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5-HT excites globus pallidus neurons by multiple receptor mechanisms
Neuroscience 151 (2008) 439 – 451
5-HT EXCITES GLOBUS PALLIDUS NEURONS BY MULTIPLE RECEPTOR MECHANISMS L. CHEN,a,b K. K. L. YUNG,c Y. S. CHANd AND W. H. YUNGa*
conclusion, we found that 5-HT could modulate the excitability of globus pallidus neurons by both pre- and post-synaptic mechanisms. In view of the extensive innervation by globus pallidus neurons on other basal ganglia nuclei, this action of 5-HT originated from the raphe may have a profound effect on the operation of the entire basal ganglia network. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved.
a
Department of Physiology, The Chinese University of Hong Kong, Shatin, Hong Kong, China
b
Department of Physiology, Qingdao University, Qingdao, China
c
Department of Biology, Hong Kong Baptist University, Hong Kong, China
Key words: 5-HT, 5-HT receptors, globus pallidus.
d
Department of Physiology, The University of Hong Kong, Hong Kong, China
The globus pallidus in rodent is homologous to the external globus pallidus in primates. More and more evidence suggests that the globus pallidus plays a critical role in the basal ganglia circuit under normal and pathological conditions. In Parkinson’s disease and its animal models, the absence of normal dopaminergic innervation leads to presumed hypoactivity and decreased output of the globus pallidus, contributing to akinesia and hypokinetic symptoms (Albin et al., 1989; Alexander and Crutcher, 1990; Filion and Tremblay, 1991; Chesselet and Delfs, 1996; Wichmann and DeLong, 1996). More recently, it was found that changes of firing pattern of pallidal neurons are closely associated with resting tremor (Bergman et al., 1998; Magnin et al., 2000; Raz et al., 2000). This is contributed by the extensive innervation of pallidal neurons on all other basal ganglia nuclei (Bolam et al., 2000). Not surprisingly, there is an increasing interest in those factors that control not only the rate but also the pattern of firing of globus pallidus neurons in normal and diseased states. Up to now, at least three factors have been considered important in determining the firing pattern of pallidal neurons. First, from the study on organotypic cultures Plenz and Kitai (1999) proposed that the reciprocally connected globus pallidus– subthalamic nucleus network could act as a central pattern generator of the basal ganglia. On the other hand, the role of descending cortical glutamatergic inputs to the generation of rhythmic firings in the globus pallidus–subthalamic nucleus network has been substantiated by in vivo extracellular recordings (Magill et al., 2000, 2001). Finally, Stanford (2003) reported that, in the absence of synaptic inputs, each globus pallidus neuron could act as an independent oscillator, which is contributed by the intrinsic membrane conductances. We believe that, in addition, 5-HT innervation from the raphe nucleus is an important determinant of the firing activities of pallidal neurons. Anatomical studies indicated that the globus pallidus receives serotonergic innervation arising from raphe nuclei, mainly the dorsal raphe nucleus (DeVito et al., 1980; Lavoie and Parent, 1990; Charara and Parent, 1994). In addition, stimulation of the dorsal raphe nucleus evoked an increase of 5-HT in globus pallidus
Abstract—Anatomical and neurochemical studies indicated that the globus pallidus receives serotonergic innervation from raphe nuclei but the membrane effects of 5-HT on globus pallidus neurons are not entirely clear. We address this question by applying whole-cell patch-clamp recordings on globus pallidus neurons in immature rat brain slices. Under current-clamp recording, 5-HT depolarized globus pallidus neurons and increased their firing rate, an action blocked by both 5-HT4 and 5-HT7 receptor antagonists and attributable to an increase in cation conductance(s). Further experiments indicated that 5-HT enhanced the hyperpolarization-activated inward conductance which is blocked by 5-HT7 receptor antagonist. To determine if 5-HT exerts any presynaptic effects on GABAergic and glutamatergic inputs, the actions of 5-HT on synaptic currents were studied. At 10 M, 5-HT increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but had no effect on both the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). However, 5-HT at a higher concentration (50 M) decreased the frequency but not the amplitude of the mIPSCs, indicating an inhibition of GABA release from the presynaptic terminals. This effect was sensitive to 5-HT1B receptor antagonist. In addition to the presynaptic effects on GABAergic neurotransmission, 5-HT at 50 M had no consistent effects on glutamatergic neurotransmission, significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in 4 of 11 neurons and decreased the frequency of mEPSCs in 3 of 11 neurons. In *Corresponding author. Tel: ⫹852-26096880; fax: ⫹852-26035022. E-mail address: [email protected] (W. H. Yung). Abbreviations: ACSF, artificial cerebrospinal fluid; AP5, (⫾)-2-amino5-phosphonopentanoic acid; CGP55845, (2S)-3-[[(1S)-1-(3,4dichlorophenyl)ethyl]amino-2-hydroxylpropyl] (phenylmethyl) phosphinic acid; CNQX, 6-cyano-7-nitroquino xaline-2,3-dione; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid; E4CPG, (RS)-␣-ethyl-4-carboxyphenylglycine; Hepes, N-2-hydroxyl piperazine-N=-2-ethane sulphonic acid; Ih, hyperpolarization-activated current; ins, instantaneous current; MDL72222, 4-[3-([1,1-dimethylethyl] amino)-2-hydroxypropul]-1H-indole-2-carbonitrile; mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; RS102221 hydrochloride, 8-[5-(2,4-dimethoxy-5-(4trifluoromethyl-phenyl-sulfonamido) phenyl-5-oxopentyl)-1,3,8triazaspiro [4.5] decane-2,4-dionehydro chloride]; SB269970, (2R)-1[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl; sIPSC, spontaneous inhibitory postsynaptic current; ss, steady-state; TTX, tetrodotoxin; ZD7288, 4-ethylphenylamino-1,2-dimethyl-6-methylamino-pyrimidinium chloride.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.11.003
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measured by microdialysis (McQuade and Sharp, 1997). Based on in situ hybridization, receptor autoradiography and immunohistochemistry, the subtypes 5-HT1B, 5-HT1D, 5-HT2, 5-HT3, 5-HT4 and 5-HT7 have been identified in globus pallidus (Hoyer et al., 1990; Sijbesma et al., 1990, 1991; Waeber et al., 1994; Compan et al., 1996; Vilaro et al., 1996; Morales et al., 1998; Sari et al., 1999; Bonaventure et al., 2000; Clemett et al., 2000; Riad et al., 2000; Li et al., 2004; Martin-Cora and Pazos, 2004). Different lines of evidence also indicate that changes in 5-HT concentration in the basal ganglia of parkinsonian subjects may contribute to the characteristic motor as well as mental impairments (Halliday et al., 1990; Van Praag, 1994; Murai et al., 2001). However, to date, the cellular effects of 5-HT on globus pallidus neurons are not well-known. Here we report that 5-HT could depolarize globus pallidus neurons and modulate synaptic transmission via post- and presynaptic mechanisms respectively, suggesting that the raphe exerts a strong command on the excitability of pallidal neurons.
EXPERIMENTAL PROCEDURES Ethical approval The experimental procedures adopted in the present work complied with the NIH guidelines for animal care and were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong. Effort was made to minimize the number of animals used and their suffering.
In vitro slice preparation Sprague–Dawley rats aged 13–14 days were used for the preparation of acute brain slices. The animals were killed by decapitation. The brains were immediately removed and placed in icecold artificial cerebrospinal fluid (ACSF) of the following composition (in mM): NaCl 125, KCl 4.0, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, glucose 11 and NaHCO3 26, which was continuously bubbled with 95% O2 and 5% CO2. Thin hemi-coronal slices (250 m) containing the globus pallidus were sectioned using a vibrating microtome (Campden Instruments, Loughborough, UK). After equilibration in ACSF, the slices were transferred to a small volume chamber mounted on an upright microscope (Zeiss Axioskop, Gottingen, Germany), and superfused with ACSF at a rate of 1.5–2.0 ml/min maintained at a temperature of 34⫾1 °C. Neuronal soma and proximal dendrites of neurons were directly visualized by a combination of differential interference contrast optics and contrast-enhanced video microscopy. Typical globus pallidus projection neurons, based on their medium size, somatodendritic morphology, as well as the clear voltage- and timedependent inward rectifier, were selected for recording. The neurons sampled were mainly from mid to caudal levels of the globus pallidus.
In order to observe the GABAA receptor–mediated synaptic current, the internal solution containing (in mM): KCl 140, Hepes 10, EGTA 1, MgCl2 2, Na2ATP 2 and Tris GTP 0.4 was used. The inclusion of 140 mM of KCl in the recording pipettes reversed the polarity of the currents from outward to inward. Monitoring through a television connected to the camera, a pipette was placed on the soma of a pallidal neuron and conventional whole-cell recording was made. Normally no series resistance compensation was applied but the cell was rejected if the series resistance increased significantly (⬎20%) during recording. To study and measure the effect of 5-HT on the membrane potential, a neuron was hyperpolarized to close to ⫺60 mV by current injection to prevent firing. To minimize the effect of receptor desensitization, in control group (5-HT alone), 5-HT was perfused only once to one neuron in each slice. The experimental groups (different 5-HT receptor antagonists) were performed in other pallidal neurons. 5-HT receptor antagonist was applied first, then 5-HT receptor antagonist together with 5-HT were co-perfused to the same neuron. Similarly, one type of 5-HT receptor antagonist was applied only once in each neuron (slice). At least 5 min of steady basal recording were collected from each cell before drug application. The duration of 5-HT exposures was 3 min. Usually the 5-HT induced pre- or postsynaptic effects occurred 1–1.5 min after 5-HT application, and reversed in about 8 min during the washout period. For 5-HT-induced postsynaptic depolarization or inward current, the peak value of change was considered as drug effect. For 5-HT-induced presynaptic effects, the synaptic currents occurring in a period of 120 s before drug application were analyzed as control. The maximal change of frequency within a 120 s period following drug application was considered as drug effect. Pre-incubation of different 5-HT receptor antagonists was at least 6 min before the co-application of 5-HT and 5-HT receptor antagonist. Hyperpolarization-activated current (Ih) was evoked under voltage-clamp recording from a holding potential of ⫺50 mV to ⫺130 mV (in 10 mV increments). Two components of voltage- and time-dependent Ih were observed, an instantaneous (ins) current and a slow-rising current, which reached a steady-state (ss) at the end of a long voltage-step. The ins were measured at the point just before the rise of the slow current while the ss were measured near the end of the voltage command. The voltage and current signals were filtered at 3 kHz and were taped using a DAT recorder (Sony, Tokyo, Japan) modified for recording AC and DC signals at a sampling rate of 32 kHz.
Analysis of synaptic currents Computer files containing information of synaptic currents were analyzed by a program developed in our laboratory. Detection of a synaptic current was based mainly on threshold detection. Once a synaptic current was detected, information on the time of occurrence, peak amplitude and kinetics was generated automatically. Statistical comparison of two cumulative probabilities was done by the Kolmogorov-Smirnov test. All detected events with the computed parameters were allowed visual inspection before acceptance. Validity of the analysis was done by cross-checking some of the results using a commercially available program MiniAnalysis (version 5, Synaptosoft, Decatur, GA, USA).
Whole-cell current- and voltage-clamp recordings Conventional tight-seal (⬎2G⍀) whole-cell patch-clamp recordings from globus pallidus neurons were obtained using a patchclamp amplifier (LM/PCA, List Medical, Darmstadt, Germany). Whole-cell pipettes (pulled by P-97, Sutter Instrument, Novato, CA, USA) typically had a resistance of 3– 4.5 M⍀. For current-clamp recording and some voltage-clamp experiments, the pipettes were filled with an internal solution of the following composition (in mM): K-gluconate 130, KCl 10, Hepes 10, EGTA 1, MgCl2 2, Na2ATP 2, Tris GTP 0.4 and the pH was adjusted to 7.25–7.30 with 1 M KOH.
