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1Department of Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, Brisbane; 2Pain Management Research Institute, University of Sydney at Royal North Shore Hospital, St Leonards NSW 2065, Sydney; and 3Prince of Wales Medical Research Institute and the University of New South Wales, Sydney, Australia
Submitted 4 June 2003; accepted in final form 6 February 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; Kalivas and Nakamura 1999; Swanson 2000). A majority of ventral pallidal neurons contain GABA (Gritti et al. 1993) and project in circuits associated with the limbic striatum, which includes the nucleus accumbens (Groenewegen et al. 1993; Joel and Weiner 2000a,b; Maurice et al. 1997; Zahm and Heimer 1990). Also present in the rat are Ch4 cholinergic neurons that are part of the magnocellular forebrain complex (Mesulam et al. 1983), which extends through a contiguous volume of basal forebrain that also encompasses the medial septum, nucleus of the diagonal band of Broca, and substantia innominata. Ch4 cholinergic neurons project to cerebral cortex and amygdala (Carlsen et al. 1985; Ingham et al. 1985; McKinney et al. 1983) and act in many cognitive functions including arousal and memory processing. This group is also homologous to cholinergic neurons that degenerate in humans suffering from Alzheimer's disease (Detari et al. 1999; Lucas-Meunier et al. 2003). It has long been recognized that imbalances in circuits encompassing the dorsal pallidum (globus pallidus) can cause motor disorders such as akinesia and dyskinesia (Bevan et al. 2002). Similar imbalances in the limbic striatopallidal circuitry, which incorporates the nucleus accumbens and ventral pallidum, are now being considered as a potential cause of attentional, motivational, and emotional dysfunction in disorders such as schizophrenia, depression, and drug addiction (Heimer et al. 1997; Joel and Weiner 2000b; Kalivas and Nakamura 1999).
The ventral pallidum is highly enriched in terminals that contain immunoreactivity for the serotonin transporter (Sur et al. 1996) and originate from midbrain 5-HT neurons in the dorsal raphe nucleus (Gasbarri et al. 1999; Jones and Cuello 1989; Vertes 1991). It is likely these terminals are functional, given that microdialysis has detected extracellular 5-HT in the ventral pallidum of anesthetized and awake rats, which is increased in a dose-dependent fashion by cocaine self-administration (Napier and Potter 1989; Sizemore et al. 2000). Several 5-HT receptor subtypes have been localized to the pallidum by autoradiography (Appel et al. 1990; To et al. 1995; Vilaro et al. 1996; Waeber and Moskowitz 1995), in situ hybridization (To et al. 1995; Ullmer et al. 1996; Wright et al. 1995), and immunohistochemistry (Neumaier et al. 2001; Oliver et al. 2000; Sari et al. 1999). However, postsynaptic somatodendritic 5-HT receptors have not been specifically localized to either the cholinergic or noncholinergic cell groups in the ventral pallidum, and the findings of many of these reports are ambiguous or controversial because of problems of low detection sensitivity, or the lack of specificity of the radioligands used (see for example Bonaventure et al. 2002).
