Neurotensin and the serotonergic system

Progress in Neurobiology Vol. 52, pp. 455 to 468, 1997 © 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0301-0082/97/$32.00

Pergamon

PIh S0301-0082(97)00025-7

NEUROTENSIN

AND

THE SEROTONERGIC

SYSTEM

THIERRY JOLAS*tJ; and GEORGE K. AGHAJANIAN*t *Department of Psychiatry, Yale University School of Medicine, Connecticut Mental Health Center, 34 Park Street, New Haven, Connecticut 06508, U.S.A. and tDepartment of Pharmacology, Yale University School of Medicine, Connecticut Mental Health Center, 34 Park Street, New Haven, Connecticut 06508, U.S.A. (Received 25 February 1997) Abstract--']F'he serotonergic system, because of very diffuse projections throughout the central nervous system, has been implicated in numerous functions including nociception, analgesia, sleep-wakefulness and autonomic regulation. Despite an abundant literature indicating the presence of neurotensin-containing (neurotensinergic) neurons, fibres and terminals in most areas containing serotonergic neurons, little is knc,wn about the possible relationship between serotonergic and neurotensinergic systems. The purpose of this review is (i) to summarize current knowledge on the anatomical relation between neurotensinergic and serotonergic system, (ii) to summarize current knowledge on the action of neurotensin on serotonergic neurons and (iii) to discuss the possible physiological relevance of this action. Neurotensin-containing cell bodies can be found in the most rostral raphe nuclei. There are neurotensin-containing fibres and terminals in all raphe nuclei. Raphe nuclei have also been shown to contain neurotensin-receptor binding sites. In the dorsal raphe nucleus, neurotensin induces a concentrationdependent increase in the firing rate of a subpopulation of serotonergic neurons. The neurotensininduced excitation, which is selectively blocked by the non-peptide neurotensin receptor antagonist SR 48692, is observed mainly in the ventral part of the nucleus. Most serotonergic neurons show marked desensitization to neurotensin, even at low concentrations. In intracellular experiments, neurotensin induces an inward current, associated in some cases with a decrease in apparent input conductance, which is occluded by supramaximal concentrations of the ~q-adrenoceptor agonist phenylephrine. In rare cases, neurotensin induces an excitation of GABAergic or glutamatergic neurons. Since the neurotensinergic system has also been implicated in nociception, analgesia, sleep-wakefulness, and autonomic regulation, the review discusses the possibility that part of this regulation could involve the activation of the serotonergic system. © 1997 Elsevier Science Ltd

CONTENTS 1. Introduction 2. Anatomical relationship between neurotensinergic and serotonergic systems 2.1. The serotonergic system 2.2. Distribution of neurotensin, its precursor's messenger RNA, and neurotensin receptor binding sites and messenger RNA in the raphe nuclei 3. Electrophysiological effect of neurotensin on serotonergic neurons of the dorsal raphe nucleus 3.1. Effect of neurotensin on serotonergic neuron electrical activity 3.2. Localization of the neurotensin responsive serotonergic neurons 3.3. Interaction between neurotensin and phenylephrine 4. Possible physiological relevance of neurotensin's action on serotonergic neurons and conclusion 4.1. Nociception 4.2. Autonomic function 4.3. Behavioural arousal 4.4. Conclusions Acknowledgemertts References

456 456 456 456 458 459 460 461 462 462 464 464 465 465 466

ABBREVIATIONS AMPA NMDA NT

ct-amino-3-hydroxy-5-methylisoxazole-4propionic acid N-methyl-D-aspartate neuroterLsin

PAG PE VTA

periaqueductal gray phenylephrine ventral tegmental area

:~Author for correspondence: Departments of Psychiatry and Pharmacology, Yale University School of Medicine, CMHC, 34 Park St., New Haven, CT 06508, U.S.A. Fax: 203-562-7079. 455

