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Age-related changes in the release and uptake activity of presynaptic axon terminals of rat locus coeruleus neurons
Neuroscience Letters 344 (2003) 212–214 www.elsevier.com/locate/neulet
Age-related changes in the release and uptake activity of presynaptic axon terminals of rat locus coeruleus neurons Tetsuya Shirokawa*, Yoshiyuki Ishida, Ken-ichi Isobe Laboratory of Physiology, Department of Basic Gerontology, National Institute for Longevity Sciences, 36-3, Morioka-cho, Obu 474-8522, Japan Received 26 March 2003; received in revised form 9 April 2003; accepted 10 April 2003
Abstract Age-related changes in the release and uptake activity of presynaptic axon terminals of rat locus coeruleus (LC) noradrenergic neurons were studied in the frontal cortex using an extracellular single unit recording technique in vivo. Clonidine, a selective a2 adrenergic agonist, and nisoxetine, a selective noradrenaline uptake inhibitor, were infused locally into the frontal cortex to examine the effects of these drugs on release and uptake activities of the axon terminals of LC neurons. Although the infusion of clonidine produced a marked suppression of release, the effect did not change with age. Infusion of nisoxetine caused an inhibition of uptake, but the effect was attenuated in aged rats. These results suggest that the release activity mediated by the presynaptic autoreceptor did not change with age, but the uptake activity mediated by the NA transporter declined with age in the axon terminals of LC neurons. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Locus coeruleus; Axon terminal; Threshold; Autoreceptor; Noradrenaline transporter
The locus coeruleus (LC) is a major noradrenergic source in the frontal cortex. The levels of noradrenaline (NA) in the frontal cortex depend on the activity of LC neurons [3]. NA release is locally regulated by presynaptic mechanisms such as autoreceptors and transporters, both located on the axon terminals of LC neurons. Past studies found that NA release was mediated by a2 adrenergic autoreceptor, and its activation leads to a decrease in NA release [1,4,8,9,11, 15,16]. The uptake of NA from the synaptic cleft is the principal mechanism by which the action of NA is terminated in the synapse, and the NA transporter is the critical protein that mediates this process. Thus, the synaptic levels of NA in the terminal field may be determined by the activity of these presynaptic mechanisms for release and uptake. Recently, we indicated that stable NA levels are maintained in the frontal cortex of aged rats despite agingrelated loss of LC innervations [5]. However, it remains unclear whether such stable NA levels are due to the release and uptake activities of the presynaptic axon terminals of LC neurons. In the present study, we electrophysiologically examined the threshold current to elicit an antidromic response as an index of the release and uptake activity, since *
Corresponding author. Tel.: þ81-562-46-2311; fax: þ 81-562-48-2373. E-mail address: [email protected] (T. Shirokawa).
the threshold current is closely related to these presynaptic activities of axon terminals of LC neurons [8]. Two drugs were infused directly into the frontal cortex to examine the effects on the threshold currents, clonidine for the suppression of NA release, and nisoxetine for the inhibition of NA uptake. Two age groups (6 and 24 months old) of male F344 rats were housed with food and water available ad libitum on a 12 h light/dark cycle. All animal procedures complied with the guidelines of the Animal Experimentation Committee of the National Institute for Longevity Sciences. For the electrophysiological assays, animals were anesthetized with urethane (1.2 g/kg, i.p.). Lidocaine (4% Xylocaine) was applied locally to all incisions. Rectal temperature was maintained at 36.5 8C by a heating pad. The electrocardiogram (ECG) and electroencephalogram (EEG) were monitored continuously during the experiments. A bipolar stimulating electrode (200 mm in diameter, tip separation 0.5 mm) was stereotaxically guided into the frontal cortex (Fr2; AP 3.0 mm, L 1.5 mm, D 1.2 mm) [10]. Electrical pulses were 0.5 ms in duration with currents ranging from 0.1 to 6.0 mA, and the cycle of stimulation was 1.5 s. The electrical activity of LC neurons was extracellularly recorded with a glass pipette microelectrode filled with 2 M NaCl. LC neurons were usually encountered 6.0 –6.5 mm