Drugs and statistics 5-HT, S(⫺)-cyanopindolol hemifumarate (4-[3-([1,1-dimethylethyl] amino)-2-hydroxypropul]-1H-indole-2-carbonitrile), MDL72222 (3tropanyl-3,5-dichloro benzoate), SDZ-205557 hydrochloride (4amino-5-chloro-2-methoxybenzoic acid 2-(diethylamino)ethyl ester hydrochloride), AP5 ((⫾)-2-amino-5-phosphonopentanoic acid), CNQX (6-cyano-7-nitroquino xaline-2,3-dione), bicuculline, picrotoxin and tetrodotoxin (TTX) were obtained from Sigma/RBI. SB269970 hydrochloride ((2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-
L. Chen et al. / Neuroscience 151 (2008) 439 – 451 [2-(4-methyl-1-piperidinyl) ethyl] pyrrolidine hydrochloride), RS102221 hydrochloride (8-[5-(2,4-dimethoxy-5-(4-trifluoromethyl-phenyl-sulfonamido) phenyl-5-oxopentyl)-1,3,8-triazaspiro [4.5] decane-2,4-dionehydro chloride], (2S)-3-[[(1S)-1-(3,4dichlorophenyl)ethyl]amino-2-hydroxylpropyl] (phenylmethyl) phosphinic acid (CGP55845), (RS)-␣-ethyl-4-carboxyphenylglycine (E4CPG) and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6methylamino-pyrimidinium chloride) were obtained from Tocris Cookson (Avonmouth, UK). The data are expressed as means⫾S.E.M. Paired Student’s t-test was used to compare the difference before and after drug application. Unpaired t-test was used to compare two groups of data. Multiple paired comparisons of the single control (5-HT) to each of the antagonist treatments were made by the analysis of variance (ANOVA) with Bonferroni-Dunn post hoc procedure. The level of significance was set at P value⫽0.05.
RESULTS 5-HT depolarizes and excites globus pallidus neurons Under current-clamp recording mode, most of the globus pallidus neurons tested in this study exhibited spontaneous action potentials. The average firing rate and resting membrane potential were 10.0⫾0.6 Hz and ⫺52.9⫾1.2 mV, respectively (n⫽52). Application of 10 M 5-HT to the superfusion medium depolarized the membrane, in a reversible manner, by 5.7⫾0.5 mV (n⫽9), which increased the firing rates of the neurons (Fig. 1A). It had been reported that 5-HT could enhance the release of glutamate from nerve terminals (Lambe and Aghajanian, 2001; Hasuo et al., 2002) or from glia (Meller et al., 2002). To determine whether the 5-HT-induced depolarization was a direct effect on postsynaptic membrane or an indirect effect on a presynaptic site, we first determined the effect of TTX on 5-HT induced depolarization. As shown in Fig. 1B, in the presence of 0.5 M TTX, 5-HT still depolarized globus pallidus neurons (6.4⫾0.8 mV, n⫽11, P⬎0.05 compared with 5-HT alone). To eliminate the possibility that 5-HT-induced depolarization was caused by affecting glutamate and GABA release at a presynaptic site, ionotropic and metabotropic receptor antagonists were used to block glutamatergic and GABAergic synaptic transmission. In the presence of 0.5 M TTX, 20 M CNQX, 50 M AP5 and 300 M E4CPG, 5-HT still induced a comparable membrane depolarization (6.4⫾0.9 mV, n⫽7, P⬎0.05 compared with 5-HT alone). Similar depolarization could still be induced by 5-HT in the presence of 0.5 M TTX, 100 M picrotoxin and 2 M CGP55845 (6.6⫾0.8 mV, n⫽7, P⬎0.05 compared with 5-HT alone). These results suggested that 5-HT exerted a direct effect by acting on the receptors expressed on the membrane of the recorded neurons. Consistent with the current-clamp data, in voltageclamp recordings in which the neurons were clamped at ⫺70 mV, 10 M 5-HT caused an inward current of 73.0⫾8.0 pA (n⫽10, P⬍0.001) and 59.0⫾7.8 pA (n⫽10, P⬍0.001) in the absence and presence of TTX respectively (Fig. 1C).
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5-HT4 and 5-HT7 receptors are involved in 5-HT-induced membrane depolarization It is known that multiple 5-HT receptor subtypes exist in the globus pallidus, including 5-HT1B, 5-HT1D, 5-HT2, 5-HT3, 5-HT4 and 5-HT7 receptors. To determine whether these receptor subtypes mediated the effect of 5-HT, subtypespecific receptor antagonists were employed. Cyanopindolol, the 5-HT1A/1B receptor antagonist, at 2 M had no effect on 5-HT-evoked depolarization (6.5⫾0.7 mV, n⫽4, P⬎0.05 compared with 5-HT alone). Similarly, the selective 5-HT3 receptor antagonist, MDL72222 (10 M), did not block the 5-HT evoked-depolarization (8.0⫾1.5 mV, n⫽3, P⬎0.05 compared with 5-HT alone). 5-HT2C receptor has been reported to depolarize subthalamic nucleus and substantia nigra pars reticulata neurons (Rick et al., 1995; Xiang et al., 2005). We found that the specific 5-HT2C receptor antagonist, RS102221, at 100 nM could not block 5-HT induced depolarization (5.8⫾1.0 mV, n⫽5, P⬎0.05 compared with 5-HT alone). On the other hand, the 5-HT4 receptor antagonist SDZ-205557 at 20 M significantly blocked the depolarization induced by 5-HT (1.2⫾0.6 mV, n⫽11, P⬍0.001 compared with 5-HT alone), indicating that 5-HT4 receptor plays a major role in mediating the depolarization. In addition, 5-HT7 receptor is also involved in 5-HT-induced depolarization. Thus, in the presence of a specific 5-HT7 receptor antagonist, SB269970 (1 M), 5-HT only induced a weak depolarization (1.5⫾0.5 mV, n⫽7, P⬍0.01 compared with 5-HT alone). Furthermore, application of SDZ-205557 together with SB269970 completely blocked 5-HT induced depolarization (n⫽4). These findings suggest that both 5-HT4 and 5-HT7 receptors contribute to the depolarizing effect of 5-HT on globus pallidus neurons. These data are summarized in Fig. 1D. 5-HT opens cationic channels 5-HT-induced membrane depolarization of pallidal neurons was associated with an increase in membrane conductance. This is indicated in Fig. 2A by a decrease in voltage deflections in response to periodic current injections, reflecting a significant decrease in input resistance (79.5⫾2.4% of control, n⫽6, P⬍0.001). To determine the reversal potential of 5-HT-induced conductance, the current–voltage relationship in the absence and presence of 5-HT was determined. The mean value obtained from seven experiments was ⫺28.7⫾7.7 mV, which suggested that 5-HT-induced depolarization resulted from an increase in cationic conductance. A typical example is shown in Fig. 2B. Role of Ih and the involvement of 5-HT7 receptor Ih has been found to be activated by 5-HT in a variety of neurons and in many cases 5-HT4 and 5-HT7 receptor are involved (Cardenas et al., 1999; Chapin and Andrade, 2001; Bickmeyer et al., 2002). As illustrated in Fig. 3A and 3B, bath application of 10 M 5-HT increased both instantaneous and ss Ih currents. In seven neurons held at ⫺100 mV, 5-HT increased the ins to 190.5⫾15.5% (P⬍0.001) and the ss to 139.1⫾10.6% (n⫽7, P⬍0.001) of control.
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Fig. 2. Activation of cationic conductance was involved in the depolarization induced by 5-HT. (A) Depolarization induced by 10 M 5-HT was associated with a decrease in input resistance. Voltage deflections were responses to periodic current injections of 100 pA for 100 ms at 0.5 Hz. (B) Current–voltage relationship revealed that the reversal potential obtained in this cell was ⫺20 mV.
The enhancement on the ins is significantly stronger than the ss (n⫽7, P⬍0.01). In the presence of 50 M ZD7288, a selective and irreversible pharmacological blocker of Ih, 10 M 5-HT did not increase the instantaneous and ss currents (Fig. 3C). 5-HT1A, 5-HT2, 5-HT4 and 5-HT7 receptors have previously been reported to modulate Ih in other brain areas (Cardenas et al., 1999; Bickmeyer et al., 2002; Iwahori et al., 2002; Liu et al., 2003). In the present studies, the selective 5-HT7 receptor antagonist, SB269970, at 1 M could block 5-HT-induced enhancement of Ih (109.0⫾4.9% and 97.0⫾2.9% of control for ins and stead-state current, respectively, n⫽7, P⬍0.01 compared with 5-HT alone, Fig. 4). These data combined together indicate that 5-HT activates both 5-HT7 and 5-HT4 receptors, with the former
action leading to augmentation of Ih currents. On the other hand, 5-HT4 receptor may lead to the activation of another non-selective cation conductance. Effects of 5-HT on inhibitory postsynaptic currents To determine if 5-HT exerts any presynaptic effects on GABAergic inputs, spontaneous inhibitory postsynaptic currents (sIPSCs) were isolated by including 20 M CNQX and 50 M AP5 in the superfusion solution to block ionotropic glutamate receptor-mediated synaptic currents. The remaining currents were sensitive to 10 M bicuculline confirming their GABAergic nature. When 10 M of 5-HT was applied to the superfusion medium, the frequency of the sIPSCs was increased significantly (control: 8.5⫾1.5 Hz; 5-HT: 11.7⫾1.9 Hz, n⫽9, P⬍0.01). This effect was par-
Fig. 1. Effect of 5-HT on globus pallidus neurons. (A) Application of 10 M 5-HT induced a reversible depolarization and increased firing frequency. a, b And c indicate the time from which the traces plotted on a fast time-base were taken. (B) Depolarization and (C) inward current induced by 5-HT were independent of action potential. (D) Summary of the effects of various 5-HT receptor antagonists on the depolarizing action of 5-HT. 5-HT1A/1B (cyanopindolol, n⫽4), 5-HT3 (MDL72222, n⫽3), 5-HT2C (RS102221, n⫽5) antagonists had no effect while 5-HT4 (SDZ-205557, n⫽11) and 5-HT7 (SB269970, n⫽7) significantly reduced the membrane effect of 5-HT, compared with the control of 5-HT (n⫽11). *** P⬍0.001.
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Fig. 3. Enhancement of Ih induced by 5-HT. (A) Application of 5-HT significantly increased the amplitude of both ins and ss currents shown in bottom evoked by a series of hyperpolarizing voltage steps shown in top. (B) Current–voltage curves showing the ins and ss in control and in the presence of 5-HT in the same neuron. (C) Blockade of Ih with ZD7288.
tially reversible when 5-HT was removed. In addition to its effect on the frequency, 5-HT increased the amplitude of sIPSCs in some of the cells tested although there was no significant difference when the data were pooled from all cells (control: 96.9⫾7.1 pA; 5-HT: 130.3⫾38.0 pA, n⫽9, P⬎0.05, Fig. 5). In another set of experiments in which TTX was added to remove action potential-dependent IPSCs, 10 M 5-HT did not affect both the frequency (control: 3.4⫾0.8 Hz; 5-HT: 3.1⫾0.9 Hz, n⫽8, P⬎0.05) and amplitude (control: 83.8⫾7.3 pA; 5-HT: 87.7⫾8.2 pA, n⫽8, P⬎0.05) of miniature inhibitory postsynaptic currents (mIPSCs). These data suggest that at a concentration of 10 M, 5-HT did not have any effect on the presynaptic GABA terminals. The increase in the frequency of the sIPSCs described above was likely to be the result of 5-HT
directly activating the globus pallidus neurons, increasing the firing frequency onto neighboring neurons via the axon collaterals. In contrast to the lack of effect at 10 M, 5-HT at a higher concentration (50 M) decreased the frequency of mIPSCs (control: 3.4⫾0.5 Hz; 5-HT: 1.6⫾0.3 Hz, n⫽11, P⬍0.001, Fig. 6). This inhibitory effect of 5-HT was selective to the frequency but not the amplitude (control: 90.4⫾6.2 pA; 5-HT: 85.1⫾7.9 pA, n⫽11, P⬎0.05) consistent with a presynaptic effect on the probability of transmitter release at the nerve terminals. This presynaptic inhibition was blocked by 5-HT1B receptor antagonist cyanopindolol (control: 3.2⫾0.7 Hz; cyanopindolol⫹5-HT: 3.0⫾0.7 Hz, n⫽8, P⬍0.001 compared with 5-HT alone, Fig. 7), which itself had no effect on mIPSCs. This finding
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Fig. 4. 5-HT7 receptor is involved in the modulation of Ih by 5-HT. In the presence of 5-HT7 receptor antagonist, SB269970, 10 M 5-HT did not induce any enhancement in Ih.
is consistent with morphological studies which demonstrated that 5-HT1B receptors are located on the striatopallidal terminals (Sari et al., 1999). Effects of 5-HT on excitatory postsynaptic currents In addition to its presynaptic effect on GABAergic neurotransmission, 5-HT has also been reported to modulate glutamatergic neurotransmission in some brain areas (Singer et al., 1996; Muramatsu et al., 1998; Funahashi et al., 2004). In the presence of 10 M bicuculline and 0.5 M TTX, we further studied the effects of 5-HT on glutamate receptor–mediated miniature excitatory postsynaptic currents (mEPSCs). These currents were sensitive to 20 M CNQX and 50 M APV, confirming their glutamatergic nature. In the 11 globus pallidus neurons with detectable mEPSCs, 50 M 5-HT significantly increased the frequency of mEPSCs in four cells (control: 1.2⫾0.3 Hz; 5-HT: 3.7⫾1.3 Hz, n⫽4, P⬍0.001). Similar to its presynaptic effects on mIPSCs, 5-HT did not change the amplitude of mEPSCs (control: 23.1⫾7.0 pA; 5-HT: 20.3⫾6.5 pA, n⫽4, P⬎0.05). However, in another 3 of the 11 neurons, 5-HT significantly decreased the frequency of mEPSCs (control: 0.9⫾0.3 Hz; 5-HT: 0.4⫾0.1 Hz, n⫽3, P⬍0.01) without any change in the amplitude (control: 16.7⫾2.4 pA; 5-HT: 16.9⫾3.1 pA, n⫽3, P⬎0.05). In the remaining four neurons, 5-HT had no significant effects on mEPSCs. These data suggest that 5-HT has no consistent effect on glutamate release in globus pallidus.