In a recent in vitro electrophysiological study we recorded from neurons in the ventral pallidum in rat brain slices, filled cells with biocytin, and identified cholinergic and noncholinergic neurons by immunostaining for choline acetyltransferase (ChAT). Analysis of the current–voltage relationships showed that cholinergic neurons had a larger conductance and exhibited fast (i.e., anomalous) inward rectification caused by a potassium current, which enabled them to be distinguished from putative GABA (noncholinergic) neurons, which had a smaller conductance and exhibited marked time-dependent inward rectification caused by an h-current (Bengtson and Osborne 2000). The present study builds on this work by assessing the presence of functional postsynaptic 5-HT receptors on cholinergic and noncholinergic neurons identified using these immunohistochemical and electrophysiological criteria. It was hypothesized that these receptors could be present on both cell groups because 5-HT terminals form close appositions on both types neurons in the basal forebrain (Gasbarri et al. 1999; Smiley et al. 1999). A previous in vivo electrophysiological study has shown 5-HT receptor agonists can alter the firing rate of neurons in the ventral pallidum, but because the drugs were administered systemically, it could not be determined whether this was attributable to direct agonist effects on neurons within the nucleus (Heidenreich and Napier 2000). We report here that 5-HT can directly induce functionally distinct postsynaptic electrophysiological responses in the 2 major cell groups in the ventral pallidum.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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All procedures involving animals were performed according to guidelines specified by the Australian National Health and Medical Research Council and approved by Animal Ethics Committees at the University of Queensland and the University of New South Wales. Transverse brain slices (200–250 µm thick) containing the ventral pallidum were prepared from 6- to 18-day-old Wistar rats that had been anesthetized by halothane inhalation and decapitated. The slices were submerged in ice-cold artificial cerebrospinal fluid (ACSF, containing in mM: NaCl, 125; KCl, 2.5; CaCl2, 2; NaH2PO4, 1.25; MgCl2, 1; glucose, 25; NaHCO3, 25) equilibrated with 95% O2–5% CO2, and cut with a tissue slicer (Campden Instruments). Before recording, slices were kept in a holding chamber submerged in ACSF at 24°C.
Electrophysiological recordings were obtained from brain slices that were continuously superfused with ACSF (32°C) while in a chamber (0.75 ml volume) mounted on a fixed-stage upright microscope (Zeiss Axioskop). Differential interference contrast optics, infrared illumination, and a CCD camera were used to view neurons on a video monitor. Whole cell patch clamp recordings were made using 4–8 M
electrodes. In the majority of experiments recordings were made using a potassium gluconate–based solution (containing in mM: K-gluconate 117.5, KCl 15, NaCl 10, HEPES 10, EGTA 0.2, Mg2-ATP 2, Na3-GTP 0.25). However, we found that recordings made from cholinergic neurons (see following text) with this solution caused a large increase in conductance to develop over time that was associated with rundown of the potassium inward rectifier current that is a characteristic of these neurons (Bengtson and Osborne 2000). These changes were minimized when a potassium methylsulfate-based internal solution was used [containing in mM: potassium methylsulfate (KCH3SO4) 135; NaCl 8; HEPES 10; Mg2-ATP 2; Na3-GTP, 0.25], which thus was used in most recordings from cholinergic neurons. Both internal solutions were adjusted to a pH of 7.3 with KOH, and an osmolarity of 270–290 mosmol/l. Neurobiotin (Vector Laboratories) was included in the internal solution in some experiments where filled neurons were later identified in fixed slices processed for choline acetyltransferase (ChAT) immunohistochemistry. Recordings were obtained with an Axopatch 1D amplifier (Axon Instruments, Union City, CA) and digitized using a Labmaster A/D converter and PClamp or Axotape software (Axon Instruments). All data were obtained when the series resistance was below 25 M, which was monitored at regular intervals throughout each experiment. A correction for the predicted liquid junction potential of the solution (–10 mV) (Barry 1994) was made in all of the data presented.
Electrophysiological criteria for identification of noncholinergic and cholinergic neurons
We previously reported that ChAT-negative and ChAT-positive neurons in the ventral pallidum have different inward rectifier currents, which can be used as electrophysiological criteria to distinguish between them (Bengtson and Osborne 2000). To do this, current–voltage (I–V) relationships for the quasi-instantaneous and steady-state currents were measured in each neuron (see Figs. 1D and 4) and used to estimate values for GHOLD (slope conductance at –60 to –80 mV), GINST ("instantaneous" conductance at –100 to –120 mV), and GSS ("steady-state" conductance at –100 to –120 mV). In cholinergic neurons the major inwardly rectifying current has fast activation kinetics and is carried by potassium inward rectifier channels, whereas in noncholinergic neurons the major current activates slowly over several hundred milliseconds and is carried by h-current channels. This can be illustrated by plotting either the conductance of the fast inward rectifier current (equal to GINST – GHOLD) or the conductance of the time-dependent inward rectifier current (equal to GSS – GINST) against GHOLD. The electrophysiological criteria used to identify noncholinergic neurons were that the conductance of the time-dependent inward rectifier current (GSS – GINST) was equal to or greater than GHOLD, and GHOLD was <5 nS. The electrophysiological criteria used to identify cholinergic neurons were that the conductance of the time-dependent inward rectifier current (GSS – GINST) was less than GHOLD, and GHOLD was >5 nS. Because some recordings in the present study were made using a potassium gluconate internal solution different from the methylsulfate-based solution used in our previous study, we confirmed the validity of our criteria by filling a subset of neurons and using ChAT immunohistochemistry to distinguish cholinergic from noncholinergic neurons.
