Release of cholecystokinin in the central nervous system

Neurochem. Int. Vol. 22, No. 6, pp. 519-527,1993 Printedin Great Britain.All rightsreserved

0197-0186/93$24.00+ 0.00 Copyright© 1993PergamonPress Ltd

INVITED REVIEW RELEASE OF CHOLECYSTOKININ IN THE CENTRAL NERVOUS SYSTEM MAURIZIO RAITERI*, PAOLO PAUDICE a n d FRANCESCO VALLEBUONA Istituto di Farmacologia e Farmacognosia, Universitfidegli Studi di Genova, Viale Cembrano 4, 16148 Genova, Italy CRITIQUE by : J. DE BELLEROCHE Abstract--The octapeptide cholecystokinin (CCK) is one of the most abundant neuropeptides of the ceritral nervous system. A number of features (for instance heterogeneity of the regional distribution, subcellular localization at the nerve terminal level, calcium-dependent release upon nervous tissue depolarization) support the candidacy of CCK as a neurotransmitter. The reported co-existenceof CCK and dopamine in some meso-limbic neurons has led to speculation that the neuropeptide may interact with the catecholamine in neuropsychopathologies linked to dopamine dysfunctions, like schizophrenia. Data from the experimental animals have so far generated conflictingresults. It should be noted that the interactions between CCK and dopamine, and, in particular, the effects of CCK and dopamine on each other release, both in vitro and in vivo, have been poorly investigated and would require special attention. Evidence is accumulating that CCK may participate in the expression of anxiety. Indeed antagonists at the central CCK receptors exhibit anxiolytic activity in the laboratory animal. An interesting linkage appears to exist in the brain between 5-hydroxytryptamine (5-HT) and CCK. Activation of 5-HT3 receptors was found to increase CCK release from rat cortical or nucleus accumbens synaptosomes. Interestingly, antagonists at 5-HT3 receptors appear to possess anxiolytic activity. Recent studies carried out in conscious unrestrained rats show that the calcium-dependent,tetrodotoxin-sensitiverelease of CCK-like immunoreactivityevoked in the rat frontal cortex by veratrine infusion can be inhibited by submicromolar concentrations of 5-HT3 receptor antagonists. It seems legitimate to conclude that 5-HT and CCK interact in the living brain, the former increasing the release of the latter through activation of receptors of the 5-HT3 type. On the basis of this interaction both 5-HT3and CCK receptor antagonists may become novel anxiolytics.

Cholecystokinin (CCK) is one of the most abundant neuropeptides of the mammalian central nervous system (Vanderhaeghen et al., 1975 ; Dockray, 1976). It is an octapeptide which occurs in brain predominantly in the sulphated form (Dodd et al., 1980; Marley et al., 1984; Rehfeld, 1985). The regional distribution of CCK is heterogeneous : the peptide is particularly concentrated in the cerebral cortex, hippocampus, basal ganglia, hypothalamus and periaqueductal grey matter (Larsson and Rehfeld, 1979; Dockray, 1980; Beinfeld et aL, 1981 ; Emson and Marley, 1983). It was estimated that approx 1% of neurones in the cerebral cortex contain CCK; in layers II, III and VI these neurones appear to be non-pyramidal cells forming synaptic contacts primarily with pyramidal cells within the cerebral cortex (McDonald et al.,

*To whom correspondence should be addressed.

1982; Parnavelus, 1986). However, a portion of cortical CCK, especially localized in the frontal cortex, is thought to be extrinsic, possibly originating from the ventral tegmental area and contained in dopaminergic neurones where the peptide has been found to co-exist with dopamine (H6kfelt et al., 1980a,b). Several CCKcontaining neurones in cerebral cortex also store GABA as indicated by immunocytochemical experiments (Hendry et al., 1984). Subcellular localization studies of CCK-like immunoreactivity (CCK-LI) in the central nervous system have identified high levels in synaptosomal preparations (Pinget et al., 1978, 1979; Dodd et al., 1980; Emson et al., 1980). Moreover, experiments in vitro have shown that CCK-LI can be released from brain slices (Emson et al., 1980) or synaptosomes (Pinget et al., 1979; Dodd et al., 1980; Paudice and Raiteri, 1991 ; Verhage et al., 1991) exposed to depolarizing stimuli. 519