DISCUSSION The present study demonstrates that 5-HT modulates the excitability of globus pallidus neurons via multiple 5-HT receptor subtypes and mechanisms. Thus, 5-HT binds to 5-HT4 and 5-HT7 receptors on pallidal neurons, activating a non-selective cation conductance and hyperpolarizationactivated conductance respectively. As a result, the pallidal neurons are depolarized and increase their firing frequency. The fact that these actions persist in the presence of TTX and glutamate and GABA receptor antagonists
indicates that they are direct effects on the postsynaptic membranes of pallidal neurons. Our findings are consistent with that reported by Bengtson et al. (2004) that 5-HT induced an inward current in homologous neurons of rat ventral pallidum mediated by 5-HT7 receptor and via modulation of the Ih current. We have further shown in globus pallidus neurons the involvement of postsynaptic 5-HT4 receptors and the presynaptic regulation of inhibitory neurotransmission by 5-HT. Although autoradiographic studies revealed relatively low 5-HT7 receptor densities in rat globus pallidus (Martin-Cora and Pazos, 2004), the present study does suggest that they are expressed and play a functional role in this area. Our data on 5-HT4 receptors are different from morphological studies showing that these receptors are mainly located on the striatopallidal terminals (Mengod et al., 1996; Vilaro et al., 1996; Bonaventure et al., 2000). The use of selective 5-HT agonists would help further clarify the role of these receptor subtypes. At a higher concentration (50 M), 5-HT also activates presynaptic 5-HT1B receptors and reduces the probability of GABA release onto the pallidal neurons. The fact that 5-HT at a low concentration (10 M) increased sIPSCs but not mIPSCs is consistent with the scenario that excitation of pallidal neurons results in action potentialdependent GABA release from their collaterals. These results are in line with morphological studies revealing that dense 5-HT1B receptors were located on axon terminals in the globus pallidus (Sari et al., 1999), and that 5-HT1B receptor mRNA was found in the striatum while 5-HT1B binding sites were found in globus pallidus (Bonaventure et al., 1998). Consistent with our findings, Kita and colleagues (2007) recently reported that 5-HT strongly suppresses GABAergic inhibition in awake monkey, probably through 5-HT1B receptors. However, in the same study, they reported the lack of clear postsynaptic effects of 5-HT on pallidal neurons in awake monkeys. The functions of other 5-HT receptor subtypes in the globus pallidus, including 5-HT1D, 5-HT2 and 5-HT3, are still unknown. Some of the discrepancies between anatomical and electrophysio-
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Fig. 5. Enhancement of sIPSCs by 10 M 5-HT. (A) Typical traces showing that 10 M 5-HT significantly and reversibly increased the frequency and amplitude of sIPSCs recorded from a globus pallidus neuron. (B) The cumulative probability distributions of the inter-event intervals and amplitudes of the sIPSCs from the experiment shown in panel A. Significant differences were found in the distributions of inter-event intervals and amplitudes. (C) Pooled data of the effect of 5-HT on the frequency and amplitude of sIPSCs. ** P⬍0.01, *** P⬍0.001.
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Fig. 6. Presynaptic suppression of GABA release by 50 M 5-HT. (A) Typical traces showing that 50 M 5-HT significantly and reversibly reduced the frequency of mIPSCs in a globus pallidus neuron. (B) The cumulative probability distributions of the inter-event intervals and amplitudes of the mIPSCs shown in panel A. (C) Pooled data of the effect of 5-HT on the mIPSCs frequency and amplitude. *** P⬍0.001, ns: not significant.
logical studies may be explained by the developmental profiles of different 5-HT receptors at different ages.
The present data indicated that 5-HT increased Ih in globus pallidus, as observed in non-cholinergic neurons in
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Fig. 7. 5-HT1B receptor is involved in the presynaptic inhibition induced by 50 M 5-HT. (A) Typical traces of mIPSCs before and after 50 M 5-HT application in the presence of 2 M 5-HT1B receptor antagonist, cyanopindolol. (B) The cumulative probability distributions of the inter-event intervals and amplitudes of the mIPSCs shown in A. (C) Mean data showing that cyanopindolol blocked the effect of 5-HT completely; ns: not significant.
the pallidal complex (Bengtson et al., 2004) and other brain areas (Bickmeyer et al., 2002; Cardenas et al., 1999; Chapin and Andrade, 2001; Iwahori et al., 2002). Four subunits of hyperpolarization-activated and cyclic-nucleotide gated nonselective cation channels (HCN1-4), which are employed in the synthesis of Ih, have been cloned. Recently, immunohistochemical studies revealed that all of the four HCN subunits are distributed in globus pallidus, with HCN2 being the dominant subunit (Notomi and Shigemoto, 2004). By using whole-cell patch clamp recordings, Chan and colleagues (2004) reported the function of HCN1
and HCN2 channels in the globus pallidus. Similar enhancement on the fast component of Ih induced by 5-HT has been reported in dorsal root ganglion neurons (Cardenas et al., 1999). In agreement with the reports in dorsal root ganglia and anterodorsal nucleus of thalamus (Cardenas et al., 1999; Chapin and Andrade, 2001), 5-HT7 receptors have also been shown to modulate Ih in the globus pallidus. Being a critical component controlling all other basal ganglia nuclei, the activity of globus pallidus has the potential to gate the output of this motor circuit (Mink and
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Thach, 1991; Chesselet and Delfs, 1996; Bolam et al., 2000). Thus, decreased pallidal activity is believed to underlie parkinsonian symptoms. Furthermore, increased synchronized firing of these neurons in Parkinson’s disease may be the origin of resting tremor (Nini et al., 1995; Bergman et al., 1998; Magnin et al., 2000; Raz et al., 2000). There is also evidence that lesioning of the globus pallidus corrects Huntington’s disease deficits (Ayalon et al., 2004). Previous studies suggest that there are at least several major factors controlling the firing rate and patterns of pallidal neurons, including (1) intrinsic membrane properties (Stanford, 2003), (2) reciprocal connections with subthalamic nucleus (Plenz and Kitai, 1999; Hanson and Jaeger, 2002) and striatum (Nakanishi et al., 1985; Bevan et al., 2002), and (3) their modulation by cortical inputs (Magill et al., 2000, 2001; Bevan et al., 2002; Goldberg et al., 2003). The present study provides evidence that, in addition to above factors, the neuromodulator 5-HT, originating from dorsal raphe nucleus, could significantly modulate the excitability of globus pallidus neurons. Changes in 5-HT innervation in the basal ganglia had been reported in both Parkinson’s and Huntington’s disease (De Deurwaerdere and Chesselet, 2000; Yohrling et al., 2002; Balcioglu et al., 2003). How does 5-HT release affect the rate and firing pattern of pallidal neurons in vivo, and therefore movement behavior in normal and pathological states? The results from the present study do not provide a complete answer because in the brain slice, the connections between globus pallidus and subthalamic nucleus, striatum, cortex and the raphe nucleus are severed. Nevertheless, in principle, the steady firing of raphe neurons found in awake subjects would mainly exert a tonic excitatory drive on globus pallidus neurons that would facilitate movements via a reduction in the output of the indirect pathway. Furthermore, computer modeling from realistic neurons of the globus pallidus–subthalamic nucleus network suggests that the level of depolarization over the long term may be an important determinant of the firing patterns of pallidal and subthalamic neurons (Bevan et al., 2002; Terman et al., 2002). Thus, the level of activity of the dorsal raphe nucleus is likely to have a direct effect on the rate as well as firing patterns of pallidal neurons. In this context, the role of 5-HT1B receptors is of particular interest. They are located pre-synaptically on striato-pallidal terminals. When activated by a relatively high concentration of 5-HT, the striato-pallidal connection is weakened, which favors the shift of the globus pallidus–subthalamic nucleus network from oscillatory, parkinsonian mode to more regular, normal mode (Terman et al., 2002). This particular action of 5-HT on 5-HT1B receptors is opposite to that of enkephalin and dynorphin which selectively weaken the collateral connections among pallidal neurons (Stanford and Cooper, 1999; Ogura and Kita, 2000). Interestingly, behavioral studies indicated that microinjection of the 5-HT1B receptor agonist, CP-93129, into the globus pallidus produced an increase in net contraversive rotations which supported that 5-HT1B receptors in the globus pallidus
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provide relief of akinesia in the reserpine-treated rat model of Parkinson’s disease (Chadha et al., 2000). In agreement with this, recent in vivo extracellular recordings revealed that activation of 5-HT1B receptor increased the firing rate of globus pallidus neurons (Querejeta et al., 2005).
CONCLUSION In conclusion, the diverse receptor mechanisms that 5-HT exerts on pallidal neurons implicate the importance of the raphe– globus pallidus connection. In view of the extensive innervation patterns of globus pallidus terminals on other basal ganglia nuclei, we speculate that the action of 5-HT may have a profound effect on the operation of the entire basal ganglia network. Furthermore, our results support the notion that increases of 5-HT tone, which mainly excite pallidal neurons directly and counteract the inhibition from the striatum selectively, will exert an anti-parkinsonian effect. Acknowledgments—This work was supported by the Research Grants Council of Hong Kong (CUHK 4175/02 M to W. H. Yung) and the grants from National Natural Science Foundation of China (30670664 to L. Chen).
REFERENCES Albin RL, Young AB, Penny JB (1989) The functional anatomy of basal ganglia disorders. Trends Neurosci 12:366 –375. Alexander GE, Crutcher MD (1990) Functional architecture of basal ganglia circuits: neural substrates of parallel processing. Trends Neurosci 13:266 –271. Ayalon L, Doron R, Weiner I, Joel D (2004) Amelioration of behavioral deficits in a rat model of Huntington’s disease by an excitotoxic lesion of the globus pallidus. Exp Neurol 186:46 –58. Balcioglu A, Zhang K, Tarazi FI (2003) Dopamine depletion abolishes apomorphine- and amphetamine-induced increases in extracellular serotonin levels in the striatum of conscious rats: a microdialysis study. Neuroscience 119:1045–1053. Bengtson CP, Lee DJ, Osborne PB (2004) Opposing electrophysiological actions of 5-HT on noncholinergic and cholinergic neurons in the rat ventral pallidum in vitro. J Neurophysiol 92:433– 443. Bergman H, Feingold A, Nini A, Raz A, Slovin H, Abeles M, Vaadia E (1998) Physiological aspects of information processing in the basal ganglia of normal and parkinsonian primates. Trends Neurosci 21:32–38. Bevan MD, Magill PJ, Terman D, Bolam JP, Wilson CJ (2002) Move to the rhythm: oscillations in the subthalamic nucleus-external globus pallidus network. Trends Neurosci 25:525–531. Bickmeyer U, Heine M, Manzke T, Richter DW (2002) Differential modulation of Ih by 5-HT receptors in mouse CA1 hippocampal neurons. Eur J Neurosci 16:209 –218. Bolam JP, Hanley JJ, Booth PAC, Bevan MD (2000) Synaptic organization of the basal ganglia. J Anat 196:527–542. Bonaventure P, Voorn P, Luyten WH, Jurzak M, Schotte A, Leysen JE (1998) Detailed mapping of serotonin 5-HT 1B and 5-HT 1D receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function. Neuroscience 82:469 – 484. Bonaventure P, Hall H, Gommeren W, Cras P, Langlois X, Jurzak M, Leysen JE (2000) Mapping of serotonin 5-HT (4) receptor mRNA and ligand binding sites in the post-mortem human brain. Synapse 36:35– 46.