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00463-4
T. Shirokawa et al. / Neuroscience Letters 344 (2003) 212–214
below the cortical surface. LC neurons antidromically activated from the frontal cortex were recorded by moving a recording electrode within about 100 mm rostrocaudally or mediolaterally to avoid sampling bias. The LC neurons were identified according to established criteria [2,6,7,13]. Briefly, the LC neurons revealed a wide spike duration (, 2 ms), a slow and tonic rate of spontaneous firing (0.5 – 6 spikes/s), and excitation by tail pinches followed by a longlasting suppression of firing. Responses of LC neurons were assumed to be antidromic when the following criteria were satisfied: (1) fixed latency; (2) ability to follow highfrequency stimulation; and (3) collision with spontaneous or orthodromic action potentials [7,13]. For individual LC neurons that were antidromically activated from the frontal cortex, the threshold currents were measured by varying the stimulating current in 0.01 mA steps. The threshold current was defined as the minimum stimulating current sufficient to elicit an antidromic response on 100% of the non-collision trials. For local infusion, a 30-gauge cannula was implanted, such that the tip of the cannula extended to the depth of the stimulating electrode tip (approximately 1.2 mm) within a lateral displacement of approximately 50 mm. Clonidine hydrochloride (10 mM) (Sigma) or nisoxetine hydrochloride (10 mM) (Tocris) was directly infused into the frontal cortex by means of an infusion pump equipped with 50 ml Hamilton syringes at a speed of 0.02 ml/min [8,9]. Recordings of LC neurons were made in three sessions (Fig. 1, recording #1– #3). Ten LC neurons were recorded from each recording session. Two control sessions were made before (recording #1) and after (recording #3) the drug treatment. The effects of clonidine on the release activity of axon terminals of LC neurons were examined. The change in threshold current was expressed as a percentage of the control threshold current (Fig. 2). Local infusion of clonidine produced a significant increase in threshold current, likely resulting in suppressed NA release in the axon terminals, both in 6-month-old (144%, n ¼ 7) and in 24-month-old rats (146%, n ¼ 4) (paired t-test, P , 0:01). This suppressive effect of clonidine did not differ between two groups (unpaired t-test, P . 0:1). The
Fig. 1. Recordings of LC neurons are composed of three sessions (recording #1–#3). Ten LC neurons that were antidromically activated from the frontal cortex were recorded during each recording session. The drug (10 mM clonidine or 10 mM nisoxetine) was infused initially at a speed of 0.2 ml/min for 30 min, and then infused at a speed of 0.02 ml/min during recording #2. Vehicle solution (0.9% saline) was infused at a speed of 0.02 ml/min during recording #1 and recording #3. The vehicle solution was infused at a speed of 0.2 ml/min after infusion of the drug.
213
Fig. 2. The effect of clonidine infusion on the threshold currents of axon terminals of LC neurons in the frontal cortex. Local infusion of clonidine into the terminal field produced a significant increase in the threshold current both in the 6- and 24-month-old rats (144% and 146%, respectively). Complete recovery from the effects of clonidine (96%) was observed in the 6-month-old rats, while no data were obtained in the 24month-old rats. Data, expressed as a percentage of the control threshold current, are the mean ^ SEM of seven 6-month-old and four 24-month-old rats. **P , 0:01 versus control (paired t-test).
increased threshold currents completely returned to the pre-drug control level (96%) in the 6-month-old rats. The effect of nisoxetine on the uptake activity of axon terminals of LC neurons was examined (Fig. 3). Infusion of
Fig. 3. The effect of nisoxetine infusion on the threshold currents of axon terminals of LC neurons in the frontal cortex. Local infusion of nisoxetine into the terminal field led to a marked increase in the threshold current in both 6-month-old rats (181%) and 24-month-old rats (141%). The difference was significant between the two groups (unpaired t-test, P , 0:05). Recovery from the effects of nisoxetine (116%) was observed in the 6-month-old rats, while no data were obtained in the 24-month-old rats. Data, expressed as a percentage of the control threshold current, are the mean ^ SEM of six 6-month-old and five 24-month-old rats. **P , 0:01, *P , 0:05 versus control (paired t-test).
