- Email: [email protected]
Serotonergic neurons projecting to hippocampus activate locomotion
Brain Research 869 (2000) 194–202 www.elsevier.com / locate / bres
Research report
Serotonergic neurons projecting to hippocampus activate locomotion Hiroshi Takahashi a , Yumiko Takada b , *, Nobuo Nagai a , Tetsumei Urano a , Akikazu Takada a b
a Department of Physiology, Hamamatsu University, School of Medicine, Hamamatsu-shi, Shizuoka-ken, 431 -3192 Japan Department of Pathophysiology, Hamamatsu University, School of Medicine, Hamamatsu-shi, Shizuoka-ken, 431 -3192 Japan
Accepted 4 April 2000
Abstract Although the role of brain serotonergic neurons in locomotion has been extensively studied, their influence may vary depending upon the terminal areas. Thus, using microdialysis and microinjection techniques, we examined the relationship between serotonin (5-HT) levels in striatum, hippocampus or prefrontal cortex (PFC) and motor activity in rats. The systemic injection (10 mg / kg i.p.) of monoamine oxidase inhibitor, tranylcypromine (TC), significantly elevated 5-HT levels in the striatum, hippocampus and PFC accompanied by a parallel increase in motor activity of the rats. This effect was mimicked by microinfusions of TC (1.0 mM) or 5-HT (1.0 mM) into the hippocampus and to some extent into PFC (the response delayed in time), but not into striatum. The increase in motor activity produced by local infusions of TC either into the hippocampus or PFC could be prevented by pretreatment with 10 mM tetrodotoxin infused into the hippocampus. However, tetrodotoxin infused to PFC failed to prevent hyperlocomotion produced by intrahippocampal infusion of TC, although the response was delayed in time. Thus, we conclude that serotonergic neurons projecting to the hippocampus are involved in locomotor activity and PFC serotonergic fibers may facilitate hippocampal control of locomotion. 2000 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Monoamines and behaviour Keywords: Serotonin; Locomotion; Hippocampus; Prefrontal cortex; MAO inhibitor
1. Introduction It is well known that central serotonergic neurons have widespread projections to the cortex and to the limbic structures, and it is believed that the serotonergic system plays an important role in the regulation of a wide variety of physiologic, emotional, and behavioral functions [5,8,15,18]. Since the involvement of serotonin (5-hydroxytryptamine; 5-HT) in human affective disorders has been well documented, much effort has been paid to elucidate the impact of this amine on various brain functions, both in humans and in laboratory animals. However, the role of the central serotonergic system in the regulation of behavior is complex [9,10]. Thus, some reports indicate that reduced central serotonergic activity is closely related to aggressive reactions [24] and exploration *Corresponding author. Tel.: 181-53-435-2247; fax: 181-53-4349225. E-mail address: [email protected] (Y. Takada)
[17]. Furthermore, the decrease in serotonin synthesis in the brain [6] or lesion of the median raphe nucleus [11,12,28,29] have been shown to produce dramatic increase in locomotion. On the other hand, it has been demonstrated that serotonergic system may participate in either generating or inhibiting motor functions related to food intake [3,14]. These different relations between central serotonergic neurons and motor responses may be due to the subtypes of the serotonergic receptors involved or to the contrariety in functions of various brain areas receiving serotonergic innervations. However, most up-todate reports utilized the systemic rather than local injections of substances related to serotonin metabolism, that can introduce greater bias and can hardly give a closer look into the relationship between serotonin levels in discrete brain areas and locomotor functions. To overcome these limitations, in the present study we examined the role of serotonergic system in motor activity by using microinfusions of 5-HT into the hippocampus, striatum, and prefrontal cortex (PFC) which receive serotonergic in-
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02385-4
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
nervations. We compared the above effects with those produced by the local infusions of monoamine oxidase inhibitor (MAO-I) in injected areas. MAO-I has been shown to elevate the levels of extracellular 5-HT and catecholamines [23] and also to induce rat hyperactivity [4]. Furthermore, we used in vivo microdialysis techniques to examine the relationship between extracellular 5-HT levels and locomotion after systemic administration of MAO-I.
2. Materials and methods
2.1. Animals Adult male Wistar rats (200–250 g) were used. They were individually housed for 2 weeks before the experiments in a temperature-controlled room (24618C) that was maintained on a 12:12 h light / dark cycle. Animals were given free access to food and water.
2.2. Surgical procedures To avoid volume-induced increase in intracerebral pressure following administration of drugs and to exclude effects of insertion of injectors on motor activity, we employed a microdialysis technique for the local infusion of 5-HT and MAO-I in free moving conditions. Three to five days before microdialysis experiments, guide cannulas were stereotaxically implanted for the penetration of a microdialysis probe in rats under sodium pentobarbital anesthesia (50 mg / kg, i.p.). The coordinates chosen according to the stereotaxic atlas of Paxinos and Watson [22] were as follows. For the striatum: AP, 0.2; ML, 3.0; DV, 3.0 (mm). For the hippocampus: AP, 25.8; ML, 5.0; DV, 3.0 (mm). For the prefrontal cortex (PFC): AP, 3.1; ML, 0.9; DV, 2.0 (mm). For the nucleus accumbens (NAC): AP, 2.1; ML, 2.5; DV, 4.5 (mm); angled at 58 perpendicularly. The microdialysis probes were implanted 2 mm for PFC and 4 mm for other regions deeper than the tips of guide cannulas. Thus coordinates of active dialyzed membranes were as follows. For the striatum: AP, 0.2; ML, 3.0; DV, 3.0–7.0 (mm). For the hippocampus: AP, 25.8; ML, 5.0; DV, 3.0–7.0 (mm). For the prefrontal cortex (PFC): AP, 3.1; ML, 0.9; DV, 2.0–4.0 (mm). The study animals displayed neither any obvious signs of bleeding or infection around the wound, nor of any postsurgical distress.
2.3. Microdialysis procedures Microdialysis probes were constructed according to the method described by Nakahara et al. [21] with an outer diameter of 220 mm and an exposed tip of 2.0 mm or 4.0 mm. These probes had an active dialyzing length of 2 mm for PFC and 4 mm for other regions and a membrane with
195
a cut-off value of molecular weight 50 000. The recovery rate of 5-HT and 5-HIAA (5-hydroxyindolo-acetic acid) were between 10 and 20%. The lactate Ringer solution used as the perfusate contained 0.1 M NaCl, 1.8 mM CaCl 2 , 4.0 mM KCl, and 0.03 M sodium lactate. The microdialysis tube was perfused continuously with Ringer solution at a rate of 2.0 ml / min using microinfusion pump, and the samples were started 3 h after probe insertion, to allow for basal-level stabilization.
2.4. HPLC procedures The contents of 5-HT and 5-HIAA in dialysates were assayed using HPLC with electrochemical detection. The electrochemical apparatus consisted of a reverse phase HPLC column (Eicom Co., Kyoto, Japan). The column was MA-5ODS and the mobile phase was composed of 0.1 M sodium acetate, 0.1 M citric acid, 18% methanol, 0.023% l-octane sulfonate, and 0.0005% EDTA, pH 3.5. The mobile phase was delivered by a pump at a flow rate of 0.3 ml / min. The column temperature was kept at 258C. The graphite electrode (WE-3G Eicom) was set at 0.6 V (an Ag /AgCl reference electrode). The dialysate was injected into the HPLC column every 20 min. by an automatic injector.
2.5. Counts of motor activity Spontaneous motor activities were measured by Supermex (Muromachi Kikai Co., Tokyo, Japan). The Supermex consists of a sensor monitor which was mounted above the cage to detect changes in heat across multiple zones of the cage through an array of Fresnel lenses. In this way, the system could monitor and count all spontaneous movements, both vertical and horizontal, including locomotion and head-movements, etc. [19,25]. All counts were automatically added and recorded at 20- or 30-min intervals.
