n t i d r o m i c latencies (Fig. 2B). In the 1 week stress rats, the frequency o f L C n e u r o n s showing the m u l t i p l e a n t i d r o m i c latencies was nearly the same as t h a t o f the c o n t r o l (38.5%). In the 2 week stress rats, m o r e t h a n h a l f o f L C n e u r o n s (57.7%) a n t i d r o m i c a l l y activated f r o m F C s t i m u l a t i o n revealed two o r m o r e discrete a n t i d r o m i c latencies (2'Z-test, P < 0.05). T o see if the increased frequency o f L C n e u r o n s a n t i d r o m i c a l l y a c t i v a t e d f r o m the cerebral cortex was due to changes in the excitability o f t e r m i n a l a x o n s o f L C neurons, t h r e s h o l d c u r r e n t s for a n t i d r o m i c a c t i v a t i o n f r o m the F C were m e a s u r e d in the non-stressed a n d stressed rats. C u r r e n t s necessary for a n t i d r o m i c a c t i v a t i o n varied f r o m cell to cell, b u t in m o s t L C n e u r o n s the t h r e s h o l d c u r r e n t s were less t h a n 2.0 m A in b o t h the non-stressed (mean_+ S.E.M., 1.7_+ 0.1 m A ) a n d stressed rats (1 week stress, 1 . 9 + 0 . 1 m A ; 2 week stress, 1.5-t-0.1 m A ) . Therefore, the changes in the P-
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Fig. 2. A: the multiple antidromic latency of a single LC neuron to FC stimulation. Slimulus currents were changed systematically in increments of 100 # A . A t a current of 0.7 m A , antidromic response w a s e v o k e d at a l a t e n c y o f 55 ms, while a small change in current (from 0.7 to 1.0 m A ) c a u s e d a small successive change of antidromic latency (2 ms) (a). Further increases in current up to 1.8 m A produced no additional latency change, but at a current of 1.9 m A the latency abruptly shifted to 23 ms (b). Further increases in stimulus currents did not cause any latency change. B: the frequency of LC neurons showing multiple antidromic latencies to FC stimulation in the control and stressed rats. The frequency of LC neurons showing multiple antidromic latencies was higher in the 2 week stress than control group, whereas there w a s no significant difference between both stressed groups (Z2-test, P < 0.05).
index for the cerebral cortex and those in the frequency of LC neurons showing the multiple antidromic !atencies were not attributable to changes in terminal excitability. In the present experiments, the density o f cortical terminal axons arising in the LC was successfully quantified by antidromic stimulation techniques. The P-index for each stimulus site is considered to reflect the frequency of LC neurons projecting to the stimulus locus, while the multiple antidromic latency is taken as a physiological index for the presence of branching of an individual neuron around a single stimulus locus. One might argue that the changes observed here are attributable to differences in the safety factor for impulse conduction along the axon, or to alterations in the excitability of terminal axons of LC neurons. Since the blockage o f impulse conduction has been reported to occur at branch points when high frequencies of stimulation were employed [6] particularly in invertebrates, we used a low frequency o f stimulation (I Hz) to minimize the occurrence o f impulse conduction failure. Furthermore, in several hundred cases of antidromically activated locus coeruleus neurons reported previously, sufficient stimulus currents have always produced 100% antidromic responses, with no cases of impulse conduction failure [11-13, 16]. In addition, from the measurements of threshold currents for antidromic activation, it was concluded that the changes in the P-index and the frequency of LC neurons showing