214
T. Shirokawa et al. / Neuroscience Letters 344 (2003) 212–214
nisoxetine produced a significant increase in threshold current, likely resulting in inhibited NA uptake in the axon terminals, both in 6-month-old rats (181%, n ¼ 6) (paired ttest, P , 0:01) and in 24-month-old rats (141%, n ¼ 5) (P , 0:05). Unlike clonidine, the inhibitory effect of nisoxetine obtained in the 6-month-old rat group was significantly greater than that obtained in the 24-month-old group (unpaired t-test, P , 0:05). The increased threshold currents returned to the pre-drug control level (116%) in the 6-month-old rats. In the present study, the electrophysiological data clearly show that the NA release activity mediated by the autoreceptor did not change with age, whereas the NA uptake activity mediated by the transporter declined with age in the axon terminals of LC neurons. Recently, we found that LC innervations decreased with age in the frontal cortex, and this decrease was followed by an increase in axonal sprouting in the presynaptic terminals of LC neurons [5,6,13]. The increase in sprouting in aged rats may be sufficient to maintain stable NA levels [5] if the NA synthesis is increased at the sprouted LC axon terminals in the aged brain. This is consistent with the finding that expression of tyrosine hydroxylase mRNA increases in the LC of aged rats [14]. A similar aging-related regulation was observed in the cholinergic vasodilator system in aging rats. Although nicotinic acetylcholine receptor activity has been shown to decline with age, muscarinic acetylcholine receptor activity and the release of acetylcholine into the extracellular space in the cortex are stable during aging [12]. If the presynaptic NA uptake activity declines with age, stable NA levels can be maintained in the aged brain. In fact, we observed just such a decrease in NA uptake activity in the aged brain. The decrease in NA uptake in the aged brain may be an adaptive response to loss of NA innervations and reduction in synaptic NA, since a decrease in NA uptake could increase synaptic levels of NA. This agrees with the finding that expression of the NA transporter mRNA decreased in the LC of aging rats [14]. Thus, we conclude that decreased NA uptake may account for the stable NA levels in the aged brain. Further studies are needed to examine changes in expression of the NA transporter in the terminal field of LC neurons during aging.
References [1] G.K. Aghajanian, C.P. VanderMaelen, Alpha 2-adrenoceptormediated hyperpolarization of locus coeruleus neurons: intracellular studies in vivo, Science 215 (1982) 1394–1396. [2] G. Aston-Jones, M. Segal, F.E. Bloom, Brain aminergic axons exhibit marked variability in conduction velocity, Brain Res. 195 (1980) 215 –222. [3] C.W. Berridge, E.D. Abercrombie, Relationship between locus coeruleus discharge rates and rates of norepinephrine release within neocortex as assessed by in vivo microdialysis, Neuroscience 93 (1999) 1263–1270. [4] T.M. Egan, G. Henderson, R.A. North, J.T. Williams, Noradrenalinemediated synaptic inhibition in rat locus coeruleus neurons, J. Physiol. 345 (1983) 477–488. [5] Y. Ishida, T. Shirokawa, Y. Komatsu, K. Isobe, Changes in cortical noradrenergic axon terminals of locus coeruleus neurons in aged F344 rats, Neurosci. Lett. 307 (2001) 197–199. [6] Y. Ishida, T. Shirokawa, O. Miyaishi, Y. Komatsu, K. Isobe, Agedependent changes in projections from locus coeruleus to hippocampus dentate gyrus and frontal cortex, Eur. J. Neurosci. 12 (2000) 1263–1270. [7] S. Nakamura, Some electrophysiological properties of neurons of rat locus coeruleus, J. Physiol. 267 (1977) 641 –658. [8] S. Nakamura, J.M. Tepper, S.J. Young, P.M. Groves, Neurophysiological consequences of presynaptic receptor activation: changes in noradrenergic terminal excitability, Brain Res. 226 (1981) 155–170. [9] S. Nakamura, J.M. Tepper, S.J. Young, P.M. Groves, Changes in noradrenergic terminal excitability induced by amphetamine and their relation to impulse traffic, Neuroscience 7 (1982) 2217–2224. [10] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 2nd Edition., Academic Press, San Diego, CA, 1986. [11] L.J. Ryan, J.M. Tepper, S.F. Sawyer, S.J. Young, P.M. Groves, Autoreceptor activation in central monoamine neurons: modulation of neurotransmitter release is not mediated by intermittent axonal conduction, Neuroscience 15 (1985) 925– 931. [12] A. Sato, Y. Sato, S. Uchida, Regulation of cerebral cortical blood flow by the basal forebrain cholinergic fibers and aging, Auton. Neurosci. 