2.6. Experimental protocols 2.6.1. Systemic administration of MAO-I In the first series of experiments, 54 rats were divided into three subgroups according to the brain area that microdialysis probes were inserted unilaterally (18 rats each for the striatum, hippocampus or prefrontal cortex). Three hours after probe insertion, 3 basal dialysates were collected every 20 min. Then, rats were further subdivided to receive intraperitoneal injection of either 1 mg / kg or 10 mg / kg of tranylcypromine (TC: Sigma, St Louis, USA), or 1 ml / kg isotonic saline as a control. Finally, 6 rats each for the striatum, hippocampus or prefrontal cortex were examined in each treatment group. Motor activity was always counted simultaneously with the measurement of 5-HT by microdialysis.
196
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
2.6.2. Local infusion of MAO-I or 5 -HT Similarly, in the second series of experiments (84 rats), rats were divided into three subgroups according to the insertion of the microdialysis probes (28 rats each for the striatum, hippocampus, or prefrontal cortex). The probes were inserted bilaterally in all groups. Three hours after probe insertions, motor activities were measured for 2 h, and then TC or 5-HT (5-hydroxytryptamine hydrochloride: Sigma, St. Louis, USA) was infused locally for 30 min through bilateral microdialysis probes (6 rats each for 0.1 mM TC, 1.0 mM TC, 0.1 mM 5-HT or 1.0 mM 5-HT per subgroup). As the control group, 18 rats (6 rats for the striatum, hippocampus, or prefrontal cortex) were perfused continuously with Ringer solution throughout the experiments. Furthermore, 0.1 or 1.0 mM of 5-HT was infused locally into the bilateral NAC (10 rats each). 0.1 or 1.0 mM of 3, 4-Dihydroxyphenethylamine hydrochloride (Dopamine5DA: Sigma, St. Louis, USA) was infused locally into the bilateral NAC (8 rats each). pH of the perfusate of TC and 5-HT was adjusted to 7.2–7.4 with sodium hydroxide. Counting of motor activities was continued more than 2 h after finishing these infusions. 2.6.3. Local infusion of tetrodotoxin ( TTX) with MAO-I In the last series of experiments (15 rats), four microdialysis probes were inserted to both bilateral hippocampus and bilateral PFC in each rat. Three hours after insertion of the probes basal levels of motor activity were measured and then 10 mM TTX was infused to the bilateral hippocampus or PFC through microdialysis probes during 1 h. Thirty minutes after starting TTX infusion, 1.0 mM TC was infused locally during 30 min through microdialysis probes to the bilateral hippocampus or PFC (5 rats each for the set). Motor activities were measured more than 3 h after finishing these infusions. 2.7. Histology Following the termination of each experiment, animals were anesthetized with pentobarbital and decapitated; brains were removed and stored in buffered formalin. Probe placement was verified microscopically, and data were discarded if any part of the dialysis probe was not in the regions intended (very small number).
2.8. Statistical analysis Serotonin (5-HT) and 5-HIAA concentrations in the dialysate (not corrected for in vitro recovery) were expressed as percent changes of basal values, 100% being defined as the average of the last three samples before injection of MAO-I. The comparison of the motor activity was performed compared with their basal values which was defined as the average of the last three samples before injection. Statistical analysis of all the results was performed by one- or two-way ANOVA with repeated mea-
sures, followed by a multicomparison test using Fisher’s PLSD (Protected Least Significant Difference). The Pvalue of less than 0.05 was considered to be statistically significant.
2.9. Ethics The study was submitted to the Ethical Committee of our University. The committee judged the experimental designs to ‘the guiding principles for the care and use of animals in the field of physiological sciences’ recommended by the Physiological Society of Japan on December 19, 1988. The guidelines were prepared to meet the requirements contained in the publications such as U.K. Animals Act, 1986, DHEW Publication No. (NIH) 85-23, 1985, and International Guiding Principle for Biomedical Research Involving Animals, CIOM, 1983. The committee approved the experiments after careful examination.
3. Results
3.1. Effect of MAO-I systemic administration on extracellular 5 -HT levels and spontaneous motor activity Motor activities considerably increased 40 or 60 min after the intraperitoneal injection of 10 mg / kg TC (P, 0.001, Fig. 1A), and remained constant throughout the experiment. During these hyperactive states, most of the growth in the motor activities was the increase in forward locomotion. At this time extracellular 5-HT level in the striatum significantly increased to 250% of the baseline (P,0.01; Fig. 1B). Conversely extracellular 5-HIAA in this brain area was markedly reduced (P,0.001; Fig. 1C). There were significant differences between the experimental group and the control group for both 5-HT (P,0.05) and 5-HIAA (P,0.001) levels. Similar changes were observed in the hippocampus at this time point. Thus, 40 min after the administration of TC, increase in extracellular 5-HT values (700% of the baseline; P,0.01; Fig. 1B) was accompanied by evident decline in 5-HIAA (P,0.05; Fig. 1C). These values differed significantly between TCtreated and control animals (P,0.05 for both parameters). In the prefrontal cortex, 40 min after TC administration, the levels of 5-HT increased to 250% of baseline (P,0.01; Fig. 1B), and continued to increase throughout the experiment. Simultaneously extracellular 5-HIAA values were significantly reduced (P,0.05; Fig. 1C). Statistically significant differences were observed between rats treated with TC and control animals (P,0.05 and P,0.005 for 5-HT and 5-HIAA, respectively). Neither the intraperitoneal administration of 1 ml / kg saline nor 1 mg / kg TC affected motor activity in any group of rats (Fig. 1A). In the rats administered with 1 mg / kg TC no significant changes either in the extracellular 5-HT or 5-HIAA were observed in any studied area (data not shown).
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
197
Fig. 1. Effects of intraperitoneal injection of isotonic saline (control; dotted line with closed circles), 1 mg / kg tranylcypromine (TC) (fine line with closed circles) or 10 mg / kg TC (bold line with open circles) on motor activity of the rats (A), extracellular 5-HT (B), and extracellular 5-HIAA levels (C) in the striatum (left graphs), hippocampus (center graphs), and prefrontal cortex (right graphs). In (B) and (C) data for 1 mg / kg TC not shown. Arrows indicate the time of TC injection. Values are expressed as mean6S.E. *P,0.05, **P,0.005, [P,0.0005 compared to the baseline value.
198
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
Fig. 2. Effects of local infusion of Ringer solution (control; dotted lines with closed circles), 0.1 mM TC (fine line with closed circles) or 1.0 mM TC (bold lines with open circles) into the striatum (left graph), hippocampus (center graph), and prefrontal cortex (right graph) on the motor activity of the rats (for 0.1 mM TC infused into the striatum data not shown). Horizontal bars indicate the term of TC infusion. Values are expressed as mean6S.E. *P,0.05, **P,0.005 compared to the baseline value.
3.2. Effects of local infusions of MAO-I or 5 -HT on spontaneous motor activity Neither the rats infused with 0.1 mM TC, 0.1 mM 5-HT nor control animals, regardless of the brain area studied, showed any changes in their motor activity (see Figs. 2 and 3 for TC and 5-HT infusions, respectively; data from 0.1 mM TC and 0.1 mM 5-HT infusions into the striatum not shown). The local infusions of either 1.0 mM TC or 1.0 mM 5-HT into the bilateral striatum did not change the motor activities (Figs. 2 and 3, respectively), but these infusions into the bilateral hippocampus increased the motor activities significantly when compared with the baseline
values (P,0.05 for both groups) or respective controls (P,0.01 for both groups; Figs. 2 and 3, respectively). These hyperactivities continued for a subsequent 90 min following infusions. During these hyperactive states, neither increases in grooming, tremor, rearing, stereotyped behavior, head shaking nor other involuntary movements were found by continuous observation. Almost all of the motor activities were increased in normal forward locomotion. Increases in components of a 5-HT-induced behavioral syndrome consisting of flat body posture, hind limb abduction, head weaving and forepaw treading [26,1,7] were not observed. Similarly, local bilateral infusions of either 1.0 mM TC or 1.0 mM 5-HT into the prefrontal cortex caused a
Fig. 3. Effects of local infusion of Ringer solution (control; dotted lines with closed circles), 0.1 mM 5-HT (fine line with closed circles) or 1.0 mM 5-HT (bold lines with open circles) into the striatum (left graph), hippocampus (center graph), and prefrontal cortex (right graph) on the motor activity of the rats (for 0.1 mM 5-HT infused into the striatum data not shown). Horizontal bars indicate the term of 5-HT infusion. Values are expressed as mean6S.E. *P,0.05, **P,0.01 compared to the baseline value.
