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Experiments using the binding of various ligands for monoamines to rat brain membranes and synaptosomal preparations for studying monoamine uptake and release have shown that d-fenfluramine is more potent than the l isomer in inhibiting 5-HT uptake, whereas d-norfenfluramine preferentially releases 5-HT from a reserpine-insensitive compartment. Studies on brain monoamine metabolism in intact animals have shown that the d and l isomers of fenfluramine at relatively low doses have a specific action on brain 5-HT and catecholamines, respectively. Based on the different ability of metergoline and ritanserin to displace 5-HT2 binding to rat brain membranes and to antagonize d-fenfluramine's anorexia, evidence has been provided that d-fenfluramine preferentially uses 5-HT1 sites in the rat brain to cause anorexia in this animal species. Finally, characteristics, regional distribution, and pharmacological characterization of a high-affinity [3H]d-fenfluramine binding to rat brain membranes have been described. This binding appears to be different from 5-HT uptake sites ([3H]imipramine binding) and 5-HT receptors and is not regionally related to the endogenous levels of 5-HT in the rat brain. It is, however, preferentially displaced by some agents using 5-HT to cause anorexia in rats, raising the possibility that it is somewhat related to 5-HT mechanisms involved in feeding control.

Using in vivo intracerebral microdialysis in conscious, freely moving rats, we examined the effect of flibanserin, a potential antidepressant drug with high affinity for human 5-HT1A receptors and four–50-fold lower affinity for 5-HT2A and D4 receptors, on basal extracellular concentrations of serotonin (5-hydroxytryptamine, 5-HT), dopamine (DA) and noradrenaline (NA) in selected regions of the rat brain. Flibanserin at 3 and 10 mg kg−1 significantly reduced extracellular 5-HT in the prefrontal cortex (by 30 and 45%) and dorsal raphe (35 and 44%), but had no effect on extracellular 5-HT in the ventral hippocampus. The 3 and 10 mg kg−1 doses raised extracellular NA to a similar extent in the prefrontal cortex (47 and 50%). In all, 10 mg kg−1 raised extracellular DA in the prefrontal cortex (63%) whereas 3 mg kg−1 had no significant effect. Pretreatment with the selective 5-HT1A receptor antagonist WAY100,635 (0.3 mg kg−1) 30 min before 10 mg kg−1 flibanserin completely antagonized the latter's effects on extracellular 5-HT, DA and NA in the prefrontal cortex. WAY100,635 by itself had no effect on cortical extracellular monoamines. The results show that the stimulation of 5-HT1A receptors plays a major role in the effect of flibanserin on brain extracellular 5-HT, DA and NA. British Journal of Pharmacology (2003) 139, 1281–1288. doi:10.1038/sj.bjp.0705341

Experiments were performed to confirm that noradrenergic terminals regulate extracellular concentrations of dopamine (DA) in the frontal cortex of rats. The effects of 20 mg/kg 1-[2-[bis(4-fluorphenyl)methoxy]-ethyl]-4-(3- phenylpropyl)piperazine (GBR 12909), a selective inhibitor of DA uptake, and 2.5 mg/kg desipramine (DMI) on the extracellular concentrations of DA in the frontal cortex and striatum were studied in rats given 6-hydroxydopamine (6 micrograms/microliters) bilaterally into the locus coeruleus to destroy noradrenergic terminals. GBR 12909 increased dialysate DA similarly in the striatum of vehicle and 6-hydroxydopamine-treated rats, whereas in the frontal cortex it raised DA concentrations only in lesioned animals. DMI raised extracellular DA concentrations in the frontal cortex but not in the striatum of controls. The effect of DMI on cortical DA was abolished by the 6-hydroxydopamine lesion. GBR 12909, at a subcutaneous dose of 20 mg/kg, further increased cortical dialysate DA in rats given DMI intraperitoneally at 20 mg/kg or through the probe at 10(-5) mol/L. The data support the hypothesis of an important regulation of the extracellular concentrations of DA in the frontal cortex by noradrenergic terminals.

After a single intraperitoneal injection of the irreversible tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA; 300 mg/kg), there was a rapid down-regulation of serotonin (5-HT) transporter mRNA levels in cell bodies. This change was significant at 1 and 2 days after PCPA administration within the ventromedial but not the dorsomedial portion of the dorsal raphe nucleus. Seven days after PCPA treatment, 5-HT transporter mRNA levels were significantly elevated compared with controls in both regions of the dorsal raphe nucleus. PCPA administration produced no change in the [3H]citalopram binding and synaptosomal [3H]5-HT uptake in terminal regions at 2 and 7 days after treatment but significantly reduced both these parameters by approximately 20% in the hippocampus and in cerebral cortex 14 days after PCPA administration. The striatum showed a lower sensitivity to this effect. No significant changes were observed in the levels of [3H]citalopram binding to 5-HT cell bodies in the dorsal raphe nucleus. In the same animals used for 5-HT transporter mRNA level measurements, levels of tryptophan hydroxylase mRNA in neurons of the ventromedial and dorsomedial portions of the dorsal raphe nucleus were increased 2 days after PCPA administration and fell to control levels 7 days after injection in the ventromedial region but not in the dorsomedial portion of the dorsal raphe nucleus, where they remained significantly higher than controls. Altogether, these results show that changes in 5-HT transporter mRNA are not temporally related to changes in 5-HT transporter protein levels. In addition, our results suggest that the 5-HT transporter and tryptophan hydroxylase genes are regulated by different mechanisms. We also provide further evidence that dorsal raphe 5-HT neurons are differentially regulated by drugs, depending on their location.