96 (2002) 13–19. [13] T. Shirokawa, Y. Ishida, K. Isobe, Age-dependent changes in axonal branching of single locus coeruleus neurons projecting to two different terminal fields, J. Neurophysiol. 84 (2000) 1120–1122. [14] M.M. Shores, S.S. White, R.C. Veith, P. Szot, Tyrosine hydroxylase mRNA is increased in old age and norepinephrine uptake transporter mRNA is decreased in middle age in locus coeruleus of BrownNorway rats, Brain Res. 826 (1999) 143 –147. [15] J.M. Tepper, P.M. Groves, S.J. Young, The neuropharmacology of the autoinhibition of monoamine release, Trends Pharmacol. Sci. 6 (1985) 251 –256. [16] M. Washburn, H.C. Moises, Electrophysiological correlates of presynaptic alpha 2-receptor-mediated inhibition of norepinephrine release at locus coeruleus synapses in dentate gyrus, J. Neurosci. 9 (1989) 2131– 2140.
Age-related changes in the release and uptake activity of presynaptic axon terminals of rat locus coeruleus neurons Tetsuya Shirokawa*, Yoshiyuki Ishida, Ken-ichi Isobe Laboratory of Physiology, Department of Basic Gerontology, National Institute for Longevity Sciences, 36-3, Morioka-cho, Obu 474-8522, Japan Received 26 March 2003; received in revised form 9 April 2003; accepted 10 April 2003
Abstract Age-related changes in the release and uptake activity of presynaptic axon terminals of rat locus coeruleus (LC) noradrenergic neurons were studied in the frontal cortex using an extracellular single unit recording technique in vivo. Clonidine, a selective a2 adrenergic agonist, and nisoxetine, a selective noradrenaline uptake inhibitor, were infused locally into the frontal cortex to examine the effects of these drugs on release and uptake activities of the axon terminals of LC neurons. Although the infusion of clonidine produced a marked suppression of release, the effect did not change with age. Infusion of nisoxetine caused an inhibition of uptake, but the effect was attenuated in aged rats. These results suggest that the release activity mediated by the presynaptic autoreceptor did not change with age, but the uptake activity mediated by the NA transporter declined with age in the axon terminals of LC neurons. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Locus coeruleus; Axon terminal; Threshold; Autoreceptor; Noradrenaline transporter
The locus coeruleus (LC) is a major noradrenergic source in the frontal cortex. The levels of noradrenaline (NA) in the frontal cortex depend on the activity of LC neurons [3]. NA release is locally regulated by presynaptic mechanisms such as autoreceptors and transporters, both located on the axon terminals of LC neurons. Past studies found that NA release was mediated by a2 adrenergic autoreceptor, and its activation leads to a decrease in NA release [1,4,8,9,11, 15,16]. The uptake of NA from the synaptic cleft is the principal mechanism by which the action of NA is terminated in the synapse, and the NA transporter is the critical protein that mediates this process. Thus, the synaptic levels of NA in the terminal field may be determined by the activity of these presynaptic mechanisms for release and uptake. Recently, we indicated that stable NA levels are maintained in the frontal cortex of aged rats despite agingrelated loss of LC innervations [5]. However, it remains unclear whether such stable NA levels are due to the release and uptake activities of the presynaptic axon terminals of LC neurons. In the present study, we electrophysiologically examined the threshold current to elicit an antidromic response as an index of the release and uptake activity, since *
Corresponding author. Tel.: þ81-562-46-2311; fax: þ 81-562-48-2373. E-mail address: [email protected] (T. Shirokawa).