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
significant increase in the motor activity when compared with the baseline values (P,0.05 and P,0.01, respectively) or control rats (P,0.01 for both groups; Figs. 2 and 3, respectively). Interestingly, these hyperactivities characterized with 2–2.5 h time delay and gained 15 to 20 times of the baseline. Most of the growth in the motor activities was the increase in forward locomotion, however, increases in grooming or head shaking were also observed. The local bilateral infusions of either 1.0 mM 5-HT or 1.0 mM DA into the NAC caused a significant increase in the motor activity when compared with the baseline values (P,0.05 and P,0.0001, respectively) or control rats (P, 0.05 and P,0.01, respectively; Fig. 4). During these hyperactive states, increases in grooming or stereotyped movements were also observed. Neither the rats locally injected with 0.1 mM 5-HT nor 0.1 mM DA into the bilateral NAC showed significant changes in their motor activities (Fig. 4).
3.3. Effects of local infusion of TTX with MAO-I on spontaneous motor activity Effects of TTX infusions on increase in the motor activities are shown in Fig. 5. The local infusion of 10 mM TTX to the bilateral hippocampus significantly inhibited baseline motor activity of the rats (P,0.05, Fig. 5A, C). In this group of animals subsequent infusion of 1.0 mM TC into the bilateral hippocampus failed to produce any increase in the motor activity (Fig. 5A). Delayed increase in the hypermotility caused by TC infusion into PFC was also inhibited by TTX infusion into the hippocampus (Fig. 5C). After the local infusion of TTX to the bilateral PFC however, subsequent local infusion of TC to the bilateral
199
hippocampus increased the motor activity significantly (P,0.05), although not immediately (Fig. 5B).
4. Discussion It was reported previously that locomotion was controlled by neurotransmitter systems, including the serotonergic neurons [6]. More recently it has been shown that the intraperitoneal injection of MAO-I, tranylcypromine (TC), significantly increased extracellular 5-HT in the PFC and dorsal raphe nucleus, and induced hyperactivity [2,4]. In the present study we demonstrated, that the intraperitoneal injection of TC caused increase in extracellular levels of 5-HT not only in PFC but also in the striatum and hippocampus along with corresponding increases in the motor activity. Although our findings suggest that TC activates motility by increasing extracellular 5-HT in the brain, the possibility can not be excluded that this effect is mediated also by an increase in noradrenalin and dopamine concentration in central nervous system. Moreover, TC could rise motor activity via effects on the peripheral nerves or blood circulation. In order to clarify these issues in the next part of the present study we infused TC or 5-HT locally into the bilateral striatum, hippocampus and PFC into which serotonergic neurons were projected. The local infusion either of 1.0 mM TC or 1.0 mM 5-HT into the bilateral striatum had no effect on the motor activity of the rats. This finding is in excellent agreement with our previous report, in which we found no relationship between motor activity and extracellular 5-HT levels in the striatum either in the daytime or nighttime [25]. Based on these findings,
Fig. 4. Effects of local infusion of 1.0 mM 5-HT into the nucleus accumbens on motor activity of the rats. The bold lines with open circles indicate the average of motor counts in the rats infused with 1.0 mM of 5-HT. Horizontal bars indicate the term of drug infusion. Values are expressed as mean6S.E.
200
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
Fig. 5. Effects of local infusions of TTX (10 mM) and TC (1.0 mM) on motor activity of the rats: (A) both TTX and TC were infused into the hippocampus, (B) TTX was infused into the PFC, and TC was infused into the hippocampus, (C) TTX was infused into the hippocampus, and TC was infused into the PFC. Bold horizontal bars show the term of TC infusions, and dotted bars show the period of TTX infusions. Values are expressed as mean6S.E. *P,0.05 compared to the baseline value.
serotonergic neurons which project the striatum may not affect motor activity. Conversely, the local infusion of TC into the bilateral hippocampus increased motor activity significantly as did the infusion of 1.0 mM 5-HT. Thus, these results are in good agreement with previous reports that serotonergic neurons in the median raphe nuclei projecting to the hippocampus are related to motor activity [8,11,12,28,29]. A clear relationship between behavioral activity and extracellular 5-HT concentrations in the hippocampus has also been reported more recently [16]. Similarly, the local infusion of 1.0 mM TC or 1.0 mM 5-HT into the bilateral PFC caused an increase in motor activity, although delayed in time and beginning from 2 h after injection. Thinking about previous reports that the medial prefrontal cortex is involved in the initiation and facilitation of voluntary movements [13], our results suggest that PFC neurons that receive serotonergic projections modulate motor activity. Serotonin is known to produce behavioral syndrome consisting of flat body posture, hind limb abduction, head weaving and forepaw treading [1,6,7,26]. This ‘5-HT-syndrome’ is known to be mediated via stimulation of postsynaptic 5-HT1A receptors, and can be induced by systemic administration of the selective 5-HT1A receptor agonist 8-OH-DPAT [26]. In the present study, however, no increases in any component of the ‘5-HT-syndrome’ were observed during and after local infusion of 5-HT to the hippocampus. Local infusion of 5-HT to the PFC, on the other hand, showed qualitatively different effects that increases in grooming or head shaking were also observed. However, the increase in motor activities induced by local infusion of 5-HT either to
the hippocampus or PFC were forward locomotion. Increase in this locomotor activity induced by TC or 5-HT infusions into the hippocampus were observed during and after infusion, but those by TC or 5-HT infusion into the PFC were characterized by a time delay. These results suggest that both of serotonergic neurons projecting to the hippocampus and PFC are somehow involved with the neural networks which regulate motor activity, but that their roles in the control of locomotion are different depending upon their terminal areas. To clarify the interrelationship between hippocampal and PFC serotonergic neurons in modulating motor activity, we infused tetrodotoxin (TTX) and / or TC into these brain structures alone or in combinations. Hyperactivities induced by the local infusion of TC either into the bilateral hippocampus or PFC were suppressed by pretreatment with 10 mM TTX infused into hippocampus. However, TTX infused into PFC failed to prevent hyperlocomotion following intrahippocampal tranylcypromine administration, although the motor response was delayed in time. These results suggest that the hippocampal neurons that receive serotonergic projections are essential for the increase in the locomotor activity, although neither the possibility that these 5-HTinduced hyper-locomotion may be secondary to other psychological processes such as anxiety nor that these TTX-induced hypoactivity may be caused by general malaise could be excluded in the present study. One plausible explanation is that the locomotor activity is regulated by neural networks between the PFC and the hippocampus, both primarily receiving serotonergic innervations from the dorsal or median raphe nuclei [20],
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
although these neural networks have not been anatomically proven. However, the mechanisms of hyperactivities mediated by hippocampal neurons remained uncertain. In the previous reports, the hippocampal-accumbens pathway was shown to initiate locomotor responses [30]. These responses were thought to be regulated by D2 receptors in the NAC. It is known that neural network from NAC to the pedunculopontine nucleus (PPN), a major component of the mesencephalic locomotor region, activate locomotor activity [27,30]. In our study, the local infusion of either 5-HT or DA into the NAC produced significant increases in motor activity. However these increases in motor activity was much smaller than those induced by hippocampal injections. Furthermore, the dramatic increases in the forward locomotion were not observed by the local injection to the NAC. Taking these observations into account, it is also possible that direct or indirect neural networks between the hippocampus and some locomotor regions including the PPN may be involved in the regulation of motility. In the present study, relatively high concentrations of 5-HT (1.0 mM) have been used. We also investigated the effects of a lower concentration (0.1 mM 5-HT), but there was no effect on motor activity. When 1.0 mM 5-HT was infused through the microdialysis probes over 30 min at a rate of 2.0 ml / min, the recovery or permeation rate of 5-HT was between 10 and 20%, thus administered 5-HT was between 5.0 and 10.0 nmol. These amounts might be lowered still further by washing out following perfusion. Because the administered drugs arrived at the neurons by diffusion, high concentrations of 5-HT might be needed for diffusion from a small microdialysis probe and for causing increase in locomotor activity. In conclusion, the local infusion of MAO-I or 5-HT into the hippocampus and PFC, but not into the striatum, induced hyperactivity. We propose that hippocampal neurons that receive serotonergic projections from the median raphe nuclei regulate locomotion.