450
L. Chen et al. / Neuroscience 151 (2008) 439 – 451
Cardenas CG, Del Mar LP, Vysokanov AV, Arnold PB, Cardenas LM, Surmeier DJ, Scroggs RS (1999) Serotonergic modulation of hyperpolarization-activated current in acutely isolated rat dorsal root ganglion neurons. J Physiol 518:507–523. Chadha A, Sur C, Atack J, Duty S (2000) The 5HT(1B) receptor agonist, CP-93129, inhibits [(3)H]-GABA release from rat globus pallidus slices and reverses akinesia following intrapallidal injection in the reserpine-treated rat. Br J Pharmacol 130:1927–1932. Chan CS, Shigemoto R, Mercer JN, Surmeier DJ (2004) HCN2 and HCN1 channels govern the regularity of autonomous pacemaking and synaptic resetting in globus pallidus neurons. J Neurosci 24:9921–9932. Chapin EM, Andrade R (2001) A 5-HT (7) receptor-mediated depolarization in the anterodorsal thalamus. II. Involvement of the hyperpolarization-activated current. Int J Pharmacol Exp Ther 297:403– 409. Charara A, Parent A (1994) Brainstem dopaminergic, cholinergic and serotoninergic afferents to the pallidum in the squirrel monkey. Brain Res 640:155–170. Chesselet MF, Delfs JM (1996) Basal ganglia and movement disorders: an update. Trends Neurosci 19:417– 422. Clemett DA, Punhani T, Duxon MS, Blackburn TP, Fone KC (2000) Immunohistochemical localisation of the 5-HT2C receptor protein in the rat CNS. Neuropharmacology 39:123–132. Compan V, Daszuta A, Salin P, Sebben M, Bockaert J, Dumuis A (1996) Lesion study of the distribution of serotonin 5-HT4 receptors in rat basal ganglia and hippocampus . Eur J Neurosci 8:2591–2598. De Deurwaerdere P, Chesselet MF (2000) Nigrostriatal lesions alter oral dyskinesia and c-Fos expression induced by the serotonin agonist 1-(m-chlorophenyl)piperazine in adult rats. J Neurosci 20:5170 –5178. DeVito JL, Anderson ME, Walsh KE (1980) A horseradish peroxidase study of afferent connections of the globus pallidus in Macaca mulatta. Exp Brain Res 38:65–73. Filion M, Tremblay L (1991) Abnormal spontaneous activity of globus pallidus neurons in monkeys with MPTP-induced parkinsonism. Brain Res 547:142–151. Funahashi M, Mitoh Y, Matsuo R (2004) Activation of presynaptic 5-HT3 receptors facilitates glutamatergic synaptic inputs to area postrema neurons in rat brain slices. Methods Find Exp Clin Pharmacol 26:615– 622. Goldberg JA, Kats SS, Jaeger D (2003) Globus pallidus discharge is coincident with striatal activity during global slow wave activity in the rat. J Neurosci 23:10058 –10063. Halliday GM, Blumbergs PC, Cotton RG, Blessing WW, Geffen LB (1990) Loss of brainstem serotonin- and substance P-containing neurons in Parkinson’s disease. Brain Res 510:104 –107. Hanson JE, Jaeger D (2002) Short-term plasticity shapes the response to simulated normal and parkinsonian input patterns in the globus pallidus. J Neurosci 22:5164 –5172. Hasuo H, Matsuoka T, Akasu T (2002) Activation of presynaptic 5-hydroxytryptamine 2A receptors facilitated excitatory synaptic transmission via protein kinase C in the dorsolateral septal nucleus. J Neurosci 22:7509 –7517. Hoyer D, Schoeffter P, Waeber C, Palacios JM (1990) Serotonin 5-HT1D receptors. Ann N Y Acad Sci 600:168 –191. Iwahori Y, Ikegaya Y, Matsuki N (2002) Hyperpolarization-activated current I(h) in nucleus of solitary tract neurons: regional difference in serotonergic modulation. Jpn J Pharmacol 88:459 – 462. Kita H, Chiken S, Tachibana Y, Nambu A (2007) Serotonin modulates pallidal neuronal activity in the awake monkey. J Neurosci 27:75– 83. Lambe EK, Aghajanian GK (2001) The role of Kv 1.2-containing potassium channels in serotonin-induced glutamate release from thalamocortical terminals in rat frontal cortex. J Neurosci 21:9955–9963.
Lavoie B, Parent A (1990) Immunohistochemical study of the serotoninergic innervation of the basal ganglia in the squirrel monkey. J Comp Neurol 299:1–16. Li QH, Nakadate K, Tanaka-Nakadate S, Nakatsuka D, Cui Y, Watanabe Y (2004) Unique expression patterns of 5-HT2A and 5-HT2C receptors in the rat brain during postnatal development: Western blot and immunohistochemical analyses. J Comp Neurol 469:128 –140. Liu Z, Bunney EB, Appel SB, Brodie MS (2003) Serotonin reduces the hyperpolarization-activated current (Ih) in ventral tegmental area dopamine neurons: involvement of 5-HT2 receptors and protein kinase C. J Neurophysiol 90:3201–3212. Magill PJ, Bolam JP, Bevan MD (2000) Relationship of activity in the subthalamic nucleus-globus pallidus network to cortical electroencephalogram. J Neurosci 20:820 – 833. Magill PJ, Bolam JP, Bevan MD (2001) Dopamine regulates the impact of the cerebral cortex on the subthalamic nucleus-globus pallidus network. Neuroscience 106:313–330. Magnin M, Morel A, Jeanmonod D (2000) Single-unit analysis of the pallidum, thalamus and subthalamic nucleus in parkinsonian patients. Neuroscience 96:549 –564. Martin-Cora FJ, Pazos A (2004) Autoradiographic distribution of 5-HT7 receptors in the human brain using [3H]mesulergine: comparison to other mammalian species. Br J Pharmacol 41:92–104. McQuade R, Sharp T (1997) Functional mapping of dorsal and median raphe 5-hydroxytryptamine pathways in forebrain of the rat using microdialysis. J Neurochem 69:791–796. Meller R, Harrison PJ, Elliott JM, Sharp T (2002) In vitro evidence that 5-hydroxytryptamine increases efflux of glial glutamate via 5-HT (2A) receptor activation. J Neurosci Res 67:399 – 405. Mengod G, Vilaro MT, Raurich A, Lopez-Gimenez JF, Cortes R, Palacios JM (1996) 5-HT receptors in mammalian brain: receptor autoradiography and in situ hybridization studies of new ligands and newly identified receptors. Histochem J 28:747–758. Mink JW, Thach WT (1991) Basal ganglia motor control. I. Nonexclusive relation of pallidal discharge to five movement modes. J Neurophysiol 65:273–300. Morales M, Battenberg E, Bloom FE (1998) Distribution of neurons expressing immunoreactivity for the 5HT-3 receptor subtype in the rat brain and spinal cord. J Comp Neurol 385:385– 401. Murai T, Müller U, Werheid K, Sorger D, Reuter M, Becker T, von Cramon DY, Barthel H (2001) In vivo evidence for differential association of striatal dopamine and midbrain serotonin systems with neuropsychiatric symptoms in Parkinson’s disease. J Neuropsychiatry Clin Neurosci 13:222–228. Muramatsu M, Lapiz MD, Tanaka E, Grenhoff J (1998) Serotonin inhibits synaptic glutamate currents in rat nucleus accumbens neurons via presynaptic 5-HT1B receptors. Eur J Neurosci 10:2371–2379. Nakanishi H, Hori N, Kastuda N (1985) Neostriatal evoked inhibition and effects of dopamine on globus pallidal neurons in rat slice preparations. Brain Res 358:282–286. Nini A, Feingold A, Slovin H, Bergman H (1995) Neurons in the globus pallidus do not show correlated activity in the normal monkey, but phase-locked oscillations appear in the MPTP model of parkinsonism. J Neurophysiol 74:1800 –1805. Notomi T, Shigemoto R (2004) Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain. J Comp Neurol 471:241–276. Ogura M, Kita H (2000) Dynorphin exerts both postsynaptic and presynaptic effects in the globus pallidus of the rat. J Neurophysiol 83:3366 –3376. Plenz D, Kitai ST (1999) A basal ganglia pacemaker formed by the subthalamic nucleus and external globus pallidus. Nature 400:677– 682. Querejeta E, Oviedo-Chavez A, Araujo-Alvarez JM, QuinonesCardenas AR, Delgado A (2005) In vivo effects of local activation
L. Chen et al. / Neuroscience 151 (2008) 439 – 451 and blockade of 5-HT1B receptors on globus pallidus neuronal spiking. Brain Res 1043:186 –194. Raz A, Vaadia E, Bergman H (2000) Firing patterns and correlations of spontaneous discharge of pallidal neurons in the normal and tremulous MPTP velvet model of parkinsonism. J Neurosci 20:8558 – 8571. Riad M, Garcia S, Watkins KC, Jodoin N, Doucet E, Langlois X, el Mestikawy S, Hamon M, Descarries L (2000) Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain. J Comp Neurol 417:181–194. Rick CE, Stanford IM, Lacey MG (1995) Excitation of rat substantia nigra pars reticulata neurons by 5-hydroxytryptamine in vitro: evidence for a direct action mediated by 5-hydroxytryptamine2C receptors. Neuroscience 69:903–913. Sari Y, Miquel MC, Brisorgueil MJ, Ruiz G, Doucet E, Hamon M, Verge D (1999) Cellular and subcellular localization of 5-hydroxytryptamine 1B receptors in the rat central nervous system: immunocytochemical, autoradiographic and lesion studies. Neuroscience 88:899 –915. Sijbesma H, Schipper J, Cornelissen JC, de Kloet ER (1990) Species differences in the distribution of central 5-HT1 binding sites: a comparative autoradiographic study between rat and guinea pig. Brain Res 555:295–304. Sijbesma H, Schipper J, de Kloet ER (1991) Eltoprazine, a drug which reduces aggressive behaviour, binds selectively to 5-HT1 receptor sites in the rat brain: an autoradiographic study. Eur J Pharmacol 177:55– 66.
451
Singer JH, Bellingham MC, Berger AJ (1996) Presynaptic inhibition of glutamatergic synaptic transmission to rat motoneurons by serotonin. J Neurophysiol 76:799 – 807. Stanford IM (2003) Independent neuronal oscillators of the rat globus pallidus. J Neurophysiol 89:1713–1717. Stanford IM, Cooper AJ (1999) Presynaptic and ␦ opioid receptor modulation of GABA-A IPSCs in the rat globus pallidus in vitro. J Neurosci 19:4796 – 4803. Terman D, Rubin JE, Yew AC, Wilson CJ (2002) Activity patterns in a model for the subthalamopallidal network of the basal ganglia. J Neurosci 22:2963–2976. Van Praag HM (1994) 5-HT-related, anxiety- and/or aggression-driven depression. Int Clin Psychopharmacol 9 (Suppl 1):5– 6. Vilaro MT, Cortes R, Gerald C, Branchek TA, Palacios JM, Mengod G (1996) Localization of 5-HT4 receptor mRNA in rat brain by in situ hybridization histochemistry. Mol Brain Res 43:356 –360. Waeber C, Sebben M, Nieoullon A, Bockaert J, Dumuis A (1994) Regional distribution and ontogeny of 5-HT 4 binding sites in rodent brain. Neuropharmacology 33:527–541. Wichmann T, DeLong MR (1996) Functional and pathophysiological models of the basal ganglia. Curr Opin Neurobiol 6:751–758. Xiang Z, Wang L, Kitai ST (2005) Modulation of spontaneous firing in rat subthalamic neurons by 5-HT receptor subtypes. J Neurophysiol 93:1145–1157. Yohrling GJ IV, Jiang GC, DeJohn MM, Robertson DJ, Vrana KE, Cha JH (2002) Inhibition of tryptophan hydroxylase activity and decreased 5-HT1A receptor binding in a mouse model of Huntington’s disease. J Neurochem 82:1416 –1423.