151 multiple antidromic latencies were not due to alterations in terminal excitability. Therefore, our findings are interpreted as morphological (axonal sprouting) rather than physiological changes in noradrenergic axon terminal of LC neurons. In the central nervous system, noradrenergic neurons are known to be among those neurons that exhibit the most reliable and robust axonal regeneration and sprouting in response to brain damage [3, 8]. Although most previous studies have shown that the plastic changes in noradrenergic LC neurons occur preferentially in early developmental stages, axonal sprouting of LC neurons has also been demonstrated in adult animals [9, 18, 20]. However, plastic changes of LC neurons have so far been reported only in animals with part of the brain damaged. The present results suggest that axonal sprouting of LC neurons can occur in adult animals even without apparent brain lesions, and support the view that neuronal elements such as synapses, and dendritic and terminal branches can dynamically change their morphology [ 15]. From the similarity of the time course between the occurrence of fl-adrenoceptor subsensitivity and adaptation of animals to stress, Stone [21] has proposed the hypothesis that subsensitivity of fl-adrenoceptors is involved in the mechanisms of adaptation to stress. It is likely that terminal sprouting of noradrenergic LC neurons is also related to stress adaptation. In chronically stressed animals, the influence of LC neurons may become more extended spatially, as their terminal fields increase in size due to newly formed axonal branches. Therefore, target cells primarily innervated by noradrenergic axon terminals of LC neurons may become more strongly affected by the supplemented noradrenergic input, while those primarily devoid of LC innervation before the occurrence of axonal sprouting may become dominated by new terminals of LC neurons. In this manner, terminal sprouting of noradrenergic LC neurons may participate in adaptive changes in repeatedly stressed animals. We are grateful to Dr. C. Yamamoto for reading the manuscript, Dr. J.M. Tepper for reviewing the manuscript and valuable comments, Dr. K. Hashimoto for valuable comments on statistical analysis, and Dr. H. Shibata for valuable discussion. We also thank Mr. T. Kida for technical assistance, Ms. S. Sawada for preparing the figures and Ms. A. Torii for typing the manuscript. 1 Anisman, H. and Zacharko, R.M., Behavioral and neurochemical consequencesassociated stressors, Ann. N.Y. Acad. Sci., 467 (1986) 205 225. 2 Bergstrom,D.A. and Keller, K.J., Effectof electronconvulsiveshock on monoaminergicreceptor binding sites in rat brain, Nature (Lond.), 278 (1979) 464~466. 3 Bj6rklund, A. and Stenevi, U., Regeneration of monoaminergicand cholinergicneurons in the mammalian central nervous system, Physiol. Rev., 59 (1979) 62-100. 4 Glavin, G.B., Stress and brain noradrenaline: a review, Neurosci. Biobehav. Rev., 9 (1985) 233 243. 5 Kvetnansky, R, Recent progress in catecholamines under stress. In Usdin, Kvetnansky and Kopin (Eds.), Catecholaminesand Stress: Recent Advances, Elsevier, Amsterdam, 1980, pp. 7 18. 6 Lipsky, J., Antidromic activation of neurones as an analytic tool in the study of the central nervous system, J. Neurosci. Methods, 4 (1987) 1-32. 7 McMahon, S.B. and Wall, P.D., Physiologicalevidence for branching of peripheral unmyelinated sensory afferent fibers in the rat, J. Comp. Neurol., 261 (1987) 13(~136.