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Brain slices containing biocytin-filled neurons were fixed overnight (at 4°C) in 4% paraformaldehyde in phosphate buffer (PB: 0.1 M, pH 7.4), rinsed. and stored up to 2 wk in 0.01% sodium azide in PB. After treatment with 0.3% triton X-100 and 0.01% sodium azide in PB for 4 days, the slices were rinsed in PB, placed for 60 min in 10% normal horse serum and 0.1% triton X-100 in PB, and then incubated for 12 h at room temperature (RT) in an affinity-purified primary antiserum raised in goat against ChAT (1:500, Chemicon, Pittsburgh, PA). They were then rinsed in PB and incubated for 1 h at RT in Cy3-conjugated donkey anti-goat IgG (1:1000, Jackson ImmunoResearch Laboratories, West Grove, PA) and FITC-conjugated streptavidin (1:100, Sigma–Aldrich, St. Louis, MO); All antisera and streptavidin conjugates were diluted in 1% normal horse serum and 0.3% Triton X-100 in PB.
Drugs used
TFMPP [N-(3-trifluoromethylphenyl)-piperazine (ICN, Cost Mesa, CA)]; 5-CT (5-carboxamidotryptamine); cesium chloride; m-CPBG, m-chlorophenyl biguanide; clozapine; DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]; 8-OH-DPAT (8-hydroxy-dipropylaminotetralin); 5-HT (5-hydroxytryptamine); mianserin hydrochloride; 5-MeOT (5-methoxytryptamine); p-MPPI {4-(2'-methoxy-)phenyl-1-[2'-(n-2"-pyridinyl)-p-iodobenzamido-]ethyl-piperazine}; methiothepin; pimozide; pindolol; TEA (tetraethylammonium); tropisetron (ICS 205-930; SigmaRBI/Sigma-Aldrich, St. Louis MO); bicuculline; QX314, lidocaine n-ethyl bromide; and tetrodotoxin citrate (Tocris Cookson, Bristol, UK) were made up as stock solutions in deionized water. Picrotoxin (SigmaRBI) and forskolin (Tocris) were made up as a stock solution in DMSO. All drugs were diluted in ACSF and applied by superfusion. The final concentration of DMSO in the superfusate was 0.1% or less, which had no direct effects. Antagonist drugs were applied by superfusion for a minimum of 10 min or until no further reduction of the agonist current was apparent.
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RESULTS |
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To test the effect of 5-HT we made whole cell patch-clamp recordings from ventral pallidal neurons in brain slices from immature rats. We identified 2 agonist-induced electrophysiological responses that were seen in separate populations of cells. In voltage-clamp recordings from 81 neurons, 5-HT induced an inward current (31 ± 2.7 pA at –60 mV) that could also be induced in the presence of tetrodotoxin (1 µM, n = 4). As shown in Fig. 1A this inward current was relatively slow to develop and required several minutes to achieve steady state. In current-clamp recordings from a further 4 neurons, 5-HT induced a depolarization (5 ± 1.3 mV) and increased the frequency of spontaneous action potential firing (Fig. 1B). In 38 neurons, 5-HT induced an outward current (112 ± 14 pA at –60 mV) that typically developed and washed out more rapidly than the inward current (Fig. 1C). Eleven neurons in the ventral pallidum did not respond to 5-HT.