The present study investigated whether 5-HT(2C) receptors in the ventrotegmental area and prefrontal cortex regulate basal and stimulus-evoked dopamine release in the prefrontal cortex. Using the in vivo microdialysis technique in conscious rats, we studied the effect of a selective 5-HT(2C) receptor agonist, Ro60-0175, on basal and immobilization stress-induced dopamine release in the prefrontal cortex. Ro60-0175 intraperitoneally (2.5 mg/kg) and into the ventrotegmental area (10 microg/0.5 microL) completely antagonized the effect of stress on extracellular dopamine without altering basal levels. Infusion of 10 microm Ro60-0175 through the cortical probe had no significant effect on basal and stress-induced dopamine release. SB242084 (10 mg/kg), a selective antagonist of 5-HT(2C) receptors, significantly increased basal extracellular dopamine and completely prevented the effect of intraperitoneal and intraventrotegmental Ro60-0175 on the stress-induced rise of extracellular dopamine, but had no effect itself in stressed rats. The results show that Ro60-0175 suppresses cortical dopamine release induced by immobilization stress through the stimulation of 5-HT(2C) receptors in the ventrotegmental area. While confirming that endogenous 5-HT acting on 5-HT(2C) receptors tonically inhibit basal dopamine release in the prefrontal cortex, the present findings suggest that the stimulation of 5-HT(2C) receptors with an exogenous agonist preferentially inhibit stimulated release.

Objective: The present study investigated the effect of stimulating 5-HT1A receptors in the dorsal raphe on the impairment of spatial learning caused by intrahippocampal 7-chloro-kynurenic acid (7-Cl-Kyn) in naive rats and in rats familiar with the general requirements of the task. Methods: A week after implantation of cannulae to give access to the dorsal raphe (DR) and the CA1 region of the dorsal hippocampus, rats started their 5 days acquisition training on a two-platform spatial discrimination task in a water maze. On each acquisition day, WAY 100635 and 8-OH-DPAT alone or in combination were injected into the dorsal raphe (DR) 5 min before intrahippocampal injections of 7-Cl-Kyn which was given 10 min before the training session. Similar experiments were conducted in rats that had been familiarized with the general requirements of the task by pretraining them in the water maze in the absence of distal cues. Results: 7-Cl-Kyn (3 µg/µl), injected bilaterally in the CA1 region of the dorsal hippocampus, impaired choice accuracy with no significant effect on choice latency. Rats treated with 7-Cl-Kyn tended to spend more time swimming close to the pool walls and made more errors of omission than controls in the first two sessions. Administered into the DR, the 5-HT1A receptor agonist 8-OH-DPAT (1 µg/0.5 µl) had no effect on any parameter of rats' performance but antagonized the impairment of choice accuracy caused by intrahippocampal 7-Cl-Kyn. Injected into the DR, 1 µg/0.5 µl WAY 100635, a 5-HT1A receptor antagonist, had no effect on rats' performance or on the impairment caused by intrahippocampal 7-Cl-Kyn, but antagonized the effect of 8-OH-DPAT on the 7-Cl-Kyn-induced deficit. The non-mnemonic behavioral disturbances shown by naive rats treated with 7-Cl-Kyn were greatly reduced in pretrained rats which, nevertheless, showed a marked impairment of choice accuracy similar to that of naive rats. As in previous experiments, administration of 1 µg/0.5 µl 8-OH-DPAT in the dorsal raphe antagonized the impairment of choice accuracy caused by intrahippocampal 7-Cl-Kyn without any effect on other parameters of rats' performance. Conclusions: The results show that stimulation of presynaptic 5-HT1A receptors in the dorsal raphe counteracts the deficit in spatial learning caused by a reduced NMDA-mediated excitatory input on pyramidal cells in the hippocampus. The possible mechanisms and the importance of these findings for the symptomatic treatment of memory disorders in man are discussed.