152 8 Moore, R.Y., Bj6rklund, A. and Stenevi, U., Growth and plasticity of adrenergic neurons. In F.O Schmitt and F.G. Worden (Eds.), The Neuroscience Third Study Program, MIT Press. Cambridge. MA, t974, pp. 961 977. 9 Moore, R.Y., Bj6rklund, A. and Stenevi, U., Plastic changes in the adrenergic innervation of rat septal area in response to denervation, Brain Res., 33 (1971) 13 35. 10 Nakamura, S., Some electrophysiological properties of neurones of rat locus coeruleus, J. Physiol. (Lond.), 267 (1977) 641 658. I I Nakamura, S., Tepper, J.M., Young, S.J. and Groves, P.M., Neurophysiological consequences of prcsynaptic receptor activation: changes in noradrenergic terminal excitability, Brain Res., 226 (198Ii I55 170. 12 Nakamura, S., Tepper, J.M., Young, S.J. and Groves, P.M., Noradrenergic terminal excitability: effects of opioids, Neurosci. Lett., 30 (1982) 57 62. 13 Nakamura, S., Tepper, J.M., Young, S.J. and Groves, P.M., Changes in noradrenergic terminal excitability induced by amphetamine and their relation to impulse traffic, Neuroscience, 7 (1982) 2217 2224. 14 Pandy, G.H., Heinze, W.J., Brown, B.D. and Davis, J.H., Electroconvulsive shock treatment decreases /#adrenergic receptor sensitivity in rat brain, Nature (Lond.), 280 (1979) 234 235. 15 Purves, D., Voyvodic, J.T., Magrassi, k. and Yawo~ H., Nerve terminal remodeling visualized in living mice by repeated examination of the same neuron, Science, 2138 (1987) 1122 1126. 16 Ryan, L.J., Tepper, J.M., Sawyer, S.F., Young, S.J. and Groves, P.M., Autoreceptor activation in central monoamine neurons: modulation of neurotransmitter release is not mediated by intermittent axonal conduction, Neuroscience, 15 (1985) 925 931. 17 Sakaguchi, T. and Nakamura, S., The mode of projections of single locus coeruleus neurons to the cerebral cortex in rats, Neuroscience, 20 (1987) 221 230. 18 Sakaguchi, T., Sirokawa, T. and Nakamura, S., Changes in projections from locus coeruleus to lateral geniculate nucleus following ablation of visual cortex in adult rats, Brain Res., 321 (1984) 319 332. 19 Stanford, C., Fillenz, M. and Ellen, R., The effect of repeated mild stress on cerebral cortical adrenoceptors and noradrenaline synthesis in the rat, Neurosci. Lett., 45 (1984) 163 167. 20 Stenevi, U., Bj6rklund, A. and Moore, R.Y., Growth of intact adrenergic axons in the denervated lateral geniculate body, Exp. Neurol., 35 (1972) 290 299. 21 Stone, E.A., Adaptation to stress and brain noradrenergic receptors, Neurosci. Biobehav Rev., 7 (1983) 503 -509. 22 Stone, E.A. and McCarty, R., Adaptation to stress: tyrosine hydroxylase activity and catecholamine release, Neurosci. Biobehav. Rev., 7 (I 983) 29 34,
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Electrophysiological evidence for terminal sprouting of locus coeruleus neurons following repeated mild stress S. Nakamura, T. Sakaguchi and F. Aoki Department of Physiology, Faculty of Medicine, Kanazawa University, Kanazawa (Japan) (Received 5 December 1988; Revisedversion received 23 January 1989; Accepted 23 January 1989)
Key words." Locus ceruleus; Stress; Terminal sprouting; Cerebral cortex; Noradrenergic neuron; Antidromic response To see if repeated mild stress causes plastic changes in central noradrenergic terminal axons, the density of terminal axons arising in locus coeruleus (LC) neurons of rats was quantified by antidromic stimulation technique. After the termination of stress treatments (immersion in warm water for 10 min daily) for 1 or 2 weeks, electrophysiologicalexperiments were performed under urethane anesthesia. The frequency of LC neurons activated antidromically from the cerebral cortex increased in rats stressed for 2 weeks but not for 1 week. Since the increased frequency of antidromic responses was not due to a change in terminal excitability, the change observed here is considered to be morphological (terminal sprouting) rather than a physiologicalconsequence. The results suggest that LC neurons dynamically alter their terminal morphology in response to environmental stimuli.
Repeated stress has been k n o w n to cause a variety o f neurochemical changes in the central n o r a d r e n e r g i c system [1, 2, 4, 5, 14, 21, 22]. C h a n g e s such as subsensitivity o f f l - a d r e n o c e p t o r s a n d e n h a n c e d n o r a d r e n a l i n e synthesis become evident after a b o u t 2 weeks from the onset o f stress t r e a t m e n t s [I 9, 21]. This suggests that repeated stress results in a delayed n e u r o c h e m i c a l response. Here we report physiological evidence that, in a n i m a l s who repeatedly received mild stress, the n u m b e r of n o r a d r e n e r g i c a x o n terminals arising in the locus coeruleus (LC) increased in the cerebral cortex. Male S p r a g u e - D a w l e y rats (8-14 weeks o f age) ( n = 2 4 ) were used. The a n i m a l s were housed 2 or 3 per cage at 2 3 + 2 ° C u n d e r a 12 h l i g h t , l a r k cycle (light on at 8.00 h). F o o d a n d water were freely available. The a n i m a l s were divided into two groups, control (n = 8) a n d stressed (n = 16). In the stress group, a n i m a l s restrained in a small cage were immersed in w a r m water (36-37°C) up to the neck for 10 m i n daily. The stress g r o u p received stress t r e a t m e n t for 1 or 2 weeks. Electrophysiological experiments were m a d e on the day following the t e r m i n a t i o n of stress treatments.