T. Jolas and G. K. Aghajanian

456 1. INTRODUCTION

The serotonergic system, because of very diffuse projections throughout the central nervous system, has been implicated in numerous functions including nociception, analgesia, sleep-wakefulness or autonomic regulation. Despite an abundant literature indicating the presence of neurotensinergic neurons fibres and terminals in most serotonergic neuronalcontaining areas, little is known about the possible relationship between serotonergic and neurotensinergic systems. Neurotensin (NT) is a tridecapeptide, originally isolated from bovine hypothalami (Carraway and Leeman, 1973), which has been reported to be involved in hypotension, hypothermia, analgesia, nociception, sleep regulation and possibly in certain kind of neuropathology such as Parkinson's disease or schizophrenia. The reported effect of NT on non-serotonergic neurons is typically an increase in firing rate (Stowe and Nemeroff, 1991; Mercuri et al., 1993; Keegan et al., 1994). This excitation is associated with a depolarization of the membrane resulting from an inward current generally associated with a decrease in input conductance in the rat (Pinnock, 1985; Audinat et al., 1989; Mercuri et al., 1993; Farkas et al., 1994; Jiang et al., 1994) or the guinea-pig (Alonso et al., 1994). The most frequently reported ionic mechanism for the NT-induced inward current is a decrease in potassium conductance (Audinat et al., 1989; Alonso et al., 1994; Farkas et al., 1994; Jiang et al., 1994). In rat substantia nigra, an activation of protein kinase C by NT via pertussis toxin (PTX)-insensitive Gproteins mediates the NT inhibition of the inward rectifying potassium currents in dopaminergic neurons (Wu and Wang, 1995). In some preparations, the NT-induced inward current has also been shown to be carried by sodium ions (Mercuri et al., 1993; Farkas et al., 1994; Jiang et al., 1994) or by the activation of a non-selective cationic conductance (Kirkpatrick and Bourque, 1995; Wu et al., 1995; Chien et al., 1996; Farkas et al., 1996). The purpose of this review is to summarize the current knowledge on (i) the anatomical relationship between neurotensinergic and serotonergic system with more focus on the dorsal raphe nucleus, (ii) the action of NT on serotonergic neurons, and (iii) the possible physiological relevance of this action.

2. ANATOMICAL RELATIONSHIP BETWEEN

NEUROTENSINERGIC AND SEROTONERGIC SYSTEMS

2.1. The Serotonergic System The serotonergic system is comprised of a group of neurons whose cell bodies are distributed from the level of the interpeduncular nucleus in the midbrain to the level of the pyramidal decussation in the medulla (T6rk, 1985). This system is divided in nine cellular groups named B1 to B9 (Dahlstr6m and Fuxe, 1964). In this review, we will use the traditional terminology for the classification of raphe nuclei (Fig. 1).

RPs

Fig. 1. Schematic representation of raphe nuclei and their neurotensin content. Asterisks indicate raphe nuclei where neurotensin immunoreactivity can be detected in cell bodies. Abbreviations: CLi, caudal linear nucleus; DRN, dorsal raphe nucleus; MR, median raphe nucleus; RMg, raphe magnus; ROb, raphe obscurus; RPa, raphe pallidus; RPn, raphe pontis,

2.2. Distribution of Neurotensin, Its Precursor's Messenger RNA, and Neurotensin Receptor Binding Sites and Messenger RNA in the Raphe Nuclei The neurotensinergic content of the different raphe nuclei (i.e. localization of the NT immunoreactivity, NT mRNA, NT receptors and mRNA) is summarized in Table 1. An abundant literature (Table 1), using immunohistochemical techniques and, in most cases, colchicine pretreatment (which maximizes peptide content in otherwise weakly-labelled cell bodies), indicates the presence of NT-like immunoreactivity in the raphe nuclei of experimental animals as well as humans (Cooper et al., 1981; Emson et al., 1982; Mai et al., 1987). NT-containing cell bodies seem to be limited in the more rostral raphe nuclei (i.e. caudal linear nucleus, dorsal raphe nucleus, and raphe pontis, except in the guinea-pig) whereas fibres and terminals can be detected in all raphe nuclei. In the dorsal raphe nucleus, NT-containing cells have been reported in high to moderate numbers in the rat, especially along the midline (Uhl et al., 1979; Seroogy et al., 1987, 1988; Shipley et al., 1987; Sutin and Jacobowitz, 1988; Van den Bergh et al., 1988), in the guinea-pig (Triepel et al., 1984) and in the cat (de Le6n et al., 1991) but seem to be absent in dorsal raphe nucleus of post m o r t e m human brains (Mai e: al., 1987). One study in the rat, reported that NT-containing cell bodies are more prominent near the fourth ventricle (Jennes et al., 1982). NTcontaining cells are rounded and fusiform cells, 10 20/~m in diameter (Uhl et al., 1979; Shipley et al., 1987; Wang et al., 1995, 1996), which first appear at day 18 of gestation (Minagawa et al., 1983). Using a triple-labelling indirect immunofluorescence procedure, two studies from the same group reported that NT could be co-localized with cholecystokinin and tyrosine hydroxylase in most NT-containing cells of the caudal linear nucleus and the dorsal raphe nucleus (Seroogy et al., 1987, 1988). NT fibres and terminals are abundant in the dorsal raphe nucleus (Uhl et al., 1979; Jennes et al., 1982). The highest density of fibres is in the dorsal part of the dorsal raphe nucleus according to one study (Shipley et al., 1987) or all along the midline