the threshold current is closely related to these presynaptic activities of axon terminals of LC neurons [8]. Two drugs were infused directly into the frontal cortex to examine the effects on the threshold currents, clonidine for the suppression of NA release, and nisoxetine for the inhibition of NA uptake. Two age groups (6 and 24 months old) of male F344 rats were housed with food and water available ad libitum on a 12 h light/dark cycle. All animal procedures complied with the guidelines of the Animal Experimentation Committee of the National Institute for Longevity Sciences. For the electrophysiological assays, animals were anesthetized with urethane (1.2 g/kg, i.p.). Lidocaine (4% Xylocaine) was applied locally to all incisions. Rectal temperature was maintained at 36.5 8C by a heating pad. The electrocardiogram (ECG) and electroencephalogram (EEG) were monitored continuously during the experiments. A bipolar stimulating electrode (200 mm in diameter, tip separation 0.5 mm) was stereotaxically guided into the frontal cortex (Fr2; AP 3.0 mm, L 1.5 mm, D 1.2 mm) [10]. Electrical pulses were 0.5 ms in duration with currents ranging from 0.1 to 6.0 mA, and the cycle of stimulation was 1.5 s. The electrical activity of LC neurons was extracellularly recorded with a glass pipette microelectrode filled with 2 M NaCl. LC neurons were usually encountered 6.0 –6.5 mm
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00463-4
T. Shirokawa et al. / Neuroscience Letters 344 (2003) 212–214
below the cortical surface. LC neurons antidromically activated from the frontal cortex were recorded by moving a recording electrode within about 100 mm rostrocaudally or mediolaterally to avoid sampling bias. The LC neurons were identified according to established criteria [2,6,7,13]. Briefly, the LC neurons revealed a wide spike duration (, 2 ms), a slow and tonic rate of spontaneous firing (0.5 – 6 spikes/s), and excitation by tail pinches followed by a longlasting suppression of firing. Responses of LC neurons were assumed to be antidromic when the following criteria were satisfied: (1) fixed latency; (2) ability to follow highfrequency stimulation; and (3) collision with spontaneous or orthodromic action potentials [7,13]. For individual LC neurons that were antidromically activated from the frontal cortex, the threshold currents were measured by varying the stimulating current in 0.01 mA steps. The threshold current was defined as the minimum stimulating current sufficient to elicit an antidromic response on 100% of the non-collision trials. For local infusion, a 30-gauge cannula was implanted, such that the tip of the cannula extended to the depth of the stimulating electrode tip (approximately 1.2 mm) within a lateral displacement of approximately 50 mm. Clonidine hydrochloride (10 mM) (Sigma) or nisoxetine hydrochloride (10 mM) (Tocris) was directly infused into the frontal cortex by means of an infusion pump equipped with 50 ml Hamilton syringes at a speed of 0.02 ml/min [8,9]. Recordings of LC neurons were made in three sessions (Fig. 1, recording #1– #3). Ten LC neurons were recorded from each recording session. Two control sessions were made before (recording #1) and after (recording #3) the drug treatment. The effects of clonidine on the release activity of axon terminals of LC neurons were examined. The change in threshold current was expressed as a percentage of the control threshold current (Fig. 2). Local infusion of clonidine produced a significant increase in threshold current, likely resulting in suppressed NA release in the axon terminals, both in 6-month-old (144%, n ¼ 7) and in 24-month-old rats (146%, n ¼ 4) (paired t-test, P , 0:01). This suppressive effect of clonidine did not differ between two groups (unpaired t-test, P . 0:1). The
Fig. 1. Recordings of LC neurons are composed of three sessions (recording #1–#3). Ten LC neurons that were antidromically activated from the frontal cortex were recorded during each recording session. The drug (10 mM clonidine or 10 mM nisoxetine) was infused initially at a speed of 0.2 ml/min for 30 min, and then infused at a speed of 0.02 ml/min during recording #2. Vehicle solution (0.9% saline) was infused at a speed of 0.02 ml/min during recording #1 and recording #3. The vehicle solution was infused at a speed of 0.2 ml/min after infusion of the drug.