[6]
[7]
[8]
[9]
[10] [11]
[12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
[20]
References [21] [1] A. Albinsson, A. Bjork, J. Svartengren, T. Klint, G. Andersson, Preclinical pharmacology of FG5893: a potential anxiolytic drug with high affinity for both 5-HT1A and 5-HT2A receptors, Eur. J. Pharmacol. 261 (1994) 285–294. [2] P. Celada, F. Artigas, Monoamine oxidase inhibitors increase preferentially extracellular 5-hydroxytryptamine in the midbrain raphe nuclei. A brain microdialysis study in the awake rat, NaunynSchmiedeberg’s Arch. Pharmacol. 343 (1993) 583–590. [3] G. Curzon, Serotonergic aspects of feeding, in: A. Takada, G. Curzon (Eds.), Serotonin in the Central Nervous System and Periphery, Elsevier Science B.V, Amsterdam, 1995, pp. 43–51. [4] A. Ferrer, F. Artigas, Effects of single treatment with tranylcypromine on extracellular serotonin in rat brain, Eur. J. Pharmacol. 263 (1994) 227–234. [5] M.A. Geyer, A. Puerto, D.B. Menkes, D.S. Segal, A.J. Mandell,
[22] [23]
[24] [25]
[26]
201
Behavioral studies following lesions of mesolimbic and mesocortical serotonergic pathway, Brain. Res. 106 (1976) 257–270. D.G. Grahame-Smith, Studies in vivo on the relationship between brain tryptophan, brain 5-HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and L-tryptophan, J. Neurochem. 18 (1971) 1053–1066. E. Hajos-Korcsok, T. Sharp, 8-OH-DPAT-induced release of hippocampal noradrenaline in vivo: evidence for a role of both 5-HT1A and dopamine D1 receptors, Eur. J Pharmacol. 314 (1996) 285–291. V. Hillegaart, S. Hjorth, Median raphe, but not dorsal raphe, application of the 5-HT1A agonist 8-OH-DPAT stimulates rat motor activity, Eur. J. Pharmacol. 160 (1989) 303–307. S. Hjorth, Functional differences between ascending 5-HT systems, in: P.B. Bradley (Ed.), Serotonin, CNS Receptors and Brain Function, Pergamon Press, Oxford, 1992, pp. 203–218. B.L. Jacobs, Serotonin and behavior: emphasis on motor control, J. Clin. Psychiat. 52 (Suppl) (1991) 17–23. B.L. Jacobs, A. Cohen, Differential behavioral effects of lesions of the median or dorsal raphe nuclei in rats: open field and pain-elicited aggression, J. Comp. Physiol. Psychol. 90 (1976) 102–108. B.L. Jacobs, W.D. Wise, K.M. Taylor, Differential behavioral and neurochemical effects following lesions of the dorsal and median raphe nuclei in rats, Brain Res. 79 (1974) 353–361. J.F. Kalasaka, D.J. Crammond, Cerebral cortical mechanisms of reaching movements, Science 255 (1992) 1517–1523. G.A. Kennett, G. Curzon, Evidence that hypophagia induced by mCPP and TFMPP requires 5-HT1C and 5-HT1B receptors; hypophagia induced by RU 24969 only requires 5-HT1B receptors, Psychopharmacology 96 (1988) 93–100. W. Kostowski, Interactions between serotonergic and catecholaminergic systems in the brain, Pol. J. Pharmacol. 27 (Suppl) (1975) 15–24. A. Linthorst, C. Flachskamm, F. Holsboer, J. Reul, Local administration of recombinant human interleukin-1 beta in the rat hippocampus increases serotonergic neurotransmission, hypothalamic-pituitary-adrenocortical axis activity, and body temperature, Endocrinology 135 (1994) 520–532. B.K. Lipska, G.E. Jaskiw, A. Arya, D.R. Weinberger, Serotonin depletion causes long-term reduction of exploration in the rat, Phamacol. Biochem. Behav. 43 (1992) 1247–1252. S.A. Lorens, H.C. Guildberg, K. Hole, C. Kohler, B. Srebro, Activity, avoidance learning and regional 5-hydroxytryptamine following intra-brain stem 5,7-dihydroxytryptamine and electrolytic midbrain raphe lesions, Brain Res. 108 (1976) 97–113. Y. Masuo, J. Noguchi, S. Morita, Y. Matsumoto, Effects of intracerebroventricular administration of pituitary adenylate cyclaseactivating polypeptide (PACAP) on the motor activity and reserpineinduced hypothermia in murines, Brain Res. 700 (1995) 219–226. M.E. Molliver, Serotonergic neuronal systems: What their anatomic organization tell us about function, J. Clin. Psychopharmacol. 7 (1987) 3S–23S. D. Nakahara, N. Ozaki, T. Nagatsu, In vivo microdialysis of neurotransmitters and their metabolites, Methods Neurotransmitter. Neuropeptide. Res. 1 (1993) 219–248. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. L.H. Price, D.S. Charney, G.R. Heninger, Effects of tranylcypromine treatment on neuroendocrine, behavioral, and autonomic responces to tryptophan in depressed patients, Life Sci. 37 (1985) 809–818. P. Soubrie, Reconciling the role of central serotonin neurons in human and animal behavior, Behav. Brain Sci. 9 (1989) 319–364. H. Takahashi, Y. Takada, N. Nagai, T. Urano, A. Takada, Extracellular serotonin in the striatum increased after immobilization stress only in the nighttime, Behav. Brain Res. 91 (1998) 185–191. M.D. Tricklebank, C. Forler, J.R. Fozard, The involvement of subtypes of the 5-HT1 receptor and catecholaminergic system in the
202
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
behavioural response to 8-hydroxy-2-(di-n-propylamino)tetralin in the rat, Eur. J. Pharmacol. 106 (1984) 271–282. [27] C. Tsai, G. Mogenson, M. Wu, C. Yang, A comparison of the effects of electrical stimulation of the amygdala and hippocampus on subparidal output neurons to the pedunculopontine nucleus, Brain Res. 494 (1989) 22–29. [28] L. Wing, K.E. Asin, D. Wirtshafter, Role of dopamine in the hyperactivity produced by electric median raphe lesions, Soc. Neurosci. Abstr. 10 (1984) 1172.
[29] D. Wirtshafter, W. Montana, K.E. Asin, Behavioral and biochemical studies of the substrates of median raphe lesion induced hyperactivity, Physiol. Behav. 38 (1986) 751–759. [30] C. Yang, G. Mogenson, Hippocampal signal transmission to the pedunculopontine nucleus and its regulation by dopamine D2 receptors in the nucleus accumbens: an electrophysiological and behavioral study, Neuroscience 23 (1987) 1041–1055.