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according to others (Sutin and Jacobowitz, 1988; de Le6n et al., 1991). A moderate to high level of NT fibres has been reported in the dorsal raphe nucleus of post mortem human brains indicating the potential importance of this neurotransmitter in human serotonergic regulation (Mai et al., 1987). Fibres and terminals appear around first postnatal day in the rat dorsal raphe nucleus (Minagawa et al., 1983). One immunohistochemical study, using electron microscopy, reports that NT axon terminals make mainly contact with dendrites on NT and non-NT-containing neurons (Wang et al., 1995). The origin of NT afferents to the dorsal raphe nucleus is not well characterized. A study, combining anterograde tracing and immunocytochemistry, reports that serotonergic and a few non-serotonergic neurons in the dorsal raphe nucleus receive NT projections from the lateral parabrachial nucleus (Petrov et al., 1992), giving the first direct anatomical evidence that NT-containing neurons may influence serotonergic neurons of the dorsal raphe nucleus. NT-containing neurons in the dorsal raphe nucleus have been shown to project at least to an area immediately dorsal to the raphe magnus (Beitz, 1982) and to the lateral hypothalamic area (Allen and Cechetto, 1995). The recent cloning of the rat gene encoding NT and neuromedin N (Kislauskis et al., 1988) has also made possible the localization of the mRNA and precursor of prepro-NT/neuromedin N in some raphe nuclei (Table 1). In particular, several studies using immunohistochemistry and in situ hybridization-histochemistry revealed the presence of prepro-NT/neuromedin N and its mRNA in cell bodies, fibres and terminals in the dorsal raphe nucleus (Sato et al., 1991; Williams and Beitz, 1993; Woulfe et al., 1994). Cells expressing prepro-NT/ neuromedin N mRNA can divided in two groups (Sato et al., 1991). In the first group, prepro-NT/ neuromedin N mRNA appears around birth, reaches a peak of expression during the first postnatal week, then decreases slightly to a plateau level of expression after about the third postnatal week. In the second group, prepro-NT/neuromedin N mRNA also appears around birth, reaches a peak of expression during the first postnatal week, but then decreases dramatically to undetectable levels. Raphe nuclei cells expressing prepro-NT/neuromedin N mRNA are among the first group (Sato et al., 1991). Murine brain, which is the most extensively studied, contains two NT binding sites with different dissociation constants (Mazella et al., 1983) and differential sensitivity to levocabastine, an antihistaminic compound (Schotte et al., 1986; Kitabgi et al., 1987). The high-affinity, low-capacity receptor, which is insensitive to levocabastine, is responsible for the pharmacological action of NT and can be found in at least three different forms in central and peripheral cells and tissues of various species (Mazella et al., 1985a,b, 1987; Mills et al., 1988; Mazella et al., 1989; Chabry et al., 1993, 1994). Almost all raphe nuclei are reported to contain NT binding sites (Table 1). Studies from the early eighties indicated a low density of NT binding sites in the dorsal raphe nucleus of the rat (Young and Kuhar, 1981; Quirion et al., 1982). More recently,

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0 5 min Fig. 2. Firing rate histogram illustrating the effect of NT on serotonergic neurons recorded in a brain slice containing the dorsal raphe nucleus. The neurons were characterized extracellularly by a regular firing rate comparable to those seen in vivo (0.5-2.5 Hz, Baraban and Aghajanian, 1980; VanderMaelen and Aghajanian, 1983) when 3 #M of phenylephrine was perfused in the slice which could be reduced by 5-HT (20pM). NT was tested when neurons ceased firing, after washout of PE. The bars and the dotted-line above the histogram represent the duration of the drug perfusions (from Jolas and Aghajanian, 1996). moderate to high density of NT-receptor binding sites have been reported in the dorsal raphe nucleus (Kessler et al., 1987; Kitabgi et al., 1987; Moyse et al., 1987; Boudin et al., 1996). NT-receptor binding is uniformly distributed over neuropil and perikarya according to one study (Kessler et al., 1987) or only over perikarya and dendrites according to another (Boudin et al., 1996). NT-receptor binding sites seem to be mainly high-affinity, low-capacity binding sites in the dorsal raphe nucleus since binding is not reduced by the antihistamine HI agent levocabastine (Kitabgi et al., 1987). Since the cloning of the rat N T receptor (Tanaka et al., 1990), to our knowledge, only one study reported the presence of N T receptor m R N A in a raphe nuclei, the caudal linear nucleus (Nicot et al., 1994). Little is known about the exact anatomical relationship between N T cell bodies, fibres and terminals and serotonergic neurons in raphe nuclei except in the dorsal raphe nucleus. Similarly, almost nothing is known about the localization of the NT-

receptor binding sites in raphe nuclei, i.e. it is not known whether N T receptors can be found on serotonergic cell bodies, non-serotonergic cells or both. One study suggests that N T receptors might be synthesized in serotonergic cells of the dorsal raphe nucleus and the median raphe nucleus and transported to terminals in the suprachiasmatic nucleus (Franqois-Bellan et al., 1992).