To determine whether cholinergic and noncholinergic neurons responded differently to 5-HT, we filled cells with biocytin and immunostained them for ChAT (Fig. 2A). In recordings from 18 ChAT-positive neurons, 5-HT induced outward currents in 15 (83%) cells and inward currents in 2 (11%) cells. We also identified another 6 ChAT-positive magnocellular neurons that were situated more dorsally in the globus pallidus, all of which showed outward currents in response to 5-HT. In recordings from 11 ChAT-negative neurons in the ventral pallidum, outward currents were induced by 5-HT in only 1 (9%) neuron, whereas inward currents were induced in 8 (73%) of these cells.
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), electrophysiological differences between ChAT-positive and ChAT-negative neurons were revealed by calculating slope-conductance values (Table 1) from the current–voltage relationships of quasi-instantaneous and steady-state currents, as described in METHODS. The difference in the conductances of the 2 groups of immunohistochemically identified neurons is illustrated by the conductance plot in Fig. 2B. When we assessed the effect of 5-HT in neurons identified by electrophysiological criteria alone, outward currents were induced in 38 (93%) out of 44 putative cholinergic neurons, whereas inward currents were induced in only 3 of these neurons. In contrast, 5-HT–induced inward currents or depolarizations were induced in 85 (92%) out of 92 noncholinergic neurons and outward currents in only 2 of these neurons. Eight neurons did not respond to 5-HT. The conductances of neurons in which outward and inward currents were induced by 5-HT are plotted separately in Fig. 2C. From these data, it is apparent that 5-HT predominantly induced outward currents in magnocellular cholinergic neurons in the ventral pallidum, whereas inward currents were primarily induced in noncholinergic basal ganglia neurons.
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To assess the conductance mediating outward currents induced by 5-HT in cholinergic neurons, we recorded current–voltage relationships as illustrated in Fig. 1D. This neuron was typical in that it expressed a fast inward rectifier current but had no h-current. The outward current measured at –60 mV in 5 neurons was associated with a conductance increase and reversed polarity at –100 ± 3 mV. The potassium equilibrium potential estimated using the Nernst equation was –102 mV, which suggested that the outward 5-HT current was mediated by the opening of potassium channels.
In noncholinergic neurons, the inward current induced by 5-HT at –60 mV did not reverse polarity when measured during a voltage step to –120 mV (Fig. 3). This step protocol also activated the characteristic h-current in these neurons, which was revealed as a slow inward relaxation current that developed during the hyperpolarizing voltage step. To determine whether 5-HT was affecting the h-current in noncholinergic ventral pallidal neurons we performed experiments using 2 mM extracellular cesium to block the current (Bengtson and Osborne 2000). In the presence of cesium, the inward current induced by 5-HT was substantially reduced, leaving a residual 5-HT current (20 ± 7.5 pA at –60 mV and 57 ± 18 pA at –120 mV, n = 6). This effect of 5-HT is illustrated in Fig. 3 by digitally subtracting records obtained in the absence and presence of cesium. As expected from our previous report (Bengtson and Osborne 2000), this procedure effectively isolated the h-current. The subtracted records show that 5-HT increased the amplitude of the cesium-sensitive current, measured both at the holding potential of –60 mV and during a hyperpolarizing step, which was accompanied by an increase in the instantaneous current seen immediately after the onset of the command step.