Research report
Serotonergic neurons projecting to hippocampus activate locomotion Hiroshi Takahashi a , Yumiko Takada b , *, Nobuo Nagai a , Tetsumei Urano a , Akikazu Takada a b
a Department of Physiology, Hamamatsu University, School of Medicine, Hamamatsu-shi, Shizuoka-ken, 431 -3192 Japan Department of Pathophysiology, Hamamatsu University, School of Medicine, Hamamatsu-shi, Shizuoka-ken, 431 -3192 Japan
Accepted 4 April 2000
Abstract Although the role of brain serotonergic neurons in locomotion has been extensively studied, their influence may vary depending upon the terminal areas. Thus, using microdialysis and microinjection techniques, we examined the relationship between serotonin (5-HT) levels in striatum, hippocampus or prefrontal cortex (PFC) and motor activity in rats. The systemic injection (10 mg / kg i.p.) of monoamine oxidase inhibitor, tranylcypromine (TC), significantly elevated 5-HT levels in the striatum, hippocampus and PFC accompanied by a parallel increase in motor activity of the rats. This effect was mimicked by microinfusions of TC (1.0 mM) or 5-HT (1.0 mM) into the hippocampus and to some extent into PFC (the response delayed in time), but not into striatum. The increase in motor activity produced by local infusions of TC either into the hippocampus or PFC could be prevented by pretreatment with 10 mM tetrodotoxin infused into the hippocampus. However, tetrodotoxin infused to PFC failed to prevent hyperlocomotion produced by intrahippocampal infusion of TC, although the response was delayed in time. Thus, we conclude that serotonergic neurons projecting to the hippocampus are involved in locomotor activity and PFC serotonergic fibers may facilitate hippocampal control of locomotion. 2000 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Monoamines and behaviour Keywords: Serotonin; Locomotion; Hippocampus; Prefrontal cortex; MAO inhibitor
1. Introduction It is well known that central serotonergic neurons have widespread projections to the cortex and to the limbic structures, and it is believed that the serotonergic system plays an important role in the regulation of a wide variety of physiologic, emotional, and behavioral functions [5,8,15,18]. Since the involvement of serotonin (5-hydroxytryptamine; 5-HT) in human affective disorders has been well documented, much effort has been paid to elucidate the impact of this amine on various brain functions, both in humans and in laboratory animals. However, the role of the central serotonergic system in the regulation of behavior is complex [9,10]. Thus, some reports indicate that reduced central serotonergic activity is closely related to aggressive reactions [24] and exploration *Corresponding author. Tel.: 181-53-435-2247; fax: 181-53-4349225. E-mail address: [email protected] (Y. Takada)
[17]. Furthermore, the decrease in serotonin synthesis in the brain [6] or lesion of the median raphe nucleus [11,12,28,29] have been shown to produce dramatic increase in locomotion. On the other hand, it has been demonstrated that serotonergic system may participate in either generating or inhibiting motor functions related to food intake [3,14]. These different relations between central serotonergic neurons and motor responses may be due to the subtypes of the serotonergic receptors involved or to the contrariety in functions of various brain areas receiving serotonergic innervations. However, most up-todate reports utilized the systemic rather than local injections of substances related to serotonin metabolism, that can introduce greater bias and can hardly give a closer look into the relationship between serotonin levels in discrete brain areas and locomotor functions. To overcome these limitations, in the present study we examined the role of serotonergic system in motor activity by using microinfusions of 5-HT into the hippocampus, striatum, and prefrontal cortex (PFC) which receive serotonergic in-
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02385-4
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
nervations. We compared the above effects with those produced by the local infusions of monoamine oxidase inhibitor (MAO-I) in injected areas. MAO-I has been shown to elevate the levels of extracellular 5-HT and catecholamines [23] and also to induce rat hyperactivity [4]. Furthermore, we used in vivo microdialysis techniques to examine the relationship between extracellular 5-HT levels and locomotion after systemic administration of MAO-I.
2. Materials and methods
2.1. Animals Adult male Wistar rats (200–250 g) were used. They were individually housed for 2 weeks before the experiments in a temperature-controlled room (24618C) that was maintained on a 12:12 h light / dark cycle. Animals were given free access to food and water.
2.2. Surgical procedures To avoid volume-induced increase in intracerebral pressure following administration of drugs and to exclude effects of insertion of injectors on motor activity, we employed a microdialysis technique for the local infusion of 5-HT and MAO-I in free moving conditions. Three to five days before microdialysis experiments, guide cannulas were stereotaxically implanted for the penetration of a microdialysis probe in rats under sodium pentobarbital anesthesia (50 mg / kg, i.p.). The coordinates chosen according to the stereotaxic atlas of Paxinos and Watson [22] were as follows. For the striatum: AP, 0.2; ML, 3.0; DV, 3.0 (mm). For the hippocampus: AP, 25.8; ML, 5.0; DV, 3.0 (mm). For the prefrontal cortex (PFC): AP, 3.1; ML, 0.9; DV, 2.0 (mm). For the nucleus accumbens (NAC): AP, 2.1; ML, 2.5; DV, 4.5 (mm); angled at 58 perpendicularly. The microdialysis probes were implanted 2 mm for PFC and 4 mm for other regions deeper than the tips of guide cannulas. Thus coordinates of active dialyzed membranes were as follows. For the striatum: AP, 0.2; ML, 3.0; DV, 3.0–7.0 (mm). For the hippocampus: AP, 25.8; ML, 5.0; DV, 3.0–7.0 (mm). For the prefrontal cortex (PFC): AP, 3.1; ML, 0.9; DV, 2.0–4.0 (mm). The study animals displayed neither any obvious signs of bleeding or infection around the wound, nor of any postsurgical distress.
2.3. Microdialysis procedures Microdialysis probes were constructed according to the method described by Nakahara et al. [21] with an outer diameter of 220 mm and an exposed tip of 2.0 mm or 4.0 mm. These probes had an active dialyzing length of 2 mm for PFC and 4 mm for other regions and a membrane with
195
a cut-off value of molecular weight 50 000. The recovery rate of 5-HT and 5-HIAA (5-hydroxyindolo-acetic acid) were between 10 and 20%. The lactate Ringer solution used as the perfusate contained 0.1 M NaCl, 1.8 mM CaCl 2 , 4.0 mM KCl, and 0.03 M sodium lactate. The microdialysis tube was perfused continuously with Ringer solution at a rate of 2.0 ml / min using microinfusion pump, and the samples were started 3 h after probe insertion, to allow for basal-level stabilization.
2.4. HPLC procedures The contents of 5-HT and 5-HIAA in dialysates were assayed using HPLC with electrochemical detection. The electrochemical apparatus consisted of a reverse phase HPLC column (Eicom Co., Kyoto, Japan). The column was MA-5ODS and the mobile phase was composed of 0.1 M sodium acetate, 0.1 M citric acid, 18% methanol, 0.023% l-octane sulfonate, and 0.0005% EDTA, pH 3.5. The mobile phase was delivered by a pump at a flow rate of 0.3 ml / min. The column temperature was kept at 258C. The graphite electrode (WE-3G Eicom) was set at 0.6 V (an Ag /AgCl reference electrode). The dialysate was injected into the HPLC column every 20 min. by an automatic injector.
2.5. Counts of motor activity Spontaneous motor activities were measured by Supermex (Muromachi Kikai Co., Tokyo, Japan). The Supermex consists of a sensor monitor which was mounted above the cage to detect changes in heat across multiple zones of the cage through an array of Fresnel lenses. In this way, the system could monitor and count all spontaneous movements, both vertical and horizontal, including locomotion and head-movements, etc. [19,25]. All counts were automatically added and recorded at 20- or 30-min intervals.