3. E L E C T R O P H Y S I O L O G I C A L E F F E C T O F N E U R O T E N S I N ON S E R O T O N E R G I C NEURONS OF THE DORSAL RAPHE NUCLEUS It was of interest to test the effect of N T on serotonergic physiological activity in the dorsal raphe nucleus since (i) this nucleus, which is located in the ventral part of the periaqueductal gray (PAG), constitutes the major source of serotonergic innervation in the rat brain, representing almost one half of the total serotonergic cell population (Descarries et al.,

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~0 5 rain Fig. 3. Firing rate histograms from serotonergic neurons recorded in a brain slice containing the dorsal raphe nucleus illustrating desensitizating (A) and non-desensitizating (B) responses to NT. (A) In this neuron, a brief application of NT produced a short duration response which was totally suppressed by a 10 rain superfusion of the neuropeptide. Note that, in this case, during the 10 min application of NT, the response strongly diminished (73%) despite the continuous presence of the neuropeptide. The apparent desensitization of the NT response was also observed with 100 nM of the neuropeptide (not shown). (B) Example of a neuron, in which a brief application of NT produced a long duration response which was negligibly reduced by a 10 min superfusion of the neuropeptide. In this neuron, there was only a slight decrease (15%) in the response to NT during the 10 min application of the neuropeptide. The bars above the histograms represent the duration of the NT perfusions (from Jolas and Aghajanian, 1996).

Neurotensin and the Serotonergic System 1982) and (ii) the dorsal raphe nucleus is the only nucleus where there is direct anatomical evidence that NT terminals make contact with serotonergic neurons (Petrov et al., 1992). All experiments were done in vitro in rat brain slices containing the dorsal raphe nucleus (Jolas and Aghajanian, 1996).

3.1. Effect of Neurotensin on Serotonergic Neuron Electrical Activity Using extracellular recording, NT was found to increase firing rate in 61% of dorsal raphe nucleus serotonergic neurons (Fig. 2). In the remaining serotonergic cells, no response to NT was detected. The direction of the response is consistent with the excitatory effects of the neuropeptide in other regions. NT has been found to induce excitation in dopaminergic neurons of title substantia nigra or the ventral tegmental area (VTA) as well as in neurons in the

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NT(8-13) (p_M) Fig. 4. Concentratio~a-response curves for NT (A) and NT8 ~3(B) in the dorsal raphe nucleus. All the serotonergic neurons tested receiw:d increasing concentrations (10 nM10#M) of NT (n = 4) or NTs-13 (n = 5). There was a 20 rain period between each perfusion to reduce the effect of a possible desensitization of the response. On the curves, the abscissa (in logarithmic scale) is the concentration of the indicated compound in #M, and the ordinate, the response to a one concentration of NT, expressed as a percentage of the maximal response recorded for this neuron. Each point on the cu~rvesrepresents a mean ___S.E.M. The theoretical lines of the concentration-curves were generated by the non-linear titling program SigmaPlot. The calculated ECso for NT and NTs 13 are respectively 142 and 154 nM (from Jolas and Aghajanian, 1996).

459

hypothalamus and PAG (for review see Stowe and Nemeroff, 1991). Serotonergic-responsive neurons in the dorsal raphe nucleus could be divided in two categories according to the duration of their response (Fig. 3). In both categories, the excitation reached its maximum rapidly after the beginning of NT perfusion. However, in the first category, the effect was of short duration due to strong desensitization (Fig. 3A). In the second category of serotonergic neurons, the NT-effect persisted more than 20 min with virtually no desensitization of the response even after prolonged application (Fig. 3B). In the dorsal raphe nucleus, the effect of NT was concentration-dependent (ECso = 142 nM) and was mimicked by NT8 13, the active fragment of the neuropeptide (Fig. 4), whereas NTI_8 was inactive (not shown). The action of NT was pharmacologically specific since it was blocked by the non-peptide NT antagonist SR 48692 (Fig. 5). The ECs0 found for NT in the dorsal raphe nucleus was higher than ECs0s found in VTA (ECso = 35 nM, Seutin et al., 1989) and the substantia nigra (ECs0 = 13nM, Keegan et al., 1994). It cannot be concluded from our study whether or not this difference corresponds to a different NT receptor in the dorsal raphe nucleus than in those other nuclei. It must be noted that contrary to neurons in the VTA and the substantia nigra, serotonergic cells are quiescent in the brain slice preparation. For this reason, we could have underestimated the ECs0s extracellularly since we only measured an effect when a neuron reached the threshold for firing. In the VTA and the substantia nigra, NT responses do not desensitize at low concentrations whereas there is marked desensitization at all concentrations in a majority of serotonergic neurons in the dorsal raphe nucleus. Our results are more comparable to those observed in the frontal cortex where pyramidal neurons are found to strongly desensitize to NT applications (Audinat et al., 1989). Thus, it is possible that the NT receptor in the dorsal raphe nucleus is different from the one found in the VTA and the substantia nigra. Indeed, several electrophysiological, behavioural and biochemical studies support the concept of receptor heterogeneity. The NT receptor can be found in at least three different forms in central and peripheral cells and tissues of various species (Mazella et al., 1987; Mills et al., 1988; Chabry et al., 1993, 1994). In the PAG, two different types of excitation to NT can be measured, one very short and the other longer reflecting the possible presence of two different types NT receptor in this structure (Behbehani et al., 1987). The new non-peptide NT receptor antagonist SR 48692 (Gully et al., 1993) varies in potency depending on the brain area examined (Pinnock and Woodruff, 1994; Steinberg et al., 1994) suggesting that NT may act on different subtypes of receptors in various regions of the central nervous system. A few papers report that NT can have an inhibitory action on neurons (Baldino et al., 1985a,b; Baldino and Wolfson, 1985; Herbison et al., 1986; Shi and Bunney, 1991). In some of these cases, NT may have acted indirectly, via the stimulation of GABAergic interneurons, for example, since synaptic blockade, by reducing extracellular Ca 2 + , sup-