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Binding sites for CCK are widely distributed throughout the central nervous system (Innis and Snyder, 1980; Saito et al., 1980). CCK receptors are classified as CCK-A and CCK-B, the majority of the brain receptors being of the latter type (Hill and Woodruff, 1990 ; Woodruff et al., 1991). Although CCK has been shown to excite central neurones (Dodd and Kelly, 1979: Boden and Hill, 1988), inhibitory postsynaptic actions have been observed (see Lopes da Silva et al., 1990). So far the physiological and behavioral significance of CCK as a putative transmitter in the central nervous system is poorly understood. The abovementioned co-existence of CCK and dopamine in a subpopulation of midbrain dopamine-containing neurones (H6kfelt et al., 1980a,b) has led to speculation that CCK may play a role in the "dopamine hypothesis of schizophrenia". Data from the experimental animals have, however, generated conflicting hypotheses (see, for a recent review, Crawley, 1991). Indeed, it is not clear whether CCK receptor agonists or antagonists may be of help m neuropsychopathologies linked to dopaminergic dysfunctions. In other words it is unknown whether an increased release of CCK in limbic areas cooperates with dopamine in the expression and maintenance of the schizophrenic condition or such a release is beneficial to the pathology. There is some evidence that C C K represents a satiety signal at the hypothalamic level where it may play a role in the control of feeding behavior (Gibbs et al., 1973: Della-Ferra and Baile, 1979: Myer and McCaleb, 1981). An involvement of the peptide in analgesia has also been proposed: CCK may act as an endogenous opioid receptor antagonist in the control of pain (Baber et al., 1989). Recent findings have linked brain CCK to anxiety (for reviews see Ravard and Dourish, 1990 ; Woodruff et al., 1991 ; Woodruff and Hughes, 1991). Such a role was first suggested by electrophysiological experiments showing that the neuronal excitation caused by CCK microiontophoretically applied to the pyramidal neurones of the rat hippocampus was suppressed by benzodiazepine anxiolytics (Bradwein and De Montigny, 1984). The number of CCK receptors in the frontal cortex was found to be increased following withdrawal from chronic diazepam treatment (Harro et al., 1990). Moreover, it has been shown that CCK at very low doses elicited panic-like attacks in patients suffering from panic disorders (De Montigny, 1989). Interestingly, selective CCK-B receptor antagonists display potent anxiolytic activity in the laboratory animal (Hughes et al., 1990; Woodruff and Hughes, 1991).

All together, the data available suggest that CCK may be a neurotransmitter in the mammalian central nervous system. A critical question centers around whether CCK is released from nerve terminals under physiological conditions. Studies of CCK release from terminal regions not only in vitro but particularly ht vivo, during drug treatments and behavioral stimuli expected to influence CCK-containing neurons, are essential to answer this question. The present article deals with in vitro and in vivo studies of the release of central CCK and of its modulation through the activation of different receptors. CHOLECYSTOKININ RELEASE IN VITRO

The release of cholecystokinin-like immunoreactivity (CCK-LI) from central nervous system tissue in vitro has been demonstrated using synaptosomal fractions of rat cerebral cortex (Pinget et al., 1979; Dodd et al., 1980; Paudice and Raiteri, 1991 ; Verhage et al., 1991) and nucleus accumbens (Paudice and Raiteri, 1991) as well as slices of rat cortex and hypothalamus (Emson et al., 1980 ; Rose et al., 1989) and of corpus striatum (Meyer and Krauss, 1983). The release was in each case induced by chemical depolarization and was calcium-dependent. The K +-evoked overflow of CCK-LI from rat brain cortex synaptosomes superfused according to Raiteri et al. (1974) is illustrated in Fig. I. Increasing conI00

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Fig. I. K +-evoked overflow of cho]ecystokinin-]ikeirnmunoreactivity (CCK-LI) From rat brain cortex synaptosomes

and its calcium-dependence. Synaptosomes were prepared and superfused as previouslydescribed (Paudice and Raiteri, 1991). A 90 s pulse of 15, 25 or 35 mM KC1 was applied after 39 min of superfusion. The Ca 2" -free medium, when used, was introduced 18 min before the depolarizing stimuli. Shown are means _+ SEM (vertical bars) of 3 experiments in triplicate. (From Paudice and Raiteri, 1991).