2.6. Experimental protocols 2.6.1. Systemic administration of MAO-I In the first series of experiments, 54 rats were divided into three subgroups according to the brain area that microdialysis probes were inserted unilaterally (18 rats each for the striatum, hippocampus or prefrontal cortex). Three hours after probe insertion, 3 basal dialysates were collected every 20 min. Then, rats were further subdivided to receive intraperitoneal injection of either 1 mg / kg or 10 mg / kg of tranylcypromine (TC: Sigma, St Louis, USA), or 1 ml / kg isotonic saline as a control. Finally, 6 rats each for the striatum, hippocampus or prefrontal cortex were examined in each treatment group. Motor activity was always counted simultaneously with the measurement of 5-HT by microdialysis.
196
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
2.6.2. Local infusion of MAO-I or 5 -HT Similarly, in the second series of experiments (84 rats), rats were divided into three subgroups according to the insertion of the microdialysis probes (28 rats each for the striatum, hippocampus, or prefrontal cortex). The probes were inserted bilaterally in all groups. Three hours after probe insertions, motor activities were measured for 2 h, and then TC or 5-HT (5-hydroxytryptamine hydrochloride: Sigma, St. Louis, USA) was infused locally for 30 min through bilateral microdialysis probes (6 rats each for 0.1 mM TC, 1.0 mM TC, 0.1 mM 5-HT or 1.0 mM 5-HT per subgroup). As the control group, 18 rats (6 rats for the striatum, hippocampus, or prefrontal cortex) were perfused continuously with Ringer solution throughout the experiments. Furthermore, 0.1 or 1.0 mM of 5-HT was infused locally into the bilateral NAC (10 rats each). 0.1 or 1.0 mM of 3, 4-Dihydroxyphenethylamine hydrochloride (Dopamine5DA: Sigma, St. Louis, USA) was infused locally into the bilateral NAC (8 rats each). pH of the perfusate of TC and 5-HT was adjusted to 7.2–7.4 with sodium hydroxide. Counting of motor activities was continued more than 2 h after finishing these infusions. 2.6.3. Local infusion of tetrodotoxin ( TTX) with MAO-I In the last series of experiments (15 rats), four microdialysis probes were inserted to both bilateral hippocampus and bilateral PFC in each rat. Three hours after insertion of the probes basal levels of motor activity were measured and then 10 mM TTX was infused to the bilateral hippocampus or PFC through microdialysis probes during 1 h. Thirty minutes after starting TTX infusion, 1.0 mM TC was infused locally during 30 min through microdialysis probes to the bilateral hippocampus or PFC (5 rats each for the set). Motor activities were measured more than 3 h after finishing these infusions. 2.7. Histology Following the termination of each experiment, animals were anesthetized with pentobarbital and decapitated; brains were removed and stored in buffered formalin. Probe placement was verified microscopically, and data were discarded if any part of the dialysis probe was not in the regions intended (very small number).
2.8. Statistical analysis Serotonin (5-HT) and 5-HIAA concentrations in the dialysate (not corrected for in vitro recovery) were expressed as percent changes of basal values, 100% being defined as the average of the last three samples before injection of MAO-I. The comparison of the motor activity was performed compared with their basal values which was defined as the average of the last three samples before injection. Statistical analysis of all the results was performed by one- or two-way ANOVA with repeated mea-
sures, followed by a multicomparison test using Fisher’s PLSD (Protected Least Significant Difference). The Pvalue of less than 0.05 was considered to be statistically significant.
2.9. Ethics The study was submitted to the Ethical Committee of our University. The committee judged the experimental designs to ‘the guiding principles for the care and use of animals in the field of physiological sciences’ recommended by the Physiological Society of Japan on December 19, 1988. The guidelines were prepared to meet the requirements contained in the publications such as U.K. Animals Act, 1986, DHEW Publication No. (NIH) 85-23, 1985, and International Guiding Principle for Biomedical Research Involving Animals, CIOM, 1983. The committee approved the experiments after careful examination.
3. Results
3.1. Effect of MAO-I systemic administration on extracellular 5 -HT levels and spontaneous motor activity Motor activities considerably increased 40 or 60 min after the intraperitoneal injection of 10 mg / kg TC (P, 0.001, Fig. 1A), and remained constant throughout the experiment. During these hyperactive states, most of the growth in the motor activities was the increase in forward locomotion. At this time extracellular 5-HT level in the striatum significantly increased to 250% of the baseline (P,0.01; Fig. 1B). Conversely extracellular 5-HIAA in this brain area was markedly reduced (P,0.001; Fig. 1C). There were significant differences between the experimental group and the control group for both 5-HT (P,0.05) and 5-HIAA (P,0.001) levels. Similar changes were observed in the hippocampus at this time point. Thus, 40 min after the administration of TC, increase in extracellular 5-HT values (700% of the baseline; P,0.01; Fig. 1B) was accompanied by evident decline in 5-HIAA (P,0.05; Fig. 1C). These values differed significantly between TCtreated and control animals (P,0.05 for both parameters). In the prefrontal cortex, 40 min after TC administration, the levels of 5-HT increased to 250% of baseline (P,0.01; Fig. 1B), and continued to increase throughout the experiment. Simultaneously extracellular 5-HIAA values were significantly reduced (P,0.05; Fig. 1C). Statistically significant differences were observed between rats treated with TC and control animals (P,0.05 and P,0.005 for 5-HT and 5-HIAA, respectively). Neither the intraperitoneal administration of 1 ml / kg saline nor 1 mg / kg TC affected motor activity in any group of rats (Fig. 1A). In the rats administered with 1 mg / kg TC no significant changes either in the extracellular 5-HT or 5-HIAA were observed in any studied area (data not shown).
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
197
Fig. 1. Effects of intraperitoneal injection of isotonic saline (control; dotted line with closed circles), 1 mg / kg tranylcypromine (TC) (fine line with closed circles) or 10 mg / kg TC (bold line with open circles) on motor activity of the rats (A), extracellular 5-HT (B), and extracellular 5-HIAA levels (C) in the striatum (left graphs), hippocampus (center graphs), and prefrontal cortex (right graphs). In (B) and (C) data for 1 mg / kg TC not shown. Arrows indicate the time of TC injection. Values are expressed as mean6S.E. *P,0.05, **P,0.005, [P,0.0005 compared to the baseline value.
198
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
Fig. 2. Effects of local infusion of Ringer solution (control; dotted lines with closed circles), 0.1 mM TC (fine line with closed circles) or 1.0 mM TC (bold lines with open circles) into the striatum (left graph), hippocampus (center graph), and prefrontal cortex (right graph) on the motor activity of the rats (for 0.1 mM TC infused into the striatum data not shown). Horizontal bars indicate the term of TC infusion. Values are expressed as mean6S.E. *P,0.05, **P,0.005 compared to the baseline value.
3.2. Effects of local infusions of MAO-I or 5 -HT on spontaneous motor activity Neither the rats infused with 0.1 mM TC, 0.1 mM 5-HT nor control animals, regardless of the brain area studied, showed any changes in their motor activity (see Figs. 2 and 3 for TC and 5-HT infusions, respectively; data from 0.1 mM TC and 0.1 mM 5-HT infusions into the striatum not shown). The local infusions of either 1.0 mM TC or 1.0 mM 5-HT into the bilateral striatum did not change the motor activities (Figs. 2 and 3, respectively), but these infusions into the bilateral hippocampus increased the motor activities significantly when compared with the baseline
values (P,0.05 for both groups) or respective controls (P,0.01 for both groups; Figs. 2 and 3, respectively). These hyperactivities continued for a subsequent 90 min following infusions. During these hyperactive states, neither increases in grooming, tremor, rearing, stereotyped behavior, head shaking nor other involuntary movements were found by continuous observation. Almost all of the motor activities were increased in normal forward locomotion. Increases in components of a 5-HT-induced behavioral syndrome consisting of flat body posture, hind limb abduction, head weaving and forepaw treading [26,1,7] were not observed. Similarly, local bilateral infusions of either 1.0 mM TC or 1.0 mM 5-HT into the prefrontal cortex caused a
Fig. 3. Effects of local infusion of Ringer solution (control; dotted lines with closed circles), 0.1 mM 5-HT (fine line with closed circles) or 1.0 mM 5-HT (bold lines with open circles) into the striatum (left graph), hippocampus (center graph), and prefrontal cortex (right graph) on the motor activity of the rats (for 0.1 mM 5-HT infused into the striatum data not shown). Horizontal bars indicate the term of 5-HT infusion. Values are expressed as mean6S.E. *P,0.05, **P,0.01 compared to the baseline value.