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Fig. 5. Blockade of the NT response by the non-peptide NT antagonist SR 48692 in the dorsal raphe nucleus. (A) Concentration-response curve of the NT effect on serotonergic neurons before (control) and after SR 48692 (100 nM). Each 1-min perfusion of NT was separated by a 20-min interval to avoid a possible desensitization of the response. The abscissa (in logarithmic scale) is the concentration of NT in /iM, and the ordinate, the response to one concentration of NT, expressed as a percentage of the maximal response recorded for this neuron. Each point on the curves represents a mean 4- S.E.M. The concentration-curve was shifted to the right after the treatment with SR 48692. (B) Firing rate histogram of a serotonergic neuron illustrating the selectivityof the SR 48692 effect. In this neuron, PE (3/tM) and NT (1 #M) were applied before and at the end of a 2-hr treatment with SR 48692 (1/~M). At this high concentration of the NT antagonist, the effect of NT was totally suppressed whereas the PE effect was unchanged. The dotted and solid lines indicate the period of perfusion of the drugs (from Jolas and Aghajanian, 1996). presses this inhibition (Bald•no and Wolfson, 1985; Herb•son et al., 1986). One study in the dorsal raphe nucleus indicates that NT fibres make contacts with non-serotonergic neurons (Petrov et al., 1992) and some preliminary results of our studies suggest an indirect inhibitory action of NT on non-serotonergic neurons in the dorsal raphe nucleus. Indeed, we found that the neuropeptide induced an increase of the frequency and the amplitude of spontaneous postsynaptic potentials in some non-serotonergic neurons recorded intracellularly (Fig. 6). These postsynaptic potentials were blocked with the GABAA antagonist bicuculline indicating their GABAergic origin (Fig. 6). A similar action of NT has been shown on pyramidal cells in the rat frontal cortex (Audinat et al., 1989). Serotonergic neurons are also known to receive inhibitory GABAergic inputs (Pan and Williams, 1989; Wang et al., 1992; Jolas and Aghajanian, 1997). Although we cannot rule out a similar action of NT on GABAergic inputs to serotonergic neurons in the dorsal raphe nucleus, we were unable to demonstrate their existence. Indeed, preliminary results from our studies indicate that NT can increase the frequency of postsynaptic cur-

rents in only a small minority of serotonergic neurons recorded in voltage-clamp mode (two out of 14 recorded serotonergic neurons), and that this effect could be blocked by LY 293558, a selective antagonist of the ~-amino-3-hydroxy-5-isoxazole-4-propionic acid (AMPA) subtype of glutamate receptor, indicating a glutamatergic origin for these NT-induced postsynaptic currents (Fig. 7). 3.2. Localization of the Neurotensin Responsive Serotonergic Neurons In preliminary experiments, we determined that some serotonergic neurons, which at first appeared to be unresponsive to NT, were excited by the neuropeptide if there was a prior induction of firing with Nomethyl-D-aspartate (NMDA; Fig. 8). Therefore, during the mapping of the NT response, NMDA was used to maximize the probability of detecting the occurrence of a NT effect. Even under these experimental conditions, the distribution of NT responses in the dorsal raphe nucleus was not homogeneous. In the ventral part of the nucleus 64% of the serotonergie neurons had a large re-

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Fig. 6. Intracellular current-clamp recordings with KC1 electrodes illustrating the effect of NT (A) and bicuculline (B) on a non-serotonergic neuron recorded in a brain slice containing the dorsal raphe nucleus. (A) NT (1/~M) induced a decrease in input resistance with an increase of the number of postsynaptic potentials. (B) Bicuculline(20/~M) totally blocked these postsynaptic potentials, indicating their GABAergic origin. sponse to NT; the remainder had either a small response (9%) or were unresponsive (27%) (Fig. 9). In the more dorsal and lateral parts of the dorsal raphe nucleus, the responses were mainly small (38.5%) or absent (38.5%) with only 15.5% of serotonergic neurons having a large response to NT. No difference in the localization of the cells with or without desensitization of the NT response was found. Often, a serotonergic neuron with a short-lasting response was recorded in the close vicinity of another with a long-lasting one.