Correspondence: S. Nakamura, Department of Physiology, Faculty of Medicine, Kanazawa University, Kanazawa 920, Japan. 0304-3940/89/$ 03.50 © 1989 Elsevier ScientificPublishers Ireland Ltd.
148
The density of noradrenergic terminal axons arising in the LC was quantified in two ways using antidromic stimulation techniques: (I) the percentage of LC neurons activated antidromically from a given stimulus site (projection (P)-index). (2) The proportion of LC neurons showing two or more discrete antidromic latencies (multiple antidromic latency) from a single stimulus site (Fig. 2A). The availability of the second physiological measure is based on the view that the multiple antidromic latency results from activation of two or more different axonal branches [7, 17]. Therefore, multiple antidromic latency can provide a physiological index of" axonal branching of an individual neuron around a single stimulus locus. For electrophysiological experiments, animals were anesthetized with urethane (1.3 g/kg, i.p.). The anesthetic was supplemented as necessary during experiments. Body temperature was maintained at 37_+ I C by a heating pad. For electrical stimulation of brain sites, stimulating electrodes consisting of two insulated stainless-steel wires (approx. diameter, 200 /~m) with an exposed tip of approximately 0.5 mm were implanted in each stimulating site. The frontal and occipital cortex (FC and OC) were subjected to electrical stimulation consisting of single square pulses of I ms duration with currents ranging from 0.1 to 5 mA. The rate of stimulation was 1 Hz in all experiments. The coordinates for the FC and OC were as follows: FC, 2.0 m m anterior to bregma, 1.5 m m lateral and 1.5 m m from the cortical surface; OC, 3.0 m m lateral to lambda and 1.0 mm from the cortical surface. The electrical activity of LC neurons was recorded extracellularly by means of a glass micropipette filled with 2 M NaCI. As described previously [10], the location of the LC was determined by appearance of field responses evoked by stimulation of the dorsal noradrenergic bundle arising in the LC. Single-unit activity of LC neurons was recorded superimposed upon the field response. When a single LC neuron was encountered, stimulation of each stimulating site was examined. Responses of LC neurons were considered to be antidromic in nature 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. In each animal, 40 70 LC neurons were recorded to assess the P-index, and 2-7 LC neurons evoked antidromically from the FC were examined to assess the presence of multiple antidromic latency. In all animals, stimulation and recording were made in the left hemisphere. The data on the P-indices were analyzed by analysis of variance and post hoc individual mean comparisons were made by Newman Keuls test. The P-index for each stimulating site in control rats varied from animal to animal (Fig. 1). The mean P-indices (+- S.E.M.) were not different between the control (FC, 38.8+3.3; OC, 23.5+2.9) and 1 week stress group (FC, 42.0+3.8; OC, 30.5+_1.9), whereas those for the FC and OC were significantly higher in the 2 week stress (FC, 50.8-+2.9; OC, 33.5 +_3.0) than the control group (Newman-Keuts test, P<0.05). The mean P-indices for the FC and OC in the 2 week stress group did not differ from those in the 1 week stress group. These results suggest that the apparent density of the projection from the LC to the cerebral cortex increases gradually, only reaching statistical significance after 2 weeks of stress. As reported previously [17], some LC neurons activated antidromically revealed
149
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Fig. 1. Physiological evidence for terminal sprouting of LC neurons in the cerebral cortex in response to 10 min restraint in warm water. The frequency of LC neurons activated antidromically from a given brain site was taken as a physiological index for the percentage of LC neurons projecting to that site (projection (P)-index). The mean P-indices for the FC and OC in 2 week stress groups were significantly higher than those in the control group (Newman-Keuls test, P<0.05). The P-indices for FC and OC did not differ between the control and 1 week stress group. Bar indicates the mean value of P-index.