Invited Review centrations of KCI in the superfusion medium (from 15 to 35 mM) resulted in a concentration-dependent increase in CCK-LI release, and the K+-evoked overflow of CCK-LI was largely calcium-dependent. Verhage et al. (1991) who studied CCK-LI release from hippocampal synaptosomes in comparison with that of glutamate or GABA, found that the release of the neuropeptide differs from that of classical transmitters. The release of CCK-LI was slower in onset and exhibited a higher apparent calcium-sensitivity. These differences may be related to differences in storage between classical transmitters and neuropeptides. The latter compounds are thought to be stored in large, dense-cored vesicles, while classical transmitters appear to be concentrated inside small clear-cored vesicles. Moreover, the neuropeptide containing vesicles are randomly distributed in the nerve terminal cytoplasm (Zhu et al., 1986), whereas the small vesicles storing classical transmitters are lined up at the active zone of the terminal (Simon and Llinas, 1985; Augustine et al., 1987 ; Smith and Augustine, 1988). These morphological differences are reflected in the finding that the release of neuroactive peptides requires higher frequencies of stimulation than the release of classical transmitters (see Bartfai et al., 1988, for a review). CHOLECYSTOKININ RELEASE IN VIVO

Using a spinal superfusion procedure, Yaksh et al. (1982) found that high-K + evoked a calcium-dependent increase of CCK-LI release which essentially consisted of CCK-8 peptide. More recently, the in vivo release of CCK-LI was investigated using brain microdialysis (Butcher et al., 1989; Takita et aL, 1989; De Mesquita et al., 1990; Maidment et al., 1991 ; Vallebuona et al., 1993). In some of these studies technical problems have been met, originating in part from the low extracellular levels of CCK-LI (see, in particular, De Mesquita et al., 1990). However, reliable basal levels and quite reproducible stimulus-evoked overflows of CCK-LI could recently be obtained in rat frontal cortex (Vallebuona et al., 1993; see below) by using the transcerebral technique described by Imperato and Di Chiara (1984), a dialysis probe allowing a particularly high recovery of CCK-LI and a traditional radioimmunoassay method (Rehfeld, 1978). The pattern of CCK-LI release observed in the frontal cortex of freely-moving rats upon infusion of veratrine is illustrated in Fig. 2. The alkaloid potently increased CCK-LI release. Starting from a basal outflow of 2.4+0.7 pg/20 min, the peak effect of CCKLI release upon veratrine depolarization amounted to

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rain Fig. 2. In vivo CCK-LI release evoked by veratrine from rat frontal cortex. A poliacrylonitrile membrane (AN69 HF, Hospal SpA) was stereotaxically inserted into the frontal cortex. The exchanging zone was 8 mm. Animals were allowed to recover from anesthesia for 24 h. Then they were placed in perspex cages and the probes were perfused with an artificial cerebrospinal fluid at a constant flow rate of 5 #1/min. Every 20 min, samples of perfusate were collected in minivials and assayed for their CCK-LI content by radioimmunoassay essentially according to Rehfeld (1978). Veratrine (150/aM) was administered for 20 min (black horizontal bar) through the dialysis probe. TTX (1/aM)-containing or Ca2+-free, Mg 2+ (20 mM)-enriched medium was applied as indicated by the arrow and perfused throughout the experiment. Each value represents the mean + SEM of 6-8 experiments. • *P < 0.01 vs basal values (Tukey's test). • veratrine; ~ veratrine+ 1 /aM TTX; • veratrine+Ca2+-free, Mg 2+ (20 mM)-enriched artificial CSF.

62.3___ 10 pg/20 min. This value was reached in the first 20 min fraction, then the overflow of CCK-LI returned to basal levels after 80 min. The overflow of CCK-LI evoked by veratrine was totally calciumdependent and tetrodotoxin(TTX)-sensitive. These characteristics are suggestive of a neuronal origin for the peptide released upon depolarization with the alkaloid. It is well known that veratrine is able to trap in the open state voltage-activated TTX-sensitive Na ÷-channels, causing a massive increase ofNa + conductance and a consequent permanent, "clamped" depolarization (Blaustein, 1975). Another depolarizing agent, often used in in vitro studies with synaptosomes or brain slices, consists of a medium enriched in K +. Solutions containing highK + depolarize excitable membranes by decreasing the K + gradient. Infusion into the rat frontal cortex of artificial cerebrospinal fluid (CSF) containing high concentrations of KC1 produced a CCK-LI overflow which was dependent on the K + concentration. Con-