3.3. lnteractiion Between Neurotensin and Phenylephrine Serotonergic neurons in the dorsal raphe nucleus are sensitive to both ~tl-adrenergic (Baraban and Aghajanian, 1980; VanderMaelen and Aghajanian, 1983; Yoshimura et al., 1985; Freedman and Aghajanian, 1987; Pan et al., 1994) and NT stimulation (Jolas and Aghajanian, 1996). As the NT receptor and the ~vadrenoceptor are members of the G-protein-coupled receptor family that are coupled to the inositol phosphate pathway, it is possible that they share part of their transduction mechanisms in the, dorsal raphe nucleus. Indeed, we noted that the NT response was very small or absent at the beginning of each experiment because of the presence of 3 #M of the ~q-agonist phenylephrine (PE), which was used to induce firing of serotonergic neurons. Thus, the prominent effect of NT described above appeared tc emerge only after the washout of PE, suggesting the possibility of an occlusion phenomenon. When compared to their actions alone, the effects of NT (1/~M) and a supramaximal concentration of PE (10/~M) were not additive in neurons recorded extracellularly (Fig. 10A) or intra-

cellularly, in voltage-clamp (Fig. 10B). These results show that, in vitro, PE occludes the NT response. Non-additivity was also obtained in serotonergic neurons by using 100/~M of the endogenous adrenoceptor agonist norepinephrine. Interestingly, when supramaximal concentrations of NT were given alone, the induction of firing obtained was always similar or lower than the one obtained with PE (see Fig. 2, Fig. 5B and Fig. I0). This limitation was not due to the fact that the maximal excitability of serotonergic neurons had been reached since NMDA (20/~M) could induce a larger activation of firing than NT or PE (not shown). In general, we did not find any significant difference between the NT and the PE responses recorded intracellularly. Each agonist induced a depolarization of the membrane with only small increase in input resistance. When the neuron was voltageclamped, NT and PE induced an inward current which, in most neurons, was associated with a decrease in input conductance (Fig. 11). At the range of potentials tested in current-clamp mode (-60 to -120 mV), we never obtained a crossing of the I-V curves, only a tendency to converge at very negative potentials. It has already been shown for dorsal raphe nucleus neurons that ~q-adrenoceptor stimulation may involve two different potassium conductances as well as an unknown factor which may prevent the observation of the reversal at potassium equilibrium potential (Pan et al., 1994). Concerning the NT response, converging I-V curves without clear reversal have also been observed in dopaminergic (Mercuri et al., 1993; Jiang et al., 1994) and cholinergic neurons (Alonso et al., 1994). These observations together with our results could be explained by a simultaneous increase in one conductance and a decrease in an other. Studies have

T. Jolas and G. K. Aghajanian

462

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Kirkpatrick and Bourque, 1995). A simultaneous decrease in a potassium and an increase of cation conductance has been shown for substance P (Shen and North, 1992b) and muscarinic (Shen and North, 1992a) stimulation in the locus coeruleus. Our results may also indicate such a dual effect. It is possible that both NT receptor and ~l-adrenoceptor stimulation induce a change in a same ionic conductances. This may be involved in the occlusion phenomenon we observed with these two compounds.

4. POSSIBLE P H Y S I O L O G I C A L RELEVANCE O F NEUROTENSIN'S ACTION ON SEROTONERGIC NEURONS AND CONCLUSION

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Both neurotensinergic and serotonergic systems have been implicated in the regulation of pain/ analgesia, autonomic and sleep-wakefulness regulation. This part of the review will evaluate the possible relationship between neurotensinergic and serotonergic systems with regard to these issues.

4.1. Nociception

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] 150 pA 200 msec Fig. 7. Neurotensin induction of postsynaptic currents in a serotonergic neuron recorded at -60 mV in a brain slice containing the dorsal raphe nucleus, in voltage-clamp mode, with KC1 electrodes. Each panel represents six of 10 successive l-see sweeps recorded in each condition. NT (1 #M) increased the number of postsynaptic currents (B) in comparison to basal activity (A). (C) The NT-induced postsynaptic currents were blocked with LY 293558 (3 #M), a selective AMPA antagonist, indicating their glutamatergic origin. (D) Recovery of the NT response 20 min after the end of LY 293558 application. reported that NT excitation can result from a decrease in a potassium conductance and an activation of a cation or a nonselective conductance (Farkas et al., 1994, 1996; Jiang et al., 1994;