two o r m o r e discrete a n t i d r o m i c latencies at different intensities o f stimulus currents (Fig. 2A). The m u l t i p l e a n t i d r o m i c latencies are c o n s i d e r e d to result f r o m a c t i v a t i o n o f different a x o n a l b r a n c h e s [7, 17]. Therefore, it is possible t h a t if i n d i v i d u a l L C neurons increase their b r a n c h i n g in response to r e p e a t e d mild stress, m o r e L C n e u r o n s m a y exhibit the m u l t i p l e a n t i d r o m i c latencies in stressed t h a n non-stressed rats. In the c o n t r o l rats, a total o f 47 L C n e u r o n s showing a n t i d r o m i c responses from F C s t i m u l a t i o n were e x a m i n e d , a n d 15 (31.9%) o f t h e m revealed the m u l t i p l e a n t i d r o m i c latencies (Fig. 2B). In the 1 week stress rats, the frequency o f L C n e u r o n s showing the m u l t i p l e a n t i d r o m i c latencies was nearly the same as t h a t o f the c o n t r o l (38.5%). In the 2 week stress rats, m o r e t h a n h a l f o f L C n e u r o n s (57.7%) a n t i d r o m i c a l l y activated f r o m F C s t i m u l a t i o n revealed two o r m o r e discrete a n t i d r o m i c latencies (2'Z-test, P < 0.05). T o see if the increased frequency o f L C n e u r o n s a n t i d r o m i c a l l y a c t i v a t e d f r o m the cerebral cortex was due to changes in the excitability o f t e r m i n a l a x o n s o f L C neurons, t h r e s h o l d c u r r e n t s for a n t i d r o m i c a c t i v a t i o n f r o m the F C were m e a s u r e d in the non-stressed a n d stressed rats. C u r r e n t s necessary for a n t i d r o m i c a c t i v a t i o n varied f r o m cell to cell, b u t in m o s t L C n e u r o n s the t h r e s h o l d c u r r e n t s were less t h a n 2.0 m A in b o t h the non-stressed (mean_+ S.E.M., 1.7_+ 0.1 m A ) a n d stressed rats (1 week stress, 1 . 9 + 0 . 1 m A ; 2 week stress, 1.5-t-0.1 m A ) . Therefore, the changes in the P-
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Fig. 2. A: the multiple antidromic latency of a single LC neuron to FC stimulation. Slimulus currents were changed systematically in increments of 100 # A . A t a current of 0.7 m A , antidromic response w a s e v o k e d at a l a t e n c y o f 55 ms, while a small change in current (from 0.7 to 1.0 m A ) c a u s e d a small successive change of antidromic latency (2 ms) (a). Further increases in current up to 1.8 m A produced no additional latency change, but at a current of 1.9 m A the latency abruptly shifted to 23 ms (b). Further increases in stimulus currents did not cause any latency change. B: the frequency of LC neurons showing multiple antidromic latencies to FC stimulation in the control and stressed rats. The frequency of LC neurons showing multiple antidromic latencies was higher in the 2 week stress than control group, whereas there w a s no significant difference between both stressed groups (Z2-test, P < 0.05).