213
Fig. 2. The effect of clonidine infusion on the threshold currents of axon terminals of LC neurons in the frontal cortex. Local infusion of clonidine into the terminal field produced a significant increase in the threshold current both in the 6- and 24-month-old rats (144% and 146%, respectively). Complete recovery from the effects of clonidine (96%) was observed in the 6-month-old rats, while no data were obtained in the 24month-old rats. Data, expressed as a percentage of the control threshold current, are the mean ^ SEM of seven 6-month-old and four 24-month-old rats. **P , 0:01 versus control (paired t-test).
increased threshold currents completely returned to the pre-drug control level (96%) in the 6-month-old rats. The effect of nisoxetine on the uptake activity of axon terminals of LC neurons was examined (Fig. 3). Infusion of
Fig. 3. The effect of nisoxetine infusion on the threshold currents of axon terminals of LC neurons in the frontal cortex. Local infusion of nisoxetine into the terminal field led to a marked increase in the threshold current in both 6-month-old rats (181%) and 24-month-old rats (141%). The difference was significant between the two groups (unpaired t-test, P , 0:05). Recovery from the effects of nisoxetine (116%) was observed in the 6-month-old rats, while no data were obtained in the 24-month-old rats. Data, expressed as a percentage of the control threshold current, are the mean ^ SEM of six 6-month-old and five 24-month-old rats. **P , 0:01, *P , 0:05 versus control (paired t-test).
214
T. Shirokawa et al. / Neuroscience Letters 344 (2003) 212–214
nisoxetine produced a significant increase in threshold current, likely resulting in inhibited NA uptake in the axon terminals, both in 6-month-old rats (181%, n ¼ 6) (paired ttest, P , 0:01) and in 24-month-old rats (141%, n ¼ 5) (P , 0:05). Unlike clonidine, the inhibitory effect of nisoxetine obtained in the 6-month-old rat group was significantly greater than that obtained in the 24-month-old group (unpaired t-test, P , 0:05). The increased threshold currents returned to the pre-drug control level (116%) in the 6-month-old rats. In the present study, the electrophysiological data clearly show that the NA release activity mediated by the autoreceptor did not change with age, whereas the NA uptake activity mediated by the transporter declined with age in the axon terminals of LC neurons. Recently, we found that LC innervations decreased with age in the frontal cortex, and this decrease was followed by an increase in axonal sprouting in the presynaptic terminals of LC neurons [5,6,13]. The increase in sprouting in aged rats may be sufficient to maintain stable NA levels [5] if the NA synthesis is increased at the sprouted LC axon terminals in the aged brain. This is consistent with the finding that expression of tyrosine hydroxylase mRNA increases in the LC of aged rats [14]. A similar aging-related regulation was observed in the cholinergic vasodilator system in aging rats. Although nicotinic acetylcholine receptor activity has been shown to decline with age, muscarinic acetylcholine receptor activity and the release of acetylcholine into the extracellular space in the cortex are stable during aging [12]. If the presynaptic NA uptake activity declines with age, stable NA levels can be maintained in the aged brain. In fact, we observed just such a decrease in NA uptake activity in the aged brain. The decrease in NA uptake in the aged brain may be an adaptive response to loss of NA innervations and reduction in synaptic NA, since a decrease in NA uptake could increase synaptic levels of NA. This agrees with the finding that expression of the NA transporter mRNA decreased in the LC of aging rats [14]. Thus, we conclude that decreased NA uptake may account for the stable NA levels in the aged brain. Further studies are needed to examine changes in expression of the NA transporter in the terminal field of LC neurons during aging.