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centrations of KCI higher than 50 mM were required to elicit CCK-LI release. The K+-evoked release of CCK-LI was calcium-dependent but TTX-insensitive (Maidment et al., 1991 ; Vallebuona et al., 1993. In/t~ vitro studies (see, for instance, Raiteri et al., 1990), the K+-evoked release of some transmitters appeared to be TTX-sensitive, provided that the concentration of K + was kept low (10 15 raM). Whether the in vivo release of CCK-LI evoked by relatively low K concentrations is sensitive to TTX remains to be established. An additional tool rarely used to depolarize excitable membranes is represented by 4-aminopyridine. The drug blocks K + channels thus leading to membrane depolarization. According to McMahon and Nicholls (1991), 4-aminopyridine causes the nerve terminals to undergo spontaneous action potentials involving a repetitive opening of TTX-sensitive Na +channels. The infusion of 4-aminopyridine (I mM for 20 min) into the dialysis probe caused release of CCKLI in rat frontal cortex. The overflow of CCK-LI evoked by 4-aminopyridine was completely TTX-sensitive, in keeping with the above idea. It was also totally calcium-dependent, suggesting an exocytotic like release of the peptide as in the case of the veratrine- or the K+-evoked release (Vallebuona et al., 1993). As to the peptide species released during microdialysis, results obtained from gel chromatography or high-performance liquid chromatography (HPLC) analysis of the dialysates show that the depolarization-evoked release of CCK-LI was largely accounted for by the CCK octapeptide sulphate (Takira et al., 1989; Maidment et al., 1991 : Vallebuona et al., 1993). Very little is known on the turnover of neuropeptides. For instance it is difficult to say whether the effect of veratrine declines (see, for instance, Fig. 2) because of compensatory mechanisms occurring at the level of ion channels or because of depletion of a "releasable pool" of CCK-LI. The question was approached in two ways. First, 150 #M veratrine was infused for 100 rain instead of 20 rain. The pattern representing the CCK-LI overflow in the two conditions was almost superimposable, a result still open to both of the above possibilities. However, when two consecutive 20-min infusions with 150/tM veratrine were applied, at min 20 and at 220 rain, respectively, the second infusion caused very little CCK-LI release (Vallebuona et al., 1993). Assuming that the time elapsed between the first and the second stimulus should be long enough to permit restoration of an ion channel mechanism possibly impaired after the first stimulus, these data suggest that one single stimulus

with 150 #M veratrine was sufficient to deplete the "'releasable pool" of the peptide and that more than 200 rain are required to replenish the depleted vesicles. M O D U L A T I O N OF" CHOLECYSTOKININ REI,EASE IN 1~TTRO

The release of CCK-LI induced by veratrine from rat striatal slices was reported to be enhanced by activation of dopaminergic receptors (possibly of the D2 type) and inhibited through receptors of the D-I type (Meyer and Krauss, 1983). Controversial results have however been reported by Hutchison et al. (1986). In particular: no effects with either D-I or D2 receptor agonists and inhibition of CCK-LI release following indirect activation of D-2 reccptors by dopamine released upon exposure of striatal slices to amphetamine. To add further confusion, a very recent work reports a D-I receptor-mediated increase of CCK-LI release (Brog and Beinfeld, 1992). As previously mentioned, dopamine and CCK have been reported to be co-localized in a subpopulation of neurons sited in the ventral tegmental area and which project to the nucleus accumbens and the frontal cortex (H6kfelt et al.. 1980a,b). Overactivity of m e s o - l i m b i c and meso-cortical dopamine systems has been hypothesized as being responsible for schizophrenia (Matthysse, 1973; Snyder, 1973; Stevens, 1973). Thus, the interaction of CCK and dopamine in these areas might be relevant in the expression of schizophrenic symptoms. Modulation by DA of CCK-LI release from slices of rat posterior nucleus accumbens was observed (Martin et al., 1986). The effect appears to be mediated through receptors of the D-2 type and to be biphasic. Low concentration of D-2 receptor agonists inhibited, while higher concentrations increased the K +-evoked CCK-LI release. Exposure to ( -)baclofen, a GABAB receptor agonist, of rat striatal slices caused inhibition of the K ~evoked release of CCK-L1, although the exact location of the G A B A , receptors, whether on the CCK-releasing terminals or on other structures, was not determined (Conzelmann et al., 1986). Morphine inhibited the K+-stimulated release of CCK-LI from cat hypothalamic slices, an effect mimicked by 0.1 #M levorphanol but not by the optical isomer dextrophan (Micevych et al., 1982). This inhibition of the release of CCK, a putative satiety factor, may represent a cellular mechanism explaining the finding that opiates stimulate, while naloxone inhibits, feeding (Holtzman, 1979; Sanger and McCarthy, 1980). Interestingly, the same authors found that opiates did not inhibit the CCK-LI release evoked by