There is some indication that the serotonergic system and in particular the dorsal raphe nucleus and the raphe magnus are involved in the control of pain (for review see Messing and Lytle, 1977; Basbaum and Fields, 1984; Fields et al., 1991; Wang and Nakai, 1994; Millan, 1995; Stamford, 1995). Indeed, stimulation of the dorsal raphe nucleus can elicit strong analgesia without aversive behavioural effects (Oliveras et al., 1979; Fardin et al., 1984). The raphe magnus has been shown to be part of the pain control descending pathway coming from the P A G and projecting to the spinal cord (Fields and Anderson, 1978; Behbehani and Fields, 1979; Dickenson et al., 1979; Pomeroy and Behbehani, 1979; Gray and Dostrovosky, 1983; Fields et al., 1991). At least part of this pain control may involve serotonin (Belcher et al., 1978; Headley et al., 1978; Griersmith and Duggan, 1980; Llewelyn et al., 1983). Another neurotransmitter involved in pain regulation is NT since central administration of NT has been shown to reduce behavioural responses to noxious stimuli in the rat and the mouse, effects blocked by thyrotrophin-releasing hormone but not by naloxone (Clineschmidt and McGuffin, 1977; Nemeroff et al., 1979; Osbahr et al., 1981; Kalivas et al., 1982; Behbehani and Pert, 1984; AI-Rodhan et al., 1991). Part of central effect of NT may involve the serotonergic system since (i) NT injected in the raphe magnus induced hyperalgesic responses at low concentrations and antinociceptive responses at higher concentrations in the tail-flick test in awake rats (Urban and Smith, 1993) and (ii) the analgesic effects of NT injected in the P A G are blocked by electrolytic lesions of the raphe magnus. (Behbehani and Pert, 1984). Furthermore, chronic pain has been shown to increases dorsal raphe nucleus proNT/neuromedin-N m R N A expression (Williams and Beitz, 1993), a raphe nucleus where we demonstrated excitatory effects of NT on serotonergic neurons. Even

Neurotensin and the Serotonergic System

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5 min Fig. 8. Firing rate histogram of a serotonergic neuron recorded in a brain slice containing the dorsal raphe nucleus illustrating the effect of NMDA on the NT response. In this continuous recording, NT (2 and 20 pM, indicated with solid line) produced a strong response only after bath application of NMDA (20 yM, indicated with dotted line). though a relationship between serotonergic and neurotensinergic systems with regard to nociception has not been established directly, such a link is suggested by our electrophysiological results as well as some other ev:idence. Depletion of serotonin in the spinal cord with neurotoxin 5,7-dihydroxytryptamine or with the inhibitor of synthesis of seroto-

nin p-chlorophenylalanine blocked for a week the antinociceptive effects of N T injected intracisternally (Naranjo et al., 1989). But another study found opposite effects, i.e. potentiation of the antinociceptive effects of NT, after p-chlorophenylalanine treatment (Long et al., 1984). The latter study also reported that central administration of N T was ac-

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T. Jolas and G. K. Aghajanian

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5 min Fig. 10. Firing rate histogram and voltage-clamp recording of two different serotonergic neurons recorded in a brain slice containing the dorsal raphe nucleus illustrating the occlusion of the NT response by PE. (A) In this neuron, recorded extracellularly, NT (1/aM) was perfused before and during a supramaximal concentration of 10 #M of PE. The excitation induced by NT and PE (in the middle) were non-additive. Different concentrations of PE (3, 10 and 30/~M) were applied to determine the maximum effect of PE. Note that this maximum effect was already obtained for the lowest concentration. The dotted and solid lines indicate the period of perfusion of the drugs. (B) In this other neuron voltageclamped at -65 mV, the inward currents induced by NT (1 #M) and PE (10 pM) are, as in (A) non additive. Note that the thickening of the trace during the PE response was due to an increase in synaptic noise (from Jolas and Aghajanian, 1996). companied by increased levels of serotonin in the nucleus tractus diagonalis, dorsal raphe nucleus (compatible with our electrophysiological results) and raphe magnus and by decreased serotonin levels in the posterior medial forebrain bundle and PAG indicating a complex effect of NT on the serotonergic system. 4.2. Autonomic Function