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; Mayer and Westbrook 1983). To investigate this further, we performed similar subtraction procedures on the current–voltage relationships shown in Fig. 4, which were measured 1) under control conditions, 2) when the 5-HT induced current had reached steady state, 3) when the 5-HT current had been reduced using 2 mM cesium, and 4) in the presence of cesium after 5-HT had been washed off and the agonist current had reversed. In this experiment, an electrode solution with cesium ions substituted for potassium ions was used to block potassium channels. In Fig. 4B current–voltage relationships measured in the absence and presence of 2 mM extracellular cesium were subtracted to obtain the current–voltage relationship of the cesium-sensitive h-current at steady-state. This curve is replotted in Fig. 4C to compare it to the equivalent subtracted current–voltage relationship measured in the presence of 5-HT. These data showed that 5-HT caused an increase in the cesium-sensitive current that was most pronounced at less-negative potentials. Because the subtracted current was now quite linear, extrapolation of the chord conductance was used to obtain an estimate of –36 mV for the reversal potential. Figure 4 also shows a small residual 5-HT current resistant to cesium, which was also relatively linear over the same potential range and reversed polarity at around –50 mV. The effect of 5-HT on the h-current in noncholinergic neurons therefore appears similar to multiple reports in neurons and cell lines in which inward currents induced by 5-HT are caused by a shift in the activation of h-currents to less-negative potentials (Bobker and Williams 1989; Cardenas et al. 1999; Chapin and Andrade 2001a; McCormick and Pape 1990; Takahashi and Berger 1990).
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It has been consistently reported that the effect of 5-HT receptors on h-currents are mediated by intracellular cAMP as a result of stimulation of adenylyl cyclase (Bobker and Williams 1989; Cardenas et al. 1999; Chapin and Andrade 2001a; McCormick and Pape 1990; Takahashi and Berger 1990). To assess whether this could also be the case in noncholinergic ventral pallidal neurons we used forskolin to stimulate adenylyl cyclase. Forskolin (10 µM) caused an inward current (40 ± 9.3 pA at –60 mV cf. 68 ± 16 pA at –120 mV, n = 14) and mimicked 5-HT (Fig. 5). A single neuron that failed to respond to forskolin was also not affected by 5-HT. Forskolin also completely or partially "occluded" the response to 5-HT such that once the current induced by forskolin (10 µM) had developed, the effects of concurrent applications of 5-HT (30 µM) were reduced or absent (77 ± 9.7% decrease, range: 54–100%, n = 5).
The 5-HT4, 5-HT6, and 5-HT7 receptors are the subtypes that are most likely to signal by stimulating adenylyl cyclase (Barnes and Sharp 1999; Raymond et al. 2001). These receptor subtypes were only identified relatively recently and the availability of selective ligands remains limited, although 2 studies have now identified 5-HT7 receptors as the subtype activating h-currents in dorsal root ganglia and thalamic neurons (Cardenas et al. 1999; Chapin and Andrade 2001b). We tested several semiselective agonists known to stimulate 5-HT7 receptors, which were subsequently found to mimic 5-HT in noncholinergic ventral pallidal neurons. 5-Carboxytryptamine (5-CT) is typically more potent than 5-HT as an agonist at 5-HT7 receptors, and this has been used as a functional screen for detecting putative 5-HT7 receptors (e.g., Chapin and Andrade 2001b). We found that 5-CT induced a maximum current of 35 ± 4.5 pA measured at –60 mV, and was 32-fold more potent than 5-HT (Fig. 6, A and B). This was determined by fitting logistic functions to the concentration–effect data shown in Fig. 6B, which provided pEC50 and Hill slope estimates of 7.3 ± 0.11 and 0.96 ± 4.5 for 5-CT (n = 4), and 5.8 ± 0.24 and 1.3 ± 0.14 for 5-HT (n = 4). We also tested 5-methoxytryptamine and 8-OH-DPAT, which like 5-CT, are 5-HT1 agonists that can also act as agonists at 5-HT7 receptors, although 8-OH-DPAT has typically been found to act as a weak partial agonist. 5-Methoxytryptamine (10–30 µM) mimicked 5-HT and induced an inward current with a mean amplitude of 29 ± 8.3pA at –60 mV (n = 4). In contrast, 8-OH-DPAT had no effect in 10 cells (1 µM, n = 5; 10 µM, n = 4; 30 µM, n = 1) and could induce only small inward (10–14%, 1 µM, n = 2; and 40%, 30 µM, n = 1) or outward currents (1–30 µM, n = 4) in the remaining neurons.