Invited Review high-K + from cortical slices, indicating a regional specificity for the effects of opiates on CCK release (Micevych et al., 1985). Opioid-CCK interactions at the spinal cord level were investigated. In fact, numerous data in the literature support the contention that CCK interacts with opioids in pain mechanisms. The overflow of CCKLI produced by K+-depolarization of rat spinal cord slices was increased by morphine. Antiopioid effects of CCK have been reported (Faris, 1985; Baber et al., 1989). Thus the increase of CCK release may counteract, at least in part, the effect(s) of morphine, thus explaining why associations of morphine and CCK receptor blockers are more analgesic than the opiate alone (Faris, 1985 ; Dourish et al., 1988). According to Bray et al., (1989) kainic acid inhibited the release of CCK-LI from rat hippocampal slices exposed to high-K +. On the other hand, the excitotoxin was reported to increase the K +-evoked CCKLI release from slices of rat cerebral cortex (Bandopadhyay and de Belleroche, 1991). The latter authors also found that quisqualic acid and N-methylD-aspartate potentiated the CCK-LI overflow. Administration of CCK can induce anxiety both in the experimental animal and in man and CCK receptor antagonists display anxiolytic activity (Hughes et al., 1990; Woodruff and Hughes, 1991). 5-Hydroxytryptamine (5-HT) has long been implicated in the control of anxiety (Iversen, 1984; Chopin and Briley, 1987). In general, it seems that stimulation of the 5-HT system results in an anxiogenic effect. Accordingly, anxiolytic activity has been reported for 5-HT3 receptor antagonists (Jones et al., 1988; Lecrubier et al., 1990). This similarity between 5-HT and CCK prompted us to investigate whether the two systems could interact in the brain. The question was first approached in vitro by examining the action of 5-HT on the release of CCK-LI from K+-depolarized rat brain synaptosomes. In both cerebrocortex and nucleus accumbens synaptosomes 5-HT potently increased (ECs0 ~ 0.45 nM; maximal effect about 60% at 10 nM 5-HT) the calcium-dependent depolarization-evoked release of CCK-LI. The 5-HT3 receptor agonist 1-phenylbiguanide (Ireland and Tyers, 1987) mimicked 5-HT (Fig. 3). The effect of 5-HT was not antagonized by methiothepin, a 5-HT~/5-HT2 receptor antagonist, but it was by ondansetron or by tropisetron, two drugs which, at concentrations in the low nanomolar range, behave as selective 5-HT3 receptor antagonists (Richardson et al., 1985 ; Butler et al., 1988) (Fig. 4). Thus 5HT, acting at 5-HT3 receptors, appears to be a potent releaser of CCK-LI in cortex and in n. accumbens.

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Fig. 3. Effects of 5-hydroxytryptamine (5-HT) or l-phenylbiguanide on the K + (15 mM)-evoked overflow of CCKLI from rat cortical or n. accumbens synaptosomes. The agonists were added concomitantly with high-K +. Each point represents the mean __+ SEM of 3-6 experiments in triplicate. (Q) 5-HT (cortex) ; (A) 5-HT (accumbens) ; (O) 1phenylbignanide (cortex). (From Paudice and Raiteri, 1991). 8O o

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Fig. 4. Antagonism by ondansetron or tropisetron of the enhancement by 5-hydroxytryptamine (5-HT) of CCK-LI release from cortical or n. accumbens synaptosomes. 5-HT (10 nM) was added as the same time as the high-K+ was applied; antagonists 8 min before 5-HT. Each point represents the mean + SEM of 3-5 experiments in triplicate. Concentrations of antagonists higher than 0.1 nM antagonized 5-HT significantly (P < 0.001) : (O) 5-HT plus tropisetron (cortex) ; (O) 5-HT plus ondansetron (cortex); ([-1) 5-HT + tropisetron (n. accumbens). (From Paudice and Raiteri, 1991).