(Accepted 30 November 2007) (Available online 7 November 2007)
5-HT EXCITES GLOBUS PALLIDUS NEURONS BY MULTIPLE RECEPTOR MECHANISMS L. CHEN,a,b K. K. L. YUNG,c Y. S. CHANd AND W. H. YUNGa*
conclusion, we found that 5-HT could modulate the excitability of globus pallidus neurons by both pre- and post-synaptic mechanisms. In view of the extensive innervation by globus pallidus neurons on other basal ganglia nuclei, this action of 5-HT originated from the raphe may have a profound effect on the operation of the entire basal ganglia network. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved.
a
Department of Physiology, The Chinese University of Hong Kong, Shatin, Hong Kong, China
b
Department of Physiology, Qingdao University, Qingdao, China
c
Department of Biology, Hong Kong Baptist University, Hong Kong, China
Key words: 5-HT, 5-HT receptors, globus pallidus.
d
Department of Physiology, The University of Hong Kong, Hong Kong, China
The globus pallidus in rodent is homologous to the external globus pallidus in primates. More and more evidence suggests that the globus pallidus plays a critical role in the basal ganglia circuit under normal and pathological conditions. In Parkinson’s disease and its animal models, the absence of normal dopaminergic innervation leads to presumed hypoactivity and decreased output of the globus pallidus, contributing to akinesia and hypokinetic symptoms (Albin et al., 1989; Alexander and Crutcher, 1990; Filion and Tremblay, 1991; Chesselet and Delfs, 1996; Wichmann and DeLong, 1996). More recently, it was found that changes of firing pattern of pallidal neurons are closely associated with resting tremor (Bergman et al., 1998; Magnin et al., 2000; Raz et al., 2000). This is contributed by the extensive innervation of pallidal neurons on all other basal ganglia nuclei (Bolam et al., 2000). Not surprisingly, there is an increasing interest in those factors that control not only the rate but also the pattern of firing of globus pallidus neurons in normal and diseased states. Up to now, at least three factors have been considered important in determining the firing pattern of pallidal neurons. First, from the study on organotypic cultures Plenz and Kitai (1999) proposed that the reciprocally connected globus pallidus– subthalamic nucleus network could act as a central pattern generator of the basal ganglia. On the other hand, the role of descending cortical glutamatergic inputs to the generation of rhythmic firings in the globus pallidus–subthalamic nucleus network has been substantiated by in vivo extracellular recordings (Magill et al., 2000, 2001). Finally, Stanford (2003) reported that, in the absence of synaptic inputs, each globus pallidus neuron could act as an independent oscillator, which is contributed by the intrinsic membrane conductances. We believe that, in addition, 5-HT innervation from the raphe nucleus is an important determinant of the firing activities of pallidal neurons. Anatomical studies indicated that the globus pallidus receives serotonergic innervation arising from raphe nuclei, mainly the dorsal raphe nucleus (DeVito et al., 1980; Lavoie and Parent, 1990; Charara and Parent, 1994). In addition, stimulation of the dorsal raphe nucleus evoked an increase of 5-HT in globus pallidus
Abstract—Anatomical and neurochemical studies indicated that the globus pallidus receives serotonergic innervation from raphe nuclei but the membrane effects of 5-HT on globus pallidus neurons are not entirely clear. We address this question by applying whole-cell patch-clamp recordings on globus pallidus neurons in immature rat brain slices. Under current-clamp recording, 5-HT depolarized globus pallidus neurons and increased their firing rate, an action blocked by both 5-HT4 and 5-HT7 receptor antagonists and attributable to an increase in cation conductance(s). Further experiments indicated that 5-HT enhanced the hyperpolarization-activated inward conductance which is blocked by 5-HT7 receptor antagonist. To determine if 5-HT exerts any presynaptic effects on GABAergic and glutamatergic inputs, the actions of 5-HT on synaptic currents were studied. At 10 M, 5-HT increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but had no effect on both the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). However, 5-HT at a higher concentration (50 M) decreased the frequency but not the amplitude of the mIPSCs, indicating an inhibition of GABA release from the presynaptic terminals. This effect was sensitive to 5-HT1B receptor antagonist. In addition to the presynaptic effects on GABAergic neurotransmission, 5-HT at 50 M had no consistent effects on glutamatergic neurotransmission, significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in 4 of 11 neurons and decreased the frequency of mEPSCs in 3 of 11 neurons. In *Corresponding author. Tel: ⫹852-26096880; fax: ⫹852-26035022. E-mail address: [email protected] (W. H. Yung). Abbreviations: ACSF, artificial cerebrospinal fluid; AP5, (⫾)-2-amino5-phosphonopentanoic acid; CGP55845, (2S)-3-[[(1S)-1-(3,4dichlorophenyl)ethyl]amino-2-hydroxylpropyl] (phenylmethyl) phosphinic acid; CNQX, 6-cyano-7-nitroquino xaline-2,3-dione; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid; E4CPG, (RS)-␣-ethyl-4-carboxyphenylglycine; Hepes, N-2-hydroxyl piperazine-N=-2-ethane sulphonic acid; Ih, hyperpolarization-activated current; ins, instantaneous current; MDL72222, 4-[3-([1,1-dimethylethyl] amino)-2-hydroxypropul]-1H-indole-2-carbonitrile; mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; RS102221 hydrochloride, 8-[5-(2,4-dimethoxy-5-(4trifluoromethyl-phenyl-sulfonamido) phenyl-5-oxopentyl)-1,3,8triazaspiro [4.5] decane-2,4-dionehydro chloride]; SB269970, (2R)-1[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl; sIPSC, spontaneous inhibitory postsynaptic current; ss, steady-state; TTX, tetrodotoxin; ZD7288, 4-ethylphenylamino-1,2-dimethyl-6-methylamino-pyrimidinium chloride.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.11.003
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measured by microdialysis (McQuade and Sharp, 1997). Based on in situ hybridization, receptor autoradiography and immunohistochemistry, the subtypes 5-HT1B, 5-HT1D, 5-HT2, 5-HT3, 5-HT4 and 5-HT7 have been identified in globus pallidus (Hoyer et al., 1990; Sijbesma et al., 1990, 1991; Waeber et al., 1994; Compan et al., 1996; Vilaro et al., 1996; Morales et al., 1998; Sari et al., 1999; Bonaventure et al., 2000; Clemett et al., 2000; Riad et al., 2000; Li et al., 2004; Martin-Cora and Pazos, 2004). Different lines of evidence also indicate that changes in 5-HT concentration in the basal ganglia of parkinsonian subjects may contribute to the characteristic motor as well as mental impairments (Halliday et al., 1990; Van Praag, 1994; Murai et al., 2001). However, to date, the cellular effects of 5-HT on globus pallidus neurons are not well-known. Here we report that 5-HT could depolarize globus pallidus neurons and modulate synaptic transmission via post- and presynaptic mechanisms respectively, suggesting that the raphe exerts a strong command on the excitability of pallidal neurons.
EXPERIMENTAL PROCEDURES Ethical approval The experimental procedures adopted in the present work complied with the NIH guidelines for animal care and were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong. Effort was made to minimize the number of animals used and their suffering.
In vitro slice preparation Sprague–Dawley rats aged 13–14 days were used for the preparation of acute brain slices. The animals were killed by decapitation. The brains were immediately removed and placed in icecold artificial cerebrospinal fluid (ACSF) of the following composition (in mM): NaCl 125, KCl 4.0, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, glucose 11 and NaHCO3 26, which was continuously bubbled with 95% O2 and 5% CO2. Thin hemi-coronal slices (250 m) containing the globus pallidus were sectioned using a vibrating microtome (Campden Instruments, Loughborough, UK). After equilibration in ACSF, the slices were transferred to a small volume chamber mounted on an upright microscope (Zeiss Axioskop, Gottingen, Germany), and superfused with ACSF at a rate of 1.5–2.0 ml/min maintained at a temperature of 34⫾1 °C. Neuronal soma and proximal dendrites of neurons were directly visualized by a combination of differential interference contrast optics and contrast-enhanced video microscopy. Typical globus pallidus projection neurons, based on their medium size, somatodendritic morphology, as well as the clear voltage- and timedependent inward rectifier, were selected for recording. The neurons sampled were mainly from mid to caudal levels of the globus pallidus.
In order to observe the GABAA receptor–mediated synaptic current, the internal solution containing (in mM): KCl 140, Hepes 10, EGTA 1, MgCl2 2, Na2ATP 2 and Tris GTP 0.4 was used. The inclusion of 140 mM of KCl in the recording pipettes reversed the polarity of the currents from outward to inward. Monitoring through a television connected to the camera, a pipette was placed on the soma of a pallidal neuron and conventional whole-cell recording was made. Normally no series resistance compensation was applied but the cell was rejected if the series resistance increased significantly (⬎20%) during recording. To study and measure the effect of 5-HT on the membrane potential, a neuron was hyperpolarized to close to ⫺60 mV by current injection to prevent firing. To minimize the effect of receptor desensitization, in control group (5-HT alone), 5-HT was perfused only once to one neuron in each slice. The experimental groups (different 5-HT receptor antagonists) were performed in other pallidal neurons. 5-HT receptor antagonist was applied first, then 5-HT receptor antagonist together with 5-HT were co-perfused to the same neuron. Similarly, one type of 5-HT receptor antagonist was applied only once in each neuron (slice). At least 5 min of steady basal recording were collected from each cell before drug application. The duration of 5-HT exposures was 3 min. Usually the 5-HT induced pre- or postsynaptic effects occurred 1–1.5 min after 5-HT application, and reversed in about 8 min during the washout period. For 5-HT-induced postsynaptic depolarization or inward current, the peak value of change was considered as drug effect. For 5-HT-induced presynaptic effects, the synaptic currents occurring in a period of 120 s before drug application were analyzed as control. The maximal change of frequency within a 120 s period following drug application was considered as drug effect. Pre-incubation of different 5-HT receptor antagonists was at least 6 min before the co-application of 5-HT and 5-HT receptor antagonist. Hyperpolarization-activated current (Ih) was evoked under voltage-clamp recording from a holding potential of ⫺50 mV to ⫺130 mV (in 10 mV increments). Two components of voltage- and time-dependent Ih were observed, an instantaneous (ins) current and a slow-rising current, which reached a steady-state (ss) at the end of a long voltage-step. The ins were measured at the point just before the rise of the slow current while the ss were measured near the end of the voltage command. The voltage and current signals were filtered at 3 kHz and were taped using a DAT recorder (Sony, Tokyo, Japan) modified for recording AC and DC signals at a sampling rate of 32 kHz.
Analysis of synaptic currents Computer files containing information of synaptic currents were analyzed by a program developed in our laboratory. Detection of a synaptic current was based mainly on threshold detection. Once a synaptic current was detected, information on the time of occurrence, peak amplitude and kinetics was generated automatically. Statistical comparison of two cumulative probabilities was done by the Kolmogorov-Smirnov test. All detected events with the computed parameters were allowed visual inspection before acceptance. Validity of the analysis was done by cross-checking some of the results using a commercially available program MiniAnalysis (version 5, Synaptosoft, Decatur, GA, USA).
Whole-cell current- and voltage-clamp recordings Conventional tight-seal (⬎2G⍀) whole-cell patch-clamp recordings from globus pallidus neurons were obtained using a patchclamp amplifier (LM/PCA, List Medical, Darmstadt, Germany). Whole-cell pipettes (pulled by P-97, Sutter Instrument, Novato, CA, USA) typically had a resistance of 3– 4.5 M⍀. For current-clamp recording and some voltage-clamp experiments, the pipettes were filled with an internal solution of the following composition (in mM): K-gluconate 130, KCl 10, Hepes 10, EGTA 1, MgCl2 2, Na2ATP 2, Tris GTP 0.4 and the pH was adjusted to 7.25–7.30 with 1 M KOH.