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). To determine whether a different 5-HT receptor subtype induced the outward current in cholinergic neurons, we retested several of the agents found to be effective in noncholinergic neurons. Forskolin (10-30 µM, n = 4) had no effect on cholinergic neurons at –60 mV, and both 8-OH-DPAT (300 nM–1 µM) and 5-CT (3–10 µM) induced outward currents (Fig. 7, A and B) that were larger than the 5-HT current (8-OH-DPAT: 124 ± 34 pA, n = 7; 5-CT: 217 ± 61 pA, n = 4) (Fig. 7C). A small inward current, 29 ± 6 pA in amplitude, was induced by TFMPP (1 µM) in these neurons (Fig. 7, A and D), and in contrast to inward agonist currents, outward currents induced by 5-HT (10 µM) were not affected by clozapine (10 µM; n = 6) (Fig. 7C).
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DISCUSSION |
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). These putative GABA neurons were depolarized by a 5-HT receptor subtype that was potently activated by 5-CT. Most of the inward current induced by 5-HT was attributed to an increase in a cesium-sensitive current that could correspond to the endogenous h-current. In contrast the outward current induced by 5-HT in cholinergic neurons was attributed to potassium channels. Although both responses could be induced by 5-CT, they are unlikely to be mediated by the same 5-HT receptor subtype. These results identify the ventral pallidum as a potential target by which 5-HT released from midbrain dorsal raphe projections could significantly influence both the basal ganglia circuitry associated with the limbic striatum as well as cholinergic corticopetal projections. Electrophysiology of the depolarizing 5-HT current in noncholinergic neurons
The inward current induced by 5-HT in putative GABA neurons was largely prevented when the h-current was blocked with 2 mM extracellular cesium. Previous studies have identified 5-HT induced depolarizations that are mediated by h-currents in numerous areas including prepositus hyperglossi, thalamus, spinal motor nucleus, and dorsal root ganglia (Bobker and Williams 1989; Cardenas et al. 1999; Chapin and Andrade 2001a,b; McCormick and Pape 1990; Takahashi and Berger 1990). Digital subtraction of records demonstrated that the isolated whole cell current, blocked by cesium, had the characteristic features of an h-current. We previously showed that 2 mM extracellular cesium completely blocks the h-current in noncholinergic neurons in the ventral pallidum but does not affect the potassium inward rectifier current in cholinergic neurons (Bengtson and Osborne 2000). We also showed that the effect of cesium on noncholinergic neurons was the same as the selective h-current blocker ZD 7288, which was not used in the present study because it can take as long as 15 min to abolish the h-current and the block is not reversible. In the present study we found in some noncholinergic neurons that 5-HT caused a large increase in the cesium-sensitive current measured at –60 mV, which was associated with an increase in the instantaneous current activated by hyperpolarization and in many cases a decrease in the relaxation attributed to time-dependent activation of the h-current. We believe these effects are similar to those produced when h-currents are activated by hyperpolarization from a potential that is not sufficiently depolarized to completely deactivate h-current channels (see Cardenas et al. 1999; Fig. 2, Mayer and Westbrook 1983). The instantaneous 5-HT current seen under these conditions is carried by h-current channels that are already open at the holding potential before hyperpolarization, which in turn can lead to a reduction in the amount of current that is activated slowly by hyperpolarization. We were unable to test this directly by holding neurons at a potential that completely deactivated the h-current because in the presence of 5-HT, this would require voltage steps from around –20 mV (–30 mV after correcting for the junction potential) and we could not completely block the voltage-dependent currents that are active at these potentials.
After block with extracellular cesium, 5-HT continued to induce a small residual 5-HT current in noncholinergic neurons. It is possible this residual 5-HT current was carried by the same channels as the cesium-sensitive h-current because it has been recently reported that HCN2 channels can produce a large instantaneous current that is not blocked by cesium (Proenza et al. 2002). Although such currents have yet to be demonstrated in native neurons in brain, HCN2 members of the h-current channel family activate relatively slowly with time constants comparable to the h-current in noncholinergic ventral pallidal neurons.