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The use of synaptosomes superfused in a thin layer makes it likely that the 5-HT3 receptors in point are sited on CCK-releasing terminals. The 5-HT/CCK interaction may provide a biological substrate encouraging the clinical development of both 5-HT 3 and CCK receptor antagonists as novel anxiolytic drugs. MODULATION OF CHOLECYSTOKININ RELEASE IN

V1VO

Very few studies have investigated in vivo the effects of possible endogenous or exogenous modulators of the release of CCK. Using a procedure to perfuse the rat spinal cord Rodriguez and Sacristan (1989) found that the release of CCK-LI was enhanced following K + depolarization or by direct activation of high threshold peripheral afferents obtained through sciatic nerve stimulation. Moreover, the efflux of CCK-LI was inhibited by a selective agonist at opioid receptors of the # type, an effect antagonized by naloxone. These data in vivo support an involvement of CCK in analgesia. In spite of the repeatedly proposed hypothesis that DA and CCK interact in limbic areas and may affect each other's contribution to the expression of the schizophrenic symptomatology, only the effects of CCK on DA release have been explored in vivo unfortunately with conflicting results (Wang et al., 1984; Phillips et al., 1986; Ruggeri et al., 1987; Crawley, 1991). On the other hand, to our knowledge, no reports have appeared so far concerning the possible effects of dopamine on the in vivo release of CCK. This appears to be due to the technical difficulties met by several authors during their studies o f in vivo CCKL1 release from limbic areas (see, in particular, De Mesquita et al., 1990). Starting from the above described observation that 5-HT, by activating receptors of the 5-HT3 type, enhanced the release of CCK-LI from rat brain cortex synaptosomes exposed to depolarizing concentrations of K + (Paudice and Raiteri, 1991) and considering that antagonists at the 5-HT3 receptors have been reported to possess anxiolytic activity in the laboratory animal (Jones et al., 1988), we investigated the effects of antagonists at 5-HT3 receptors on the release of CCK-LI from the rat frontal cortex using intracerebral microdialysis (Raitori et al., 1993). Assuming that an increase of CCK release by 5-HT is anxiogenic, 5-HT3 receptor antagonists would be expected to decrease CCK-LI release. Two 5-HT3 receptor antagonists, ondansetron and tropisetron, decreased the release of CCK-LI evoked

by veratrine infusion. As mentioned above, these two drugs are known to behave as selective 5-HT~ receptor antagonists when used at concentrations lower than micromolar (Richardson et al., 1985; Butler et al., 1988). Figure 5 shows the results obtained when ondansetron (I or 0.1 #M) was infused through the dialysis probe. Also on the basis of the previous work carried out in vitro (Paudice and Raiteri, 1991), the following model could be proposed: depolarization with veratrine causes in vivo release of CCK as well as of 5-HT : endogenous 5-HT reaches 5-HT~ receptors possibly sited on CCK-containing neurons and potentiates the veratrine-induced release of CCK. In other words, the highest peak observed in Fig. 5 would include the peptide released by veratrine and that released by 5HT through activation of 5-HT~ receptors. Infusion of 5-HT~ receptor antagonists prevents the effect of endogenous 5-HT thus causing decrease of CCK-L1 release. This effect might be at the basis of the reported anxiolytic activity of 5-HT3 receptor antagonists. Interestingly, CCK receptor antagonists also are endowed with anxiolytic properties (Hughes et al., 6O

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TIME OF PERFUSION (min) Fig. 5. Effect of ondansetron on the release of CCK-LI evoked by veratrine in frontal cortex of conscious rats. For technical details see legend to Fig. 2. Veratrine (150 #M) was added when indicated by the arrow and was present for 20 min. Ondansetron (0. l or 1 #M) was perfused from t = 0 throughout. Each value represents the mean + SEM of 6 experiments (two-way ANOVA and Tukey's test on significant effects). *P < 0.01 vs veratrine. + P < 0.05 vs 0.1 ILM ondansetron. (Q) veratrine; (V) veratrine+0.1 BM ondansetron ; (V)veratrine +1 I~M ondansetron.

Invited Review 1990; W o o d r u f f and Hughes, 1991), thus supporting the idea that a reduction of C C K release (for instance following administration of 5-HT3 receptor antagonists) may reflect anxiolysis. Antagonists at both C C K and 5-HT3 receptors may thus become novel anxiolytics. These drugs have been proposed to be effective also to prevent opiate tolerance (Dourish et al., 1988), or the anxiogenic effect of withdrawal from morphine (Costall et al., 1990), two phenomena in which the interaction between 5H T and C C K first observed in synaptosomes (Paudice and Raiteri, 1991) might also be involved. Acknowledgements--The authors wish to thank Mrs Maura Agate for her expert secretarial assistance. This work was supported by grants from the Italian M.U.R.S.T. and from the Italian C.N.R.

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