Drugs and statistics 5-HT, S(⫺)-cyanopindolol hemifumarate (4-[3-([1,1-dimethylethyl] amino)-2-hydroxypropul]-1H-indole-2-carbonitrile), MDL72222 (3tropanyl-3,5-dichloro benzoate), SDZ-205557 hydrochloride (4amino-5-chloro-2-methoxybenzoic acid 2-(diethylamino)ethyl ester hydrochloride), AP5 ((⫾)-2-amino-5-phosphonopentanoic acid), CNQX (6-cyano-7-nitroquino xaline-2,3-dione), bicuculline, picrotoxin and tetrodotoxin (TTX) were obtained from Sigma/RBI. SB269970 hydrochloride ((2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-
L. Chen et al. / Neuroscience 151 (2008) 439 – 451 [2-(4-methyl-1-piperidinyl) ethyl] pyrrolidine hydrochloride), RS102221 hydrochloride (8-[5-(2,4-dimethoxy-5-(4-trifluoromethyl-phenyl-sulfonamido) phenyl-5-oxopentyl)-1,3,8-triazaspiro [4.5] decane-2,4-dionehydro chloride], (2S)-3-[[(1S)-1-(3,4dichlorophenyl)ethyl]amino-2-hydroxylpropyl] (phenylmethyl) phosphinic acid (CGP55845), (RS)-␣-ethyl-4-carboxyphenylglycine (E4CPG) and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6methylamino-pyrimidinium chloride) were obtained from Tocris Cookson (Avonmouth, UK). The data are expressed as means⫾S.E.M. Paired Student’s t-test was used to compare the difference before and after drug application. Unpaired t-test was used to compare two groups of data. Multiple paired comparisons of the single control (5-HT) to each of the antagonist treatments were made by the analysis of variance (ANOVA) with Bonferroni-Dunn post hoc procedure. The level of significance was set at P value⫽0.05.
RESULTS 5-HT depolarizes and excites globus pallidus neurons Under current-clamp recording mode, most of the globus pallidus neurons tested in this study exhibited spontaneous action potentials. The average firing rate and resting membrane potential were 10.0⫾0.6 Hz and ⫺52.9⫾1.2 mV, respectively (n⫽52). Application of 10 M 5-HT to the superfusion medium depolarized the membrane, in a reversible manner, by 5.7⫾0.5 mV (n⫽9), which increased the firing rates of the neurons (Fig. 1A). It had been reported that 5-HT could enhance the release of glutamate from nerve terminals (Lambe and Aghajanian, 2001; Hasuo et al., 2002) or from glia (Meller et al., 2002). To determine whether the 5-HT-induced depolarization was a direct effect on postsynaptic membrane or an indirect effect on a presynaptic site, we first determined the effect of TTX on 5-HT induced depolarization. As shown in Fig. 1B, in the presence of 0.5 M TTX, 5-HT still depolarized globus pallidus neurons (6.4⫾0.8 mV, n⫽11, P⬎0.05 compared with 5-HT alone). To eliminate the possibility that 5-HT-induced depolarization was caused by affecting glutamate and GABA release at a presynaptic site, ionotropic and metabotropic receptor antagonists were used to block glutamatergic and GABAergic synaptic transmission. In the presence of 0.5 M TTX, 20 M CNQX, 50 M AP5 and 300 M E4CPG, 5-HT still induced a comparable membrane depolarization (6.4⫾0.9 mV, n⫽7, P⬎0.05 compared with 5-HT alone). Similar depolarization could still be induced by 5-HT in the presence of 0.5 M TTX, 100 M picrotoxin and 2 M CGP55845 (6.6⫾0.8 mV, n⫽7, P⬎0.05 compared with 5-HT alone). These results suggested that 5-HT exerted a direct effect by acting on the receptors expressed on the membrane of the recorded neurons. Consistent with the current-clamp data, in voltageclamp recordings in which the neurons were clamped at ⫺70 mV, 10 M 5-HT caused an inward current of 73.0⫾8.0 pA (n⫽10, P⬍0.001) and 59.0⫾7.8 pA (n⫽10, P⬍0.001) in the absence and presence of TTX respectively (Fig. 1C).
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5-HT4 and 5-HT7 receptors are involved in 5-HT-induced membrane depolarization It is known that multiple 5-HT receptor subtypes exist in the globus pallidus, including 5-HT1B, 5-HT1D, 5-HT2, 5-HT3, 5-HT4 and 5-HT7 receptors. To determine whether these receptor subtypes mediated the effect of 5-HT, subtypespecific receptor antagonists were employed. Cyanopindolol, the 5-HT1A/1B receptor antagonist, at 2 M had no effect on 5-HT-evoked depolarization (6.5⫾0.7 mV, n⫽4, P⬎0.05 compared with 5-HT alone). Similarly, the selective 5-HT3 receptor antagonist, MDL72222 (10 M), did not block the 5-HT evoked-depolarization (8.0⫾1.5 mV, n⫽3, P⬎0.05 compared with 5-HT alone). 5-HT2C receptor has been reported to depolarize subthalamic nucleus and substantia nigra pars reticulata neurons (Rick et al., 1995; Xiang et al., 2005). We found that the specific 5-HT2C receptor antagonist, RS102221, at 100 nM could not block 5-HT induced depolarization (5.8⫾1.0 mV, n⫽5, P⬎0.05 compared with 5-HT alone). On the other hand, the 5-HT4 receptor antagonist SDZ-205557 at 20 M significantly blocked the depolarization induced by 5-HT (1.2⫾0.6 mV, n⫽11, P⬍0.001 compared with 5-HT alone), indicating that 5-HT4 receptor plays a major role in mediating the depolarization. In addition, 5-HT7 receptor is also involved in 5-HT-induced depolarization. Thus, in the presence of a specific 5-HT7 receptor antagonist, SB269970 (1 M), 5-HT only induced a weak depolarization (1.5⫾0.5 mV, n⫽7, P⬍0.01 compared with 5-HT alone). Furthermore, application of SDZ-205557 together with SB269970 completely blocked 5-HT induced depolarization (n⫽4). These findings suggest that both 5-HT4 and 5-HT7 receptors contribute to the depolarizing effect of 5-HT on globus pallidus neurons. These data are summarized in Fig. 1D. 5-HT opens cationic channels 5-HT-induced membrane depolarization of pallidal neurons was associated with an increase in membrane conductance. This is indicated in Fig. 2A by a decrease in voltage deflections in response to periodic current injections, reflecting a significant decrease in input resistance (79.5⫾2.4% of control, n⫽6, P⬍0.001). To determine the reversal potential of 5-HT-induced conductance, the current–voltage relationship in the absence and presence of 5-HT was determined. The mean value obtained from seven experiments was ⫺28.7⫾7.7 mV, which suggested that 5-HT-induced depolarization resulted from an increase in cationic conductance. A typical example is shown in Fig. 2B. Role of Ih and the involvement of 5-HT7 receptor Ih has been found to be activated by 5-HT in a variety of neurons and in many cases 5-HT4 and 5-HT7 receptor are involved (Cardenas et al., 1999; Chapin and Andrade, 2001; Bickmeyer et al., 2002). As illustrated in Fig. 3A and 3B, bath application of 10 M 5-HT increased both instantaneous and ss Ih currents. In seven neurons held at ⫺100 mV, 5-HT increased the ins to 190.5⫾15.5% (P⬍0.001) and the ss to 139.1⫾10.6% (n⫽7, P⬍0.001) of control.
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Fig. 2. Activation of cationic conductance was involved in the depolarization induced by 5-HT. (A) Depolarization induced by 10 M 5-HT was associated with a decrease in input resistance. Voltage deflections were responses to periodic current injections of 100 pA for 100 ms at 0.5 Hz. (B) Current–voltage relationship revealed that the reversal potential obtained in this cell was ⫺20 mV.
The enhancement on the ins is significantly stronger than the ss (n⫽7, P⬍0.01). In the presence of 50 M ZD7288, a selective and irreversible pharmacological blocker of Ih, 10 M 5-HT did not increase the instantaneous and ss currents (Fig. 3C). 5-HT1A, 5-HT2, 5-HT4 and 5-HT7 receptors have previously been reported to modulate Ih in other brain areas (Cardenas et al., 1999; Bickmeyer et al., 2002; Iwahori et al., 2002; Liu et al., 2003). In the present studies, the selective 5-HT7 receptor antagonist, SB269970, at 1 M could block 5-HT-induced enhancement of Ih (109.0⫾4.9% and 97.0⫾2.9% of control for ins and stead-state current, respectively, n⫽7, P⬍0.01 compared with 5-HT alone, Fig. 4). These data combined together indicate that 5-HT activates both 5-HT7 and 5-HT4 receptors, with the former
action leading to augmentation of Ih currents. On the other hand, 5-HT4 receptor may lead to the activation of another non-selective cation conductance. Effects of 5-HT on inhibitory postsynaptic currents To determine if 5-HT exerts any presynaptic effects on GABAergic inputs, spontaneous inhibitory postsynaptic currents (sIPSCs) were isolated by including 20 M CNQX and 50 M AP5 in the superfusion solution to block ionotropic glutamate receptor-mediated synaptic currents. The remaining currents were sensitive to 10 M bicuculline confirming their GABAergic nature. When 10 M of 5-HT was applied to the superfusion medium, the frequency of the sIPSCs was increased significantly (control: 8.5⫾1.5 Hz; 5-HT: 11.7⫾1.9 Hz, n⫽9, P⬍0.01). This effect was par-
Fig. 1. Effect of 5-HT on globus pallidus neurons. (A) Application of 10 M 5-HT induced a reversible depolarization and increased firing frequency. a, b And c indicate the time from which the traces plotted on a fast time-base were taken. (B) Depolarization and (C) inward current induced by 5-HT were independent of action potential. (D) Summary of the effects of various 5-HT receptor antagonists on the depolarizing action of 5-HT. 5-HT1A/1B (cyanopindolol, n⫽4), 5-HT3 (MDL72222, n⫽3), 5-HT2C (RS102221, n⫽5) antagonists had no effect while 5-HT4 (SDZ-205557, n⫽11) and 5-HT7 (SB269970, n⫽7) significantly reduced the membrane effect of 5-HT, compared with the control of 5-HT (n⫽11). *** P⬍0.001.
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Fig. 3. Enhancement of Ih induced by 5-HT. (A) Application of 5-HT significantly increased the amplitude of both ins and ss currents shown in bottom evoked by a series of hyperpolarizing voltage steps shown in top. (B) Current–voltage curves showing the ins and ss in control and in the presence of 5-HT in the same neuron. (C) Blockade of Ih with ZD7288.
tially reversible when 5-HT was removed. In addition to its effect on the frequency, 5-HT increased the amplitude of sIPSCs in some of the cells tested although there was no significant difference when the data were pooled from all cells (control: 96.9⫾7.1 pA; 5-HT: 130.3⫾38.0 pA, n⫽9, P⬎0.05, Fig. 5). In another set of experiments in which TTX was added to remove action potential-dependent IPSCs, 10 M 5-HT did not affect both the frequency (control: 3.4⫾0.8 Hz; 5-HT: 3.1⫾0.9 Hz, n⫽8, P⬎0.05) and amplitude (control: 83.8⫾7.3 pA; 5-HT: 87.7⫾8.2 pA, n⫽8, P⬎0.05) of miniature inhibitory postsynaptic currents (mIPSCs). These data suggest that at a concentration of 10 M, 5-HT did not have any effect on the presynaptic GABA terminals. The increase in the frequency of the sIPSCs described above was likely to be the result of 5-HT
directly activating the globus pallidus neurons, increasing the firing frequency onto neighboring neurons via the axon collaterals. In contrast to the lack of effect at 10 M, 5-HT at a higher concentration (50 M) decreased the frequency of mIPSCs (control: 3.4⫾0.5 Hz; 5-HT: 1.6⫾0.3 Hz, n⫽11, P⬍0.001, Fig. 6). This inhibitory effect of 5-HT was selective to the frequency but not the amplitude (control: 90.4⫾6.2 pA; 5-HT: 85.1⫾7.9 pA, n⫽11, P⬎0.05) consistent with a presynaptic effect on the probability of transmitter release at the nerve terminals. This presynaptic inhibition was blocked by 5-HT1B receptor antagonist cyanopindolol (control: 3.2⫾0.7 Hz; cyanopindolol⫹5-HT: 3.0⫾0.7 Hz, n⫽8, P⬍0.001 compared with 5-HT alone, Fig. 7), which itself had no effect on mIPSCs. This finding
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Fig. 4. 5-HT7 receptor is involved in the modulation of Ih by 5-HT. In the presence of 5-HT7 receptor antagonist, SB269970, 10 M 5-HT did not induce any enhancement in Ih.
is consistent with morphological studies which demonstrated that 5-HT1B receptors are located on the striatopallidal terminals (Sari et al., 1999). Effects of 5-HT on excitatory postsynaptic currents In addition to its presynaptic effect on GABAergic neurotransmission, 5-HT has also been reported to modulate glutamatergic neurotransmission in some brain areas (Singer et al., 1996; Muramatsu et al., 1998; Funahashi et al., 2004). In the presence of 10 M bicuculline and 0.5 M TTX, we further studied the effects of 5-HT on glutamate receptor–mediated miniature excitatory postsynaptic currents (mEPSCs). These currents were sensitive to 20 M CNQX and 50 M APV, confirming their glutamatergic nature. In the 11 globus pallidus neurons with detectable mEPSCs, 50 M 5-HT significantly increased the frequency of mEPSCs in four cells (control: 1.2⫾0.3 Hz; 5-HT: 3.7⫾1.3 Hz, n⫽4, P⬍0.001). Similar to its presynaptic effects on mIPSCs, 5-HT did not change the amplitude of mEPSCs (control: 23.1⫾7.0 pA; 5-HT: 20.3⫾6.5 pA, n⫽4, P⬎0.05). However, in another 3 of the 11 neurons, 5-HT significantly decreased the frequency of mEPSCs (control: 0.9⫾0.3 Hz; 5-HT: 0.4⫾0.1 Hz, n⫽3, P⬍0.01) without any change in the amplitude (control: 16.7⫾2.4 pA; 5-HT: 16.9⫾3.1 pA, n⫽3, P⬎0.05). In the remaining four neurons, 5-HT had no significant effects on mEPSCs. These data suggest that 5-HT has no consistent effect on glutamate release in globus pallidus.