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
significant increase in the motor activity when compared with the baseline values (P,0.05 and P,0.01, respectively) or control rats (P,0.01 for both groups; Figs. 2 and 3, respectively). Interestingly, these hyperactivities characterized with 2–2.5 h time delay and gained 15 to 20 times of the baseline. Most of the growth in the motor activities was the increase in forward locomotion, however, increases in grooming or head shaking were also observed. The local bilateral infusions of either 1.0 mM 5-HT or 1.0 mM DA into the NAC caused a significant increase in the motor activity when compared with the baseline values (P,0.05 and P,0.0001, respectively) or control rats (P, 0.05 and P,0.01, respectively; Fig. 4). During these hyperactive states, increases in grooming or stereotyped movements were also observed. Neither the rats locally injected with 0.1 mM 5-HT nor 0.1 mM DA into the bilateral NAC showed significant changes in their motor activities (Fig. 4).
3.3. Effects of local infusion of TTX with MAO-I on spontaneous motor activity Effects of TTX infusions on increase in the motor activities are shown in Fig. 5. The local infusion of 10 mM TTX to the bilateral hippocampus significantly inhibited baseline motor activity of the rats (P,0.05, Fig. 5A, C). In this group of animals subsequent infusion of 1.0 mM TC into the bilateral hippocampus failed to produce any increase in the motor activity (Fig. 5A). Delayed increase in the hypermotility caused by TC infusion into PFC was also inhibited by TTX infusion into the hippocampus (Fig. 5C). After the local infusion of TTX to the bilateral PFC however, subsequent local infusion of TC to the bilateral
199
hippocampus increased the motor activity significantly (P,0.05), although not immediately (Fig. 5B).
4. Discussion It was reported previously that locomotion was controlled by neurotransmitter systems, including the serotonergic neurons [6]. More recently it has been shown that the intraperitoneal injection of MAO-I, tranylcypromine (TC), significantly increased extracellular 5-HT in the PFC and dorsal raphe nucleus, and induced hyperactivity [2,4]. In the present study we demonstrated, that the intraperitoneal injection of TC caused increase in extracellular levels of 5-HT not only in PFC but also in the striatum and hippocampus along with corresponding increases in the motor activity. Although our findings suggest that TC activates motility by increasing extracellular 5-HT in the brain, the possibility can not be excluded that this effect is mediated also by an increase in noradrenalin and dopamine concentration in central nervous system. Moreover, TC could rise motor activity via effects on the peripheral nerves or blood circulation. In order to clarify these issues in the next part of the present study we infused TC or 5-HT locally into the bilateral striatum, hippocampus and PFC into which serotonergic neurons were projected. The local infusion either of 1.0 mM TC or 1.0 mM 5-HT into the bilateral striatum had no effect on the motor activity of the rats. This finding is in excellent agreement with our previous report, in which we found no relationship between motor activity and extracellular 5-HT levels in the striatum either in the daytime or nighttime [25]. Based on these findings,
Fig. 4. Effects of local infusion of 1.0 mM 5-HT into the nucleus accumbens on motor activity of the rats. The bold lines with open circles indicate the average of motor counts in the rats infused with 1.0 mM of 5-HT. Horizontal bars indicate the term of drug infusion. Values are expressed as mean6S.E.
200
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
Fig. 5. Effects of local infusions of TTX (10 mM) and TC (1.0 mM) on motor activity of the rats: (A) both TTX and TC were infused into the hippocampus, (B) TTX was infused into the PFC, and TC was infused into the hippocampus, (C) TTX was infused into the hippocampus, and TC was infused into the PFC. Bold horizontal bars show the term of TC infusions, and dotted bars show the period of TTX infusions. Values are expressed as mean6S.E. *P,0.05 compared to the baseline value.
serotonergic neurons which project the striatum may not affect motor activity. Conversely, the local infusion of TC into the bilateral hippocampus increased motor activity significantly as did the infusion of 1.0 mM 5-HT. Thus, these results are in good agreement with previous reports that serotonergic neurons in the median raphe nuclei projecting to the hippocampus are related to motor activity [8,11,12,28,29]. A clear relationship between behavioral activity and extracellular 5-HT concentrations in the hippocampus has also been reported more recently [16]. Similarly, the local infusion of 1.0 mM TC or 1.0 mM 5-HT into the bilateral PFC caused an increase in motor activity, although delayed in time and beginning from 2 h after injection. Thinking about previous reports that the medial prefrontal cortex is involved in the initiation and facilitation of voluntary movements [13], our results suggest that PFC neurons that receive serotonergic projections modulate motor activity. Serotonin is known to produce behavioral syndrome consisting of flat body posture, hind limb abduction, head weaving and forepaw treading [1,6,7,26]. This ‘5-HT-syndrome’ is known to be mediated via stimulation of postsynaptic 5-HT1A receptors, and can be induced by systemic administration of the selective 5-HT1A receptor agonist 8-OH-DPAT [26]. In the present study, however, no increases in any component of the ‘5-HT-syndrome’ were observed during and after local infusion of 5-HT to the hippocampus. Local infusion of 5-HT to the PFC, on the other hand, showed qualitatively different effects that increases in grooming or head shaking were also observed. However, the increase in motor activities induced by local infusion of 5-HT either to
the hippocampus or PFC were forward locomotion. Increase in this locomotor activity induced by TC or 5-HT infusions into the hippocampus were observed during and after infusion, but those by TC or 5-HT infusion into the PFC were characterized by a time delay. These results suggest that both of serotonergic neurons projecting to the hippocampus and PFC are somehow involved with the neural networks which regulate motor activity, but that their roles in the control of locomotion are different depending upon their terminal areas. To clarify the interrelationship between hippocampal and PFC serotonergic neurons in modulating motor activity, we infused tetrodotoxin (TTX) and / or TC into these brain structures alone or in combinations. Hyperactivities induced by the local infusion of TC either into the bilateral hippocampus or PFC were suppressed by pretreatment with 10 mM TTX infused into hippocampus. However, TTX infused into PFC failed to prevent hyperlocomotion following intrahippocampal tranylcypromine administration, although the motor response was delayed in time. These results suggest that the hippocampal neurons that receive serotonergic projections are essential for the increase in the locomotor activity, although neither the possibility that these 5-HTinduced hyper-locomotion may be secondary to other psychological processes such as anxiety nor that these TTX-induced hypoactivity may be caused by general malaise could be excluded in the present study. One plausible explanation is that the locomotor activity is regulated by neural networks between the PFC and the hippocampus, both primarily receiving serotonergic innervations from the dorsal or median raphe nuclei [20],
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
although these neural networks have not been anatomically proven. However, the mechanisms of hyperactivities mediated by hippocampal neurons remained uncertain. In the previous reports, the hippocampal-accumbens pathway was shown to initiate locomotor responses [30]. These responses were thought to be regulated by D2 receptors in the NAC. It is known that neural network from NAC to the pedunculopontine nucleus (PPN), a major component of the mesencephalic locomotor region, activate locomotor activity [27,30]. In our study, the local infusion of either 5-HT or DA into the NAC produced significant increases in motor activity. However these increases in motor activity was much smaller than those induced by hippocampal injections. Furthermore, the dramatic increases in the forward locomotion were not observed by the local injection to the NAC. Taking these observations into account, it is also possible that direct or indirect neural networks between the hippocampus and some locomotor regions including the PPN may be involved in the regulation of motility. In the present study, relatively high concentrations of 5-HT (1.0 mM) have been used. We also investigated the effects of a lower concentration (0.1 mM 5-HT), but there was no effect on motor activity. When 1.0 mM 5-HT was infused through the microdialysis probes over 30 min at a rate of 2.0 ml / min, the recovery or permeation rate of 5-HT was between 10 and 20%, thus administered 5-HT was between 5.0 and 10.0 nmol. These amounts might be lowered still further by washing out following perfusion. Because the administered drugs arrived at the neurons by diffusion, high concentrations of 5-HT might be needed for diffusion from a small microdialysis probe and for causing increase in locomotor activity. In conclusion, the local infusion of MAO-I or 5-HT into the hippocampus and PFC, but not into the striatum, induced hyperactivity. We propose that hippocampal neurons that receive serotonergic projections from the median raphe nuclei regulate locomotion.