References [1] G.K. Aghajanian, C.P. VanderMaelen, Alpha 2-adrenoceptormediated hyperpolarization of locus coeruleus neurons: intracellular studies in vivo, Science 215 (1982) 1394–1396. [2] G. Aston-Jones, M. Segal, F.E. Bloom, Brain aminergic axons exhibit marked variability in conduction velocity, Brain Res. 195 (1980) 215 –222. [3] C.W. Berridge, E.D. Abercrombie, Relationship between locus coeruleus discharge rates and rates of norepinephrine release within neocortex as assessed by in vivo microdialysis, Neuroscience 93 (1999) 1263–1270. [4] T.M. Egan, G. Henderson, R.A. North, J.T. Williams, Noradrenalinemediated synaptic inhibition in rat locus coeruleus neurons, J. Physiol. 345 (1983) 477–488. [5] Y. Ishida, T. Shirokawa, Y. Komatsu, K. Isobe, Changes in cortical noradrenergic axon terminals of locus coeruleus neurons in aged F344 rats, Neurosci. Lett. 307 (2001) 197–199. [6] Y. Ishida, T. Shirokawa, O. Miyaishi, Y. Komatsu, K. Isobe, Agedependent changes in projections from locus coeruleus to hippocampus dentate gyrus and frontal cortex, Eur. J. Neurosci. 12 (2000) 1263–1270. [7] S. Nakamura, Some electrophysiological properties of neurons of rat locus coeruleus, J. Physiol. 267 (1977) 641 –658. [8] S. Nakamura, J.M. Tepper, S.J. Young, P.M. Groves, Neurophysiological consequences of presynaptic receptor activation: changes in noradrenergic terminal excitability, Brain Res. 226 (1981) 155–170. [9] S. Nakamura, J.M. Tepper, S.J. Young, P.M. Groves, Changes in noradrenergic terminal excitability induced by amphetamine and their relation to impulse traffic, Neuroscience 7 (1982) 2217–2224. [10] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 2nd Edition., Academic Press, San Diego, CA, 1986. [11] L.J. Ryan, J.M. Tepper, S.F. Sawyer, S.J. Young, P.M. Groves, Autoreceptor activation in central monoamine neurons: modulation of neurotransmitter release is not mediated by intermittent axonal conduction, Neuroscience 15 (1985) 925– 931. [12] A. Sato, Y. Sato, S. Uchida, Regulation of cerebral cortical blood flow by the basal forebrain cholinergic fibers and aging, Auton. Neurosci. 96 (2002) 13–19. [13] T. Shirokawa, Y. Ishida, K. Isobe, Age-dependent changes in axonal branching of single locus coeruleus neurons projecting to two different terminal fields, J. Neurophysiol. 84 (2000) 1120–1122. [14] M.M. Shores, S.S. White, R.C. Veith, P. Szot, Tyrosine hydroxylase mRNA is increased in old age and norepinephrine uptake transporter mRNA is decreased in middle age in locus coeruleus of BrownNorway rats, Brain Res. 826 (1999) 143 –147. [15] J.M. Tepper, P.M. Groves, S.J. Young, The neuropharmacology of the autoinhibition of monoamine release, Trends Pharmacol. Sci. 6 (1985) 251 –256. [16] M. Washburn, H.C. Moises, Electrophysiological correlates of presynaptic alpha 2-receptor-mediated inhibition of norepinephrine release at locus coeruleus synapses in dentate gyrus, J. Neurosci. 9 (1989) 2131– 2140.