Central serotonergic neurons project to regions of the brain involved in autonomic regulation such as the hypothalamic area (Van De Kar and Lorens, 1979; Sawchenko et al., 1983), the lateral parabrachial nucleus (T6rk, 1985) and the nucleus tractus solitarius (Schaffar et al., 1988). Central administration of serotonin or serotonin agonists has been shown to have hypotensive (for review see Mueller et al., 1982) or hypertensive effects in animals (Merahi et al., 1992; Merahi and Laguzzi, 1995). Hypotensive effects of serotonin might be due to 5HT2 stimulation since local injection in the nucleus tractus solitarius of the 5-HT 2 agonist 2,5dimethoxy-3-bromo-amphetamine (DOB) induced a dose-dependent hypotension and bradycardia blocked by ketanserin in rats (Merahi and Laguzzi, 1995). Central administration of NT has also been shown to have hypotensive effect in the rat (Rioux et al., 1981; see also discussion of Allen and Cechetto, 1995). The dorsal raphe nucleus receives feedback neurotensinergic inputs from the lateral parabrachial nucleus (Petrov et al., 1992). For this reason, part of the serotonin hypotensive action could involve a neurotensinergic stimulation seroto-

nergic neurons in the dorsal raphe nucleus. Nevertheless, direct evidence for a link between serotonergic and neurotensinergic systems in regard to autonomic regulation remain to be determined. 4.3. Behaviourai Arousal

There is strong evidence for an involvement of the serotonergic system in sleep-wakefulness regulation (Adrien, 1995) and that the activity of brain serotonergic neurons is linked to behavioural states (McGinty and Harper, 1976; Trulson and Jacobs, 1979). In particular, it has been shown in the cat that the electrical activity of serotonergic dorsal raphe nucleus neurons is maximal during wake or arousal and progressively decreases in slow wave sleep and paradoxical sleep (McGinty and Harper, 1976; Trulson and Jacobs, 1979). In the dorsal raphe nucleus, NT excitatory effects on serotonergic neurons in vitro are not measurable when phenylephrine, an :q-adrenoceptor agonist, or norepinephrine is used to induce firing in serotonergic neurons (Jolas and Aghajanian, 1996). Phenylephrine occluded the NT response in serotonergic neurons recorded extraceilularly and intracellularly indicating a possible commonality between the transduction pathways of the NT receptor and the ~l-adrenoceptor (Jolas and Aghajanian, 1996). If this occlusion also occurs in vivo, NT should have the greatest effect on the firing of serotonergic neurons when the noradrenergic influence is suppressed or reduced. This could be the case for example during sleep states, when the activity of noradrenergic neurons is reduced (for review see Jacobs,

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5 min Fig. 11. Intracellular current- and voltage-clamp recordings of two serotonergic neurons recorded in a brain slice containing the dorsal raphe nucleus before and after a 1 min perfusion of PE (3/~M) and NT (1/tM). A, B, C, D, E, F are recordings of the same neuron and G of a different one. (A) and (D) Evolution of the voltage with time. (B) and (E) Superimposed traces illustrating the effect on the voltage of increasing hyperpolarizing current steps followed by a constant depolarizing step before (B and E left) and at the peak of action of PE and NT (B and E right). These data were used to plot the I-V curves in (C) and (F). Both PE and NT induced a depolarization of the membrane. Only in the case of PE, the I-V curve converged, but it did not cross between -70 and -120 mV. In this neuron, NT did not induce any change of input resistance. (G) In this neuron, voltage-clamped at -60 mV, PE (3/zM) and NT (1 #M) induced an inward current of 80 pA. These inward currents were accompanied by a small reduction of the input conductance for both PE and NT. The input conductance was measured by giving constant 20 mV hyperpolarizing pulses every 20 sec (from Jolas and Aghajanian, 1996). 1986; Aston-Jones et al., 1991). There is one report indicating that intracerebroventricular administration of N T has an awaking effect on rats and increases the latency of the first episode of intermediate and slow wave sleep stages (Castel et al., 1988). Thus, it would be of interest to determine if local injection of N T in the dorsal raphe nucleus could have simil~Lr effect on sleep-wakefulness stages. 4.4.

Conclusions

Anatomical as well as electrophysiological studies have shown a relationship between the neurotensinergic and the serotonergic system. Rostral raphe nuclei contain N T neurons and in all raphe nuclei N T axons, termir~als and binding sites can be detected. The iden~tity of the neurons upon which N T binding sites are localized remain to be determined. Serotonergic neurons are good candidates since we demonstrated that N T excites a subpopu-

lation of serotonergic neurons. Further investigations are needed to determine the exact ionic mechanism of N T action on serotonergic neurons (e.g. involvement of potassium, sodium and/or nonselective cationic conductances). We demonstrated the existence of two different responses to N T in serotonergic neurons, one which desensitizes and another one which does not. The mechanism of this desensitization and whether or not different responses to N T could be due to the existence of different N T receptors in the dorsal raphe nucleus is still unknown. The development of new selective agonists or antagonists of N T receptors should help to answer the latter point. Finally, the physiological relevance of the action of N T on serotonergic neurons remains to be determined. This review gives three possible tracks for future research based on our results and the literature.

Acknowledgements--The authors would like to thank Sanofi Recherche for the gift of SR 48692. This work was

T. Jolas and G. K. A g h a j a n i a n

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s u p p o r t e d by PHS g r a n t s MH17871, M H I 4 2 7 6 and the state o f Connecticut.

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