DISCUSSION The present study demonstrates that 5-HT modulates the excitability of globus pallidus neurons via multiple 5-HT receptor subtypes and mechanisms. Thus, 5-HT binds to 5-HT4 and 5-HT7 receptors on pallidal neurons, activating a non-selective cation conductance and hyperpolarizationactivated conductance respectively. As a result, the pallidal neurons are depolarized and increase their firing frequency. The fact that these actions persist in the presence of TTX and glutamate and GABA receptor antagonists
indicates that they are direct effects on the postsynaptic membranes of pallidal neurons. Our findings are consistent with that reported by Bengtson et al. (2004) that 5-HT induced an inward current in homologous neurons of rat ventral pallidum mediated by 5-HT7 receptor and via modulation of the Ih current. We have further shown in globus pallidus neurons the involvement of postsynaptic 5-HT4 receptors and the presynaptic regulation of inhibitory neurotransmission by 5-HT. Although autoradiographic studies revealed relatively low 5-HT7 receptor densities in rat globus pallidus (Martin-Cora and Pazos, 2004), the present study does suggest that they are expressed and play a functional role in this area. Our data on 5-HT4 receptors are different from morphological studies showing that these receptors are mainly located on the striatopallidal terminals (Mengod et al., 1996; Vilaro et al., 1996; Bonaventure et al., 2000). The use of selective 5-HT agonists would help further clarify the role of these receptor subtypes. At a higher concentration (50 M), 5-HT also activates presynaptic 5-HT1B receptors and reduces the probability of GABA release onto the pallidal neurons. The fact that 5-HT at a low concentration (10 M) increased sIPSCs but not mIPSCs is consistent with the scenario that excitation of pallidal neurons results in action potentialdependent GABA release from their collaterals. These results are in line with morphological studies revealing that dense 5-HT1B receptors were located on axon terminals in the globus pallidus (Sari et al., 1999), and that 5-HT1B receptor mRNA was found in the striatum while 5-HT1B binding sites were found in globus pallidus (Bonaventure et al., 1998). Consistent with our findings, Kita and colleagues (2007) recently reported that 5-HT strongly suppresses GABAergic inhibition in awake monkey, probably through 5-HT1B receptors. However, in the same study, they reported the lack of clear postsynaptic effects of 5-HT on pallidal neurons in awake monkeys. The functions of other 5-HT receptor subtypes in the globus pallidus, including 5-HT1D, 5-HT2 and 5-HT3, are still unknown. Some of the discrepancies between anatomical and electrophysio-
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Fig. 5. Enhancement of sIPSCs by 10 M 5-HT. (A) Typical traces showing that 10 M 5-HT significantly and reversibly increased the frequency and amplitude of sIPSCs recorded from a globus pallidus neuron. (B) The cumulative probability distributions of the inter-event intervals and amplitudes of the sIPSCs from the experiment shown in panel A. Significant differences were found in the distributions of inter-event intervals and amplitudes. (C) Pooled data of the effect of 5-HT on the frequency and amplitude of sIPSCs. ** P⬍0.01, *** P⬍0.001.
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Fig. 6. Presynaptic suppression of GABA release by 50 M 5-HT. (A) Typical traces showing that 50 M 5-HT significantly and reversibly reduced the frequency of mIPSCs in a globus pallidus neuron. (B) The cumulative probability distributions of the inter-event intervals and amplitudes of the mIPSCs shown in panel A. (C) Pooled data of the effect of 5-HT on the mIPSCs frequency and amplitude. *** P⬍0.001, ns: not significant.
logical studies may be explained by the developmental profiles of different 5-HT receptors at different ages.
The present data indicated that 5-HT increased Ih in globus pallidus, as observed in non-cholinergic neurons in
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Fig. 7. 5-HT1B receptor is involved in the presynaptic inhibition induced by 50 M 5-HT. (A) Typical traces of mIPSCs before and after 50 M 5-HT application in the presence of 2 M 5-HT1B receptor antagonist, cyanopindolol. (B) The cumulative probability distributions of the inter-event intervals and amplitudes of the mIPSCs shown in A. (C) Mean data showing that cyanopindolol blocked the effect of 5-HT completely; ns: not significant.
the pallidal complex (Bengtson et al., 2004) and other brain areas (Bickmeyer et al., 2002; Cardenas et al., 1999; Chapin and Andrade, 2001; Iwahori et al., 2002). Four subunits of hyperpolarization-activated and cyclic-nucleotide gated nonselective cation channels (HCN1-4), which are employed in the synthesis of Ih, have been cloned. Recently, immunohistochemical studies revealed that all of the four HCN subunits are distributed in globus pallidus, with HCN2 being the dominant subunit (Notomi and Shigemoto, 2004). By using whole-cell patch clamp recordings, Chan and colleagues (2004) reported the function of HCN1
and HCN2 channels in the globus pallidus. Similar enhancement on the fast component of Ih induced by 5-HT has been reported in dorsal root ganglion neurons (Cardenas et al., 1999). In agreement with the reports in dorsal root ganglia and anterodorsal nucleus of thalamus (Cardenas et al., 1999; Chapin and Andrade, 2001), 5-HT7 receptors have also been shown to modulate Ih in the globus pallidus. Being a critical component controlling all other basal ganglia nuclei, the activity of globus pallidus has the potential to gate the output of this motor circuit (Mink and
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Thach, 1991; Chesselet and Delfs, 1996; Bolam et al., 2000). Thus, decreased pallidal activity is believed to underlie parkinsonian symptoms. Furthermore, increased synchronized firing of these neurons in Parkinson’s disease may be the origin of resting tremor (Nini et al., 1995; Bergman et al., 1998; Magnin et al., 2000; Raz et al., 2000). There is also evidence that lesioning of the globus pallidus corrects Huntington’s disease deficits (Ayalon et al., 2004). Previous studies suggest that there are at least several major factors controlling the firing rate and patterns of pallidal neurons, including (1) intrinsic membrane properties (Stanford, 2003), (2) reciprocal connections with subthalamic nucleus (Plenz and Kitai, 1999; Hanson and Jaeger, 2002) and striatum (Nakanishi et al., 1985; Bevan et al., 2002), and (3) their modulation by cortical inputs (Magill et al., 2000, 2001; Bevan et al., 2002; Goldberg et al., 2003). The present study provides evidence that, in addition to above factors, the neuromodulator 5-HT, originating from dorsal raphe nucleus, could significantly modulate the excitability of globus pallidus neurons. Changes in 5-HT innervation in the basal ganglia had been reported in both Parkinson’s and Huntington’s disease (De Deurwaerdere and Chesselet, 2000; Yohrling et al., 2002; Balcioglu et al., 2003). How does 5-HT release affect the rate and firing pattern of pallidal neurons in vivo, and therefore movement behavior in normal and pathological states? The results from the present study do not provide a complete answer because in the brain slice, the connections between globus pallidus and subthalamic nucleus, striatum, cortex and the raphe nucleus are severed. Nevertheless, in principle, the steady firing of raphe neurons found in awake subjects would mainly exert a tonic excitatory drive on globus pallidus neurons that would facilitate movements via a reduction in the output of the indirect pathway. Furthermore, computer modeling from realistic neurons of the globus pallidus–subthalamic nucleus network suggests that the level of depolarization over the long term may be an important determinant of the firing patterns of pallidal and subthalamic neurons (Bevan et al., 2002; Terman et al., 2002). Thus, the level of activity of the dorsal raphe nucleus is likely to have a direct effect on the rate as well as firing patterns of pallidal neurons. In this context, the role of 5-HT1B receptors is of particular interest. They are located pre-synaptically on striato-pallidal terminals. When activated by a relatively high concentration of 5-HT, the striato-pallidal connection is weakened, which favors the shift of the globus pallidus–subthalamic nucleus network from oscillatory, parkinsonian mode to more regular, normal mode (Terman et al., 2002). This particular action of 5-HT on 5-HT1B receptors is opposite to that of enkephalin and dynorphin which selectively weaken the collateral connections among pallidal neurons (Stanford and Cooper, 1999; Ogura and Kita, 2000). Interestingly, behavioral studies indicated that microinjection of the 5-HT1B receptor agonist, CP-93129, into the globus pallidus produced an increase in net contraversive rotations which supported that 5-HT1B receptors in the globus pallidus
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provide relief of akinesia in the reserpine-treated rat model of Parkinson’s disease (Chadha et al., 2000). In agreement with this, recent in vivo extracellular recordings revealed that activation of 5-HT1B receptor increased the firing rate of globus pallidus neurons (Querejeta et al., 2005).
CONCLUSION In conclusion, the diverse receptor mechanisms that 5-HT exerts on pallidal neurons implicate the importance of the raphe– globus pallidus connection. In view of the extensive innervation patterns of globus pallidus terminals on other basal ganglia nuclei, we speculate that the action of 5-HT may have a profound effect on the operation of the entire basal ganglia network. Furthermore, our results support the notion that increases of 5-HT tone, which mainly excite pallidal neurons directly and counteract the inhibition from the striatum selectively, will exert an anti-parkinsonian effect. Acknowledgments—This work was supported by the Research Grants Council of Hong Kong (CUHK 4175/02 M to W. H. Yung) and the grants from National Natural Science Foundation of China (30670664 to L. Chen).
REFERENCES Albin RL, Young AB, Penny JB (1989) The functional anatomy of basal ganglia disorders. Trends Neurosci 12:366 –375. Alexander GE, Crutcher MD (1990) Functional architecture of basal ganglia circuits: neural substrates of parallel processing. Trends Neurosci 13:266 –271. Ayalon L, Doron R, Weiner I, Joel D (2004) Amelioration of behavioral deficits in a rat model of Huntington’s disease by an excitotoxic lesion of the globus pallidus. Exp Neurol 186:46 –58. Balcioglu A, Zhang K, Tarazi FI (2003) Dopamine depletion abolishes apomorphine- and amphetamine-induced increases in extracellular serotonin levels in the striatum of conscious rats: a microdialysis study. Neuroscience 119:1045–1053. Bengtson CP, Lee DJ, Osborne PB (2004) Opposing electrophysiological actions of 5-HT on noncholinergic and cholinergic neurons in the rat ventral pallidum in vitro. J Neurophysiol 92:433– 443. Bergman H, Feingold A, Nini A, Raz A, Slovin H, Abeles M, Vaadia E (1998) Physiological aspects of information processing in the basal ganglia of normal and parkinsonian primates. Trends Neurosci 21:32–38. Bevan MD, Magill PJ, Terman D, Bolam JP, Wilson CJ (2002) Move to the rhythm: oscillations in the subthalamic nucleus-external globus pallidus network. Trends Neurosci 25:525–531. Bickmeyer U, Heine M, Manzke T, Richter DW (2002) Differential modulation of Ih by 5-HT receptors in mouse CA1 hippocampal neurons. Eur J Neurosci 16:209 –218. Bolam JP, Hanley JJ, Booth PAC, Bevan MD (2000) Synaptic organization of the basal ganglia. J Anat 196:527–542. Bonaventure P, Voorn P, Luyten WH, Jurzak M, Schotte A, Leysen JE (1998) Detailed mapping of serotonin 5-HT 1B and 5-HT 1D receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function. Neuroscience 82:469 – 484. Bonaventure P, Hall H, Gommeren W, Cras P, Langlois X, Jurzak M, Leysen JE (2000) Mapping of serotonin 5-HT (4) receptor mRNA and ligand binding sites in the post-mortem human brain. Synapse 36:35– 46.