Pharmacology of the 5-HT receptor that depolarizes noncholinergic neurons
In the present study 5-CT behaved as a full agonist and was more potent than 5-HT (note that 5-HT uptake was not blocked). Binding sites labeled by [3H]5-CT have been localized to the pallidal complex by receptor autoradiography. A moderate density of sites in guinea-pig ventral pallidum are labeled by [3H]5-CT when (-)-cyanopindolol and sumatriptan are used to mask 5-HT1A and 5-HT1D receptors (To et al. 1995). In rat, [3H]5-CT combined with PAPP and (-)-pindolol labels sites in the globus pallidus and substantia innominata (Gustafson et al. 1996), which may also extend into the ventral pallidum. In accordance with these binding studies, the currents induced by 5-CT or 5-HT in noncholinergic neurons were not reduced by pindolol (or by p-MPPI) and the nonselective 5-HT1/5-HT2 receptor agonists TFMPP were also ineffective. Although 8-OH-DPAT has been used as a selective 5-HT1A receptor agonist, it is now known to also behave as a partial agonist of 5-HT7 receptors (Chapin and Andrade 2001b). This is consistent with our findings that only relatively high concentrations of 8-OH-DPAT were effective at inducing inward currents and these were always small in amplitude.
Further pharmacological studies are required to identify the 5-HT receptor subtype expressed by noncholinergic neurons in the ventral pallidum. The ability of forskolin to mimic and occlude the 5-HT current implicated receptor subtypes that preferentially signal by stimulating adenylyl cyclase (i.e., 5-HT4, 5-HT6, and 5-HT7) (Barnes and Sharp 1999). Two studies have identified the 5-HT7 receptor as the subtype that activates the h-current channels in rat dorsal root ganglion and thalamic neurons (Cardenas et al. 1999; Chapin and Andrade 2001b). Although, receptor identification using relative potency is not reliable, it has been consistently found in most functional assays that 5-CT is a more potent agonist of the 5-HT7 receptor subtype than 5-HT (Adham et al. 1998; Jasper et al. 1997) but is less potent than 5-HT at increasing cAMP levels in intact cells expressing rat 5-HT4 (Gerald et al. 1995) or 5-HT6 receptors (Boess et al. 1997; Grimaldi et al. 1998; Sleight et al. 1998). The possibility that 5-HT7 receptors could mediate inward 5-HT current in ventral pallidal neurons is consistent with our findings that 5-CT was more potent than 5-HT, and 5-methoxytryptamine was a full agonist, whereas 8-OH-DPAT was a weak partial agonist. However, we also found that several antagonists that bind with relatively high affinity to cloned 5-HT7 receptors (Table 2) had only relatively weak effects (SB-269770, pKi = 8.9; clozapine, pKi = 8.2) or were ineffective (pimozide, pKi = 9.3; mianserin, pKi = 7.1). In fact, the 5-CT binding site previously identified in the pallidum is also inconsistent with a 5-HT7 receptor. In contrast to putative autoradiographic binding to 5-HT7 receptors elsewhere in the brain, [3H]5-CT binding in the globus pallidus and substantia nigra is not masked by low concentrations of methiothepin (Gustafson et al. 1996), which has high affinity for recombinant 5-HT7 receptors (Table 2) (Eglen et al. 1997; To et al. 1995). Furthermore, in situ hybridization studies have reported either low (Neumaier et al. 2001) or undetectable levels of 5-HT7 mRNA in the pallidal complex (Heidmann et al. 1998; To et al. 1995). Therefore the identity of the 5-HT receptor subtype that mediates the effect of 5-CT in the ventral pallidum remains to be determined.