[6]
[7]
[8]
[9]
[10] [11]
[12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
[20]
References [21] [1] A. Albinsson, A. Bjork, J. Svartengren, T. Klint, G. Andersson, Preclinical pharmacology of FG5893: a potential anxiolytic drug with high affinity for both 5-HT1A and 5-HT2A receptors, Eur. J. Pharmacol. 261 (1994) 285–294. [2] P. Celada, F. Artigas, Monoamine oxidase inhibitors increase preferentially extracellular 5-hydroxytryptamine in the midbrain raphe nuclei. A brain microdialysis study in the awake rat, NaunynSchmiedeberg’s Arch. Pharmacol. 343 (1993) 583–590. [3] G. Curzon, Serotonergic aspects of feeding, in: A. Takada, G. Curzon (Eds.), Serotonin in the Central Nervous System and Periphery, Elsevier Science B.V, Amsterdam, 1995, pp. 43–51. [4] A. Ferrer, F. Artigas, Effects of single treatment with tranylcypromine on extracellular serotonin in rat brain, Eur. J. Pharmacol. 263 (1994) 227–234. [5] M.A. Geyer, A. Puerto, D.B. Menkes, D.S. Segal, A.J. Mandell,
[22] [23]
[24] [25]
[26]
201
Behavioral studies following lesions of mesolimbic and mesocortical serotonergic pathway, Brain. Res. 106 (1976) 257–270. D.G. Grahame-Smith, Studies in vivo on the relationship between brain tryptophan, brain 5-HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and L-tryptophan, J. Neurochem. 18 (1971) 1053–1066. E. Hajos-Korcsok, T. Sharp, 8-OH-DPAT-induced release of hippocampal noradrenaline in vivo: evidence for a role of both 5-HT1A and dopamine D1 receptors, Eur. J Pharmacol. 314 (1996) 285–291. V. Hillegaart, S. Hjorth, Median raphe, but not dorsal raphe, application of the 5-HT1A agonist 8-OH-DPAT stimulates rat motor activity, Eur. J. Pharmacol. 160 (1989) 303–307. S. Hjorth, Functional differences between ascending 5-HT systems, in: P.B. Bradley (Ed.), Serotonin, CNS Receptors and Brain Function, Pergamon Press, Oxford, 1992, pp. 203–218. B.L. Jacobs, Serotonin and behavior: emphasis on motor control, J. Clin. Psychiat. 52 (Suppl) (1991) 17–23. B.L. Jacobs, A. Cohen, Differential behavioral effects of lesions of the median or dorsal raphe nuclei in rats: open field and pain-elicited aggression, J. Comp. Physiol. Psychol. 90 (1976) 102–108. B.L. Jacobs, W.D. Wise, K.M. Taylor, Differential behavioral and neurochemical effects following lesions of the dorsal and median raphe nuclei in rats, Brain Res. 79 (1974) 353–361. J.F. Kalasaka, D.J. Crammond, Cerebral cortical mechanisms of reaching movements, Science 255 (1992) 1517–1523. G.A. Kennett, G. Curzon, Evidence that hypophagia induced by mCPP and TFMPP requires 5-HT1C and 5-HT1B receptors; hypophagia induced by RU 24969 only requires 5-HT1B receptors, Psychopharmacology 96 (1988) 93–100. W. Kostowski, Interactions between serotonergic and catecholaminergic systems in the brain, Pol. J. Pharmacol. 27 (Suppl) (1975) 15–24. A. Linthorst, C. Flachskamm, F. Holsboer, J. Reul, Local administration of recombinant human interleukin-1 beta in the rat hippocampus increases serotonergic neurotransmission, hypothalamic-pituitary-adrenocortical axis activity, and body temperature, Endocrinology 135 (1994) 520–532. B.K. Lipska, G.E. Jaskiw, A. Arya, D.R. Weinberger, Serotonin depletion causes long-term reduction of exploration in the rat, Phamacol. Biochem. Behav. 43 (1992) 1247–1252. S.A. Lorens, H.C. Guildberg, K. Hole, C. Kohler, B. Srebro, Activity, avoidance learning and regional 5-hydroxytryptamine following intra-brain stem 5,7-dihydroxytryptamine and electrolytic midbrain raphe lesions, Brain Res. 108 (1976) 97–113. Y. Masuo, J. Noguchi, S. Morita, Y. Matsumoto, Effects of intracerebroventricular administration of pituitary adenylate cyclaseactivating polypeptide (PACAP) on the motor activity and reserpineinduced hypothermia in murines, Brain Res. 700 (1995) 219–226. M.E. Molliver, Serotonergic neuronal systems: What their anatomic organization tell us about function, J. Clin. Psychopharmacol. 7 (1987) 3S–23S. D. Nakahara, N. Ozaki, T. Nagatsu, In vivo microdialysis of neurotransmitters and their metabolites, Methods Neurotransmitter. Neuropeptide. Res. 1 (1993) 219–248. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. L.H. Price, D.S. Charney, G.R. Heninger, Effects of tranylcypromine treatment on neuroendocrine, behavioral, and autonomic responces to tryptophan in depressed patients, Life Sci. 37 (1985) 809–818. P. Soubrie, Reconciling the role of central serotonin neurons in human and animal behavior, Behav. Brain Sci. 9 (1989) 319–364. H. Takahashi, Y. Takada, N. Nagai, T. Urano, A. Takada, Extracellular serotonin in the striatum increased after immobilization stress only in the nighttime, Behav. Brain Res. 91 (1998) 185–191. M.D. Tricklebank, C. Forler, J.R. Fozard, The involvement of subtypes of the 5-HT1 receptor and catecholaminergic system in the
202
H. Takahashi et al. / Brain Research 869 (2000) 194 – 202
behavioural response to 8-hydroxy-2-(di-n-propylamino)tetralin in the rat, Eur. J. Pharmacol. 106 (1984) 271–282. [27] C. Tsai, G. Mogenson, M. Wu, C. Yang, A comparison of the effects of electrical stimulation of the amygdala and hippocampus on subparidal output neurons to the pedunculopontine nucleus, Brain Res. 494 (1989) 22–29. [28] L. Wing, K.E. Asin, D. Wirtshafter, Role of dopamine in the hyperactivity produced by electric median raphe lesions, Soc. Neurosci. Abstr. 10 (1984) 1172.
[29] D. Wirtshafter, W. Montana, K.E. Asin, Behavioral and biochemical studies of the substrates of median raphe lesion induced hyperactivity, Physiol. Behav. 38 (1986) 751–759. [30] C. Yang, G. Mogenson, Hippocampal signal transmission to the pedunculopontine nucleus and its regulation by dopamine D2 receptors in the nucleus accumbens: an electrophysiological and behavioral study, Neuroscience 23 (1987) 1041–1055.