450
L. Chen et al. / Neuroscience 151 (2008) 439 – 451
Cardenas CG, Del Mar LP, Vysokanov AV, Arnold PB, Cardenas LM, Surmeier DJ, Scroggs RS (1999) Serotonergic modulation of hyperpolarization-activated current in acutely isolated rat dorsal root ganglion neurons. J Physiol 518:507–523. Chadha A, Sur C, Atack J, Duty S (2000) The 5HT(1B) receptor agonist, CP-93129, inhibits [(3)H]-GABA release from rat globus pallidus slices and reverses akinesia following intrapallidal injection in the reserpine-treated rat. Br J Pharmacol 130:1927–1932. Chan CS, Shigemoto R, Mercer JN, Surmeier DJ (2004) HCN2 and HCN1 channels govern the regularity of autonomous pacemaking and synaptic resetting in globus pallidus neurons. J Neurosci 24:9921–9932. Chapin EM, Andrade R (2001) A 5-HT (7) receptor-mediated depolarization in the anterodorsal thalamus. II. Involvement of the hyperpolarization-activated current. Int J Pharmacol Exp Ther 297:403– 409. Charara A, Parent A (1994) Brainstem dopaminergic, cholinergic and serotoninergic afferents to the pallidum in the squirrel monkey. Brain Res 640:155–170. Chesselet MF, Delfs JM (1996) Basal ganglia and movement disorders: an update. Trends Neurosci 19:417– 422. Clemett DA, Punhani T, Duxon MS, Blackburn TP, Fone KC (2000) Immunohistochemical localisation of the 5-HT2C receptor protein in the rat CNS. Neuropharmacology 39:123–132. Compan V, Daszuta A, Salin P, Sebben M, Bockaert J, Dumuis A (1996) Lesion study of the distribution of serotonin 5-HT4 receptors in rat basal ganglia and hippocampus . Eur J Neurosci 8:2591–2598. De Deurwaerdere P, Chesselet MF (2000) Nigrostriatal lesions alter oral dyskinesia and c-Fos expression induced by the serotonin agonist 1-(m-chlorophenyl)piperazine in adult rats. J Neurosci 20:5170 –5178. DeVito JL, Anderson ME, Walsh KE (1980) A horseradish peroxidase study of afferent connections of the globus pallidus in Macaca mulatta. Exp Brain Res 38:65–73. Filion M, Tremblay L (1991) Abnormal spontaneous activity of globus pallidus neurons in monkeys with MPTP-induced parkinsonism. Brain Res 547:142–151. Funahashi M, Mitoh Y, Matsuo R (2004) Activation of presynaptic 5-HT3 receptors facilitates glutamatergic synaptic inputs to area postrema neurons in rat brain slices. Methods Find Exp Clin Pharmacol 26:615– 622. Goldberg JA, Kats SS, Jaeger D (2003) Globus pallidus discharge is coincident with striatal activity during global slow wave activity in the rat. J Neurosci 23:10058 –10063. Halliday GM, Blumbergs PC, Cotton RG, Blessing WW, Geffen LB (1990) Loss of brainstem serotonin- and substance P-containing neurons in Parkinson’s disease. Brain Res 510:104 –107. Hanson JE, Jaeger D (2002) Short-term plasticity shapes the response to simulated normal and parkinsonian input patterns in the globus pallidus. J Neurosci 22:5164 –5172. Hasuo H, Matsuoka T, Akasu T (2002) Activation of presynaptic 5-hydroxytryptamine 2A receptors facilitated excitatory synaptic transmission via protein kinase C in the dorsolateral septal nucleus. J Neurosci 22:7509 –7517. Hoyer D, Schoeffter P, Waeber C, Palacios JM (1990) Serotonin 5-HT1D receptors. Ann N Y Acad Sci 600:168 –191. Iwahori Y, Ikegaya Y, Matsuki N (2002) Hyperpolarization-activated current I(h) in nucleus of solitary tract neurons: regional difference in serotonergic modulation. Jpn J Pharmacol 88:459 – 462. Kita H, Chiken S, Tachibana Y, Nambu A (2007) Serotonin modulates pallidal neuronal activity in the awake monkey. J Neurosci 27:75– 83. Lambe EK, Aghajanian GK (2001) The role of Kv 1.2-containing potassium channels in serotonin-induced glutamate release from thalamocortical terminals in rat frontal cortex. J Neurosci 21:9955–9963.
Lavoie B, Parent A (1990) Immunohistochemical study of the serotoninergic innervation of the basal ganglia in the squirrel monkey. J Comp Neurol 299:1–16. Li QH, Nakadate K, Tanaka-Nakadate S, Nakatsuka D, Cui Y, Watanabe Y (2004) Unique expression patterns of 5-HT2A and 5-HT2C receptors in the rat brain during postnatal development: Western blot and immunohistochemical analyses. J Comp Neurol 469:128 –140. Liu Z, Bunney EB, Appel SB, Brodie MS (2003) Serotonin reduces the hyperpolarization-activated current (Ih) in ventral tegmental area dopamine neurons: involvement of 5-HT2 receptors and protein kinase C. J Neurophysiol 90:3201–3212. Magill PJ, Bolam JP, Bevan MD (2000) Relationship of activity in the subthalamic nucleus-globus pallidus network to cortical electroencephalogram. J Neurosci 20:820 – 833. Magill PJ, Bolam JP, Bevan MD (2001) Dopamine regulates the impact of the cerebral cortex on the subthalamic nucleus-globus pallidus network. Neuroscience 106:313–330. Magnin M, Morel A, Jeanmonod D (2000) Single-unit analysis of the pallidum, thalamus and subthalamic nucleus in parkinsonian patients. Neuroscience 96:549 –564. Martin-Cora FJ, Pazos A (2004) Autoradiographic distribution of 5-HT7 receptors in the human brain using [3H]mesulergine: comparison to other mammalian species. Br J Pharmacol 41:92–104. McQuade R, Sharp T (1997) Functional mapping of dorsal and median raphe 5-hydroxytryptamine pathways in forebrain of the rat using microdialysis. J Neurochem 69:791–796. Meller R, Harrison PJ, Elliott JM, Sharp T (2002) In vitro evidence that 5-hydroxytryptamine increases efflux of glial glutamate via 5-HT (2A) receptor activation. J Neurosci Res 67:399 – 405. Mengod G, Vilaro MT, Raurich A, Lopez-Gimenez JF, Cortes R, Palacios JM (1996) 5-HT receptors in mammalian brain: receptor autoradiography and in situ hybridization studies of new ligands and newly identified receptors. Histochem J 28:747–758. Mink JW, Thach WT (1991) Basal ganglia motor control. I. Nonexclusive relation of pallidal discharge to five movement modes. J Neurophysiol 65:273–300. Morales M, Battenberg E, Bloom FE (1998) Distribution of neurons expressing immunoreactivity for the 5HT-3 receptor subtype in the rat brain and spinal cord. J Comp Neurol 385:385– 401. Murai T, Müller U, Werheid K, Sorger D, Reuter M, Becker T, von Cramon DY, Barthel H (2001) In vivo evidence for differential association of striatal dopamine and midbrain serotonin systems with neuropsychiatric symptoms in Parkinson’s disease. J Neuropsychiatry Clin Neurosci 13:222–228. Muramatsu M, Lapiz MD, Tanaka E, Grenhoff J (1998) Serotonin inhibits synaptic glutamate currents in rat nucleus accumbens neurons via presynaptic 5-HT1B receptors. Eur J Neurosci 10:2371–2379. Nakanishi H, Hori N, Kastuda N (1985) Neostriatal evoked inhibition and effects of dopamine on globus pallidal neurons in rat slice preparations. Brain Res 358:282–286. Nini A, Feingold A, Slovin H, Bergman H (1995) Neurons in the globus pallidus do not show correlated activity in the normal monkey, but phase-locked oscillations appear in the MPTP model of parkinsonism. J Neurophysiol 74:1800 –1805. Notomi T, Shigemoto R (2004) Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain. J Comp Neurol 471:241–276. Ogura M, Kita H (2000) Dynorphin exerts both postsynaptic and presynaptic effects in the globus pallidus of the rat. J Neurophysiol 83:3366 –3376. Plenz D, Kitai ST (1999) A basal ganglia pacemaker formed by the subthalamic nucleus and external globus pallidus. Nature 400:677– 682. Querejeta E, Oviedo-Chavez A, Araujo-Alvarez JM, QuinonesCardenas AR, Delgado A (2005) In vivo effects of local activation
L. Chen et al. / Neuroscience 151 (2008) 439 – 451 and blockade of 5-HT1B receptors on globus pallidus neuronal spiking. Brain Res 1043:186 –194. Raz A, Vaadia E, Bergman H (2000) Firing patterns and correlations of spontaneous discharge of pallidal neurons in the normal and tremulous MPTP velvet model of parkinsonism. J Neurosci 20:8558 – 8571. Riad M, Garcia S, Watkins KC, Jodoin N, Doucet E, Langlois X, el Mestikawy S, Hamon M, Descarries L (2000) Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain. J Comp Neurol 417:181–194. Rick CE, Stanford IM, Lacey MG (1995) Excitation of rat substantia nigra pars reticulata neurons by 5-hydroxytryptamine in vitro: evidence for a direct action mediated by 5-hydroxytryptamine2C receptors. Neuroscience 69:903–913. Sari Y, Miquel MC, Brisorgueil MJ, Ruiz G, Doucet E, Hamon M, Verge D (1999) Cellular and subcellular localization of 5-hydroxytryptamine 1B receptors in the rat central nervous system: immunocytochemical, autoradiographic and lesion studies. Neuroscience 88:899 –915. Sijbesma H, Schipper J, Cornelissen JC, de Kloet ER (1990) Species differences in the distribution of central 5-HT1 binding sites: a comparative autoradiographic study between rat and guinea pig. Brain Res 555:295–304. Sijbesma H, Schipper J, de Kloet ER (1991) Eltoprazine, a drug which reduces aggressive behaviour, binds selectively to 5-HT1 receptor sites in the rat brain: an autoradiographic study. Eur J Pharmacol 177:55– 66.
451
Singer JH, Bellingham MC, Berger AJ (1996) Presynaptic inhibition of glutamatergic synaptic transmission to rat motoneurons by serotonin. J Neurophysiol 76:799 – 807. Stanford IM (2003) Independent neuronal oscillators of the rat globus pallidus. J Neurophysiol 89:1713–1717. Stanford IM, Cooper AJ (1999) Presynaptic and ␦ opioid receptor modulation of GABA-A IPSCs in the rat globus pallidus in vitro. J Neurosci 19:4796 – 4803. Terman D, Rubin JE, Yew AC, Wilson CJ (2002) Activity patterns in a model for the subthalamopallidal network of the basal ganglia. J Neurosci 22:2963–2976. Van Praag HM (1994) 5-HT-related, anxiety- and/or aggression-driven depression. Int Clin Psychopharmacol 9 (Suppl 1):5– 6. Vilaro MT, Cortes R, Gerald C, Branchek TA, Palacios JM, Mengod G (1996) Localization of 5-HT4 receptor mRNA in rat brain by in situ hybridization histochemistry. Mol Brain Res 43:356 –360. Waeber C, Sebben M, Nieoullon A, Bockaert J, Dumuis A (1994) Regional distribution and ontogeny of 5-HT 4 binding sites in rodent brain. Neuropharmacology 33:527–541. Wichmann T, DeLong MR (1996) Functional and pathophysiological models of the basal ganglia. Curr Opin Neurobiol 6:751–758. Xiang Z, Wang L, Kitai ST (2005) Modulation of spontaneous firing in rat subthalamic neurons by 5-HT receptor subtypes. J Neurophysiol 93:1145–1157. Yohrling GJ IV, Jiang GC, DeJohn MM, Robertson DJ, Vrana KE, Cha JH (2002) Inhibition of tryptophan hydroxylase activity and decreased 5-HT1A receptor binding in a mouse model of Huntington’s disease. J Neurochem 82:1416 –1423.
(Accepted 30 November 2007) (Available online 7 November 2007)