Electrophysiology and pharmacology of 5-HT induced hyperpolarization of cholinergic neurons
We found that the major effect of 5-HT on magnocellular cholinergic neurons in the ventral pallidum was to induce a hyperpolarizing current, possibly by opening G protein–gated inward rectifier potassium (GIRK) channels. TFMPP, which can act as a partial agonist of the 5-HT1A, 5-HT1B, and 5-HT2 receptor subtypes, also revealed a small depolarizing effect but this was not characterized further. Previous in vitro studies of magnocellular cholinergic neurons located in other nuclei have reported electrophysiological responses to 5-HT. In rat, both hyperpolarizing and depolarizing responses were induced by 5-HT in putative cholinergic neurons identified by electrophysiological criteria in the medial septum and diagonal band of Broca (Gorelova and Reiner 1996). However, in these neurons, 5-HT consistently reduced the slow afterhyperpolarization after an action potential and caused a related enhancement in spike frequency adaptation that was independent of the direction of the change in membrane potential. In guinea pig, 5-HT has been reported to inhibit cholinergic neurons in the caudal substantia innominata and preoptic nucleus adjacent to the ventral pallidum, but the electrophysiological mechanism was not identified (Khateb et al. 1993). 5-HT also inhibits N-type calcium channels in cholinergic neurons located in these guinea pig nuclei and the horizontal limb of the diagonal band of Broca (Williams et al. 1998).
The 5-HT inhibition of magnocellular cholinergic neurons reported in guinea pig was mimicked by 5-HT1A selective agonists (Khateb et al. 1993; Williams et al. 1998). 5-HT1A receptors have been detected by in situ hybridization for 5-HT1A mRNA and high-affinity [H3]8-OH-DPAT receptor binding is present in neurons scattered through basal forebrain regions where the magnocellular cholinergic cell group is located (Nyakas et al. 1997; Pompeiano et al. 1992). Although not conclusive, our results were consistent with the possibility that a 5-HT1A receptor mediated the hyperpolarizing effect of 5-HT on pallidal cholinergic neurons. In contrast to noncholinergic neurons, the effect of 5-HT in cholinergic neurons was mimicked by both 8-OH-DPAT and 5-CT, which hyperpolarized the neurons more strongly than 5-HT itself. Furthermore, these effects were not mimicked when adenylyl cyclase was stimulated with forskolin, nor were they blocked by clozapine.
Implications
We have shown that virtually all noncholinergic neurons in both the lateral and medial areas of the ventral pallidum are excited by 5-HT. This effect appeared to be primarily mediated by changes in an h-current, which can function as a pacemaker current or can also shape the pattern of action potential firing in neurons. In thalamocortical neurons, activity-dependent modulation of h-current channels can produce prolonged changes in the rhythmicity and periodicity of spike discharge (see review by Luthi 1998). The frequency and pattern of action potential firing in noncholinergic ventral pallidal neurons are similarly highly voltage dependent and the complex patterns of discharge observed in vivo and in vitro in these neurons (Bengtson and Osborne 2000; Lavin and Grace 1996) could therefore be regulated in a similar way by 5-HT receptors that couple to adenylyl cyclase and modulate the h-current. We predict that such an effect would not be restricted to 5-HT, given that afferent nerve terminals projecting to the ventral pallidum contain other endogenous agonists (e.g., dopamine and opioid peptides) that are known to affect cAMP signaling and ventral pallidal activity in vivo (Mitrovic and Napier 1995, 1996; Napier and Maslowski-Cobuzzi 1994; Napier et al. 1991).
In conclusion, our study has shown that 5-HT projections from the dorsal raphe could have opposing functional effects on basal ganglia neurons in the limbic striatal–related circuitry and cholinergic corticopetal neurons in the rat ventral pallidum. If this is the case then the ability of the atypical antipsychotic drug clozapine to selectively block 5-HT effects on putative GABA neurons in the ventral pallidum warrants further investigation as the clinical actions of this drug on basal forebrain circuits are relevant to the treatment of schizophrenia and other psychiatric and mood disorders.
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Address for reprint requests and other correspondence: P. Osborne, Pain Management Research Institute, University of Sydney, Royal North Shore Hospital, St Leonards NSW 2065, Australia (E-mail p.osborne{at}usyd.edu.au).
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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