index for the cerebral cortex and those in the frequency of LC neurons showing the multiple antidromic !atencies were not attributable to changes in terminal excitability. In the present experiments, the density o f cortical terminal axons arising in the LC was successfully quantified by antidromic stimulation techniques. The P-index for each stimulus site is considered to reflect the frequency of LC neurons projecting to the stimulus locus, while the multiple antidromic latency is taken as a physiological index for the presence of branching of an individual neuron around a single stimulus locus. One might argue that the changes observed here are attributable to differences in the safety factor for impulse conduction along the axon, or to alterations in the excitability of terminal axons of LC neurons. Since the blockage o f impulse conduction has been reported to occur at branch points when high frequencies of stimulation were employed [6] particularly in invertebrates, we used a low frequency o f stimulation (I Hz) to minimize the occurrence o f impulse conduction failure. Furthermore, in several hundred cases of antidromically activated locus coeruleus neurons reported previously, sufficient stimulus currents have always produced 100% antidromic responses, with no cases of impulse conduction failure [11-13, 16]. In addition, from the measurements of threshold currents for antidromic activation, it was concluded that the changes in the P-index and the frequency of LC neurons showing
151 multiple antidromic latencies were not due to alterations in terminal excitability. Therefore, our findings are interpreted as morphological (axonal sprouting) rather than physiological changes in noradrenergic axon terminal of LC neurons. In the central nervous system, noradrenergic neurons are known to be among those neurons that exhibit the most reliable and robust axonal regeneration and sprouting in response to brain damage [3, 8]. Although most previous studies have shown that the plastic changes in noradrenergic LC neurons occur preferentially in early developmental stages, axonal sprouting of LC neurons has also been demonstrated in adult animals [9, 18, 20]. However, plastic changes of LC neurons have so far been reported only in animals with part of the brain damaged. The present results suggest that axonal sprouting of LC neurons can occur in adult animals even without apparent brain lesions, and support the view that neuronal elements such as synapses, and dendritic and terminal branches can dynamically change their morphology [ 15]. From the similarity of the time course between the occurrence of fl-adrenoceptor subsensitivity and adaptation of animals to stress, Stone [21] has proposed the hypothesis that subsensitivity of fl-adrenoceptors is involved in the mechanisms of adaptation to stress. It is likely that terminal sprouting of noradrenergic LC neurons is also related to stress adaptation. In chronically stressed animals, the influence of LC neurons may become more extended spatially, as their terminal fields increase in size due to newly formed axonal branches. Therefore, target cells primarily innervated by noradrenergic axon terminals of LC neurons may become more strongly affected by the supplemented noradrenergic input, while those primarily devoid of LC innervation before the occurrence of axonal sprouting may become dominated by new terminals of LC neurons. In this manner, terminal sprouting of noradrenergic LC neurons may participate in adaptive changes in repeatedly stressed animals. We are grateful to Dr. C. Yamamoto for reading the manuscript, Dr. J.M. Tepper for reviewing the manuscript and valuable comments, Dr. K. Hashimoto for valuable comments on statistical analysis, and Dr. H. Shibata for valuable discussion. We also thank Mr. T. Kida for technical assistance, Ms. S. Sawada for preparing the figures and Ms. A. Torii for typing the manuscript. 1 Anisman, H. and Zacharko, R.M., Behavioral and neurochemical consequencesassociated stressors, Ann. N.Y. Acad. Sci., 467 (1986) 205 225. 2 Bergstrom,D.A. and Keller, K.J., Effectof electronconvulsiveshock on monoaminergicreceptor binding sites in rat brain, Nature (Lond.), 278 (1979) 464~466. 3 Bj6rklund, A. and Stenevi, U., Regeneration of monoaminergicand cholinergicneurons in the mammalian central nervous system, Physiol. Rev., 59 (1979) 62-100. 4 Glavin, G.B., Stress and brain noradrenaline: a review, Neurosci. Biobehav. Rev., 9 (1985) 233 243. 5 Kvetnansky, R, Recent progress in catecholamines under stress. In Usdin, Kvetnansky and Kopin (Eds.), Catecholaminesand Stress: Recent Advances, Elsevier, Amsterdam, 1980, pp. 7 18. 6 Lipsky, J., Antidromic activation of neurones as an analytic tool in the study of the central nervous system, J. Neurosci. Methods, 4 (1987) 1-32. 7 McMahon, S.B. and Wall, P.D., Physiologicalevidence for branching of peripheral unmyelinated sensory afferent fibers in the rat, J. Comp. Neurol., 261 (1987) 13(~136.
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