1. This study investigated the effect of acute (2 days) and chronic (14 days) treatment with a selective inhibitor of noradrenaline uptake, reboxetine (10 mg kg(-1) day(-1)) by osmotic pumps, on extracellular noradrenaline and the sensitivity of alpha(2)-adrenoceptors in the prefrontal cortex of rats. 2. The effect of continuous infusion of reboxetine for 14 days on cortical extracellular noradrenaline was significantly higher (599% of vehicle levels) than after 2 days (263% of vehicle levels). 3. Brain concentrations of reboxetine after 2 and 14 days of infusion were 37.9+/-17.8 and 37.1+/-7.7 ng g(-1), respectively. 4. Reboxetine infused for 2 and 14 days significantly increased extracellular dopamine in the prefrontal cortex, to a similar extent (257 and 342% of vehicle levels, respectively), whereas extracellular 5-HT was not modified by either treatment. 5. Clonidine (10 and 30 microg kg(-1) i.p.) reduced cortical extracellular noradrenaline similarly in animals treated with reboxetine or vehicle for 2 days whereas the effects in rats infused with reboxetine for 14 days were markedly less than in vehicle-treated animals. 6. Clonidine (0.05 and 0.2 microM), infused through the dialysis probe into the prefrontal cortex, reduced cortical extracellular noradrenaline much less in rats treated with reboxetine for 14 days than in vehicle-treated animals. 7. Reboxetine's effect on extracellular noradrenaline in the prefrontal cortex was greater after chronic treatment and could be associated with desensitization of terminal alpha(2)-adrenoceptors that normally serve to inhibit noradrenaline release.

We studied the effects of low doses of 8-OH-DPAT, a 5-HT1A receptor agonist, on the impairment of spatial learning caused by scopolamine injected into the CA1 region of the dorsal hippocampus of rats performing a two-platform spatial discrimination task. Bilateral injections of 4 μg (in 1 μl) of scopolamine into the CA1 region of the dorsal hippocampus 10 min before each training session impaired choice accuracy with no effect on choice latency and errors of omission. Administered subcutaneously 20 min before each training session, 8-OH-DPAT 10 and 30 (but not 3) μg kg−1 did not modify choice accuracy but prevented the impairment by intrahippocampal scopolamine Injection of 1.0 μg (in 0.5 μl) of WAY 100635, a 5-HT1A receptor antagonist, into the dorsal raphe 5 min before scopolamine had no effect on choice accuracy and latency or errors of omission and did not modify the effect of scopolamine, but completely antagonized the effect of 10 and 30 μg kg−1 8-OH-DPAT on scopolamine-induced impairment of choice accuracy The results confirm previous findings that stimulation of presynaptic 5-HT1A receptors in the dorsal raphe attenuates the deficit of spatial learning caused by blockade of cholinergic excitatory input on hippocampal pyramidal cells. Drugs that stimulate presynaptic 5-HT1A receptors such as 5-HT1A receptor partial agonists may be useful in the symptomatic treatment of human memory disturbances associated with loss of cholinergic innervation to the hippocampus. British Journal of Pharmacology (2000) 131, 375–381; doi:10.1038/sj.bjp.0703567

8-OH-DPAT [8-hydroxy-2-(di-N-propylamino)tetralin], a 5-HT(1A) receptor agonist, and S 15535 (4-benzodioxan-5-yl)1-(indan-2-yl)piperazine, a partial agonist at 5-HT(1A) receptors, were administered into the dorsal raphe nucleus and dorsal hippocampus and their behavioral effects were assessed in a modified Geller-Seifter conflict model. Injected into the dorsal raphe nucleus 8-OH-DPAT, 1 microg but not 0.04 or 0.2 microg 0.5 microl(-1), and S 15535, 2.5 microg but not 0.1 or 0.5 microg 0.5 microl(-1), significantly increased punished responding with no effect on rates of unpunished or time-out responding. WAY 100635, a selective 5-HT(1A) receptor antagonist, injected subcutaneously at 0. 3 mg kg(-1) 30 min before 1 microg 8-OH-DPAT or 2.5 microg S 15535 in the dorsal raphe, completely antagonized their effects on punished responding. At doses ranging from 1 to 10 microg microl(-1) injected into the CA1 region of the dorsal hippocampus neither 8-OH-DPAT nor S 15535 modified punished responding or the rates of time-out. At the highest doses, 8-OH-DPAT significantly reduced unpunished responding whereas S 15535 had the opposite effect. The results suggest that stimulation of 5-HT(1A) receptors in the dorsal raphe nucleus has anxiolytic-like effects whereas stimulation of postsynaptic receptors in the dorsal hippocampus has no anxiolytic or anxiogenic effects, at least judging from changes in rates of punished responding. These results are compatible with the hypothesis that 5-HT(1A) receptor agonists and partial agonists attenuate anxiety by reducing serotonergic transmission in brain areas innervated by the dorsal raphe nucleus.

Rationale: Whilst several studies have investigated the role of serotonergic receptor subtypes in learning and memory, relatively few studies have examined their role in attentional processes. Objective: The present study investigated the role of pre- and postsynaptic 5-HT1A receptors on rats’ attentional performance in the five-choice serial reaction time task (5-CSRT). Methods: Hungry rats were trained in the 5-CSRT task to detect brief (0.5 s) flashes of light presented randomly in one of five locations with a fixed intertrial interval of 5 s paced by the rat. We studied the effects of 8-OH-DPAT, a 5-HT1A receptor agonist, at various subcutaneous (SC) doses (10–100 µg/kg) on measures of rats’ discriminative accuracy (the index of attentional functioning) and various behavioural indices of response control and motivation. Manipulations of basic task parameters, intracerebroventricular (ICV) injections of 5,7-dihydroxytryptamine (5,7-DHT) to deplete forebrain 5-HT and treatments with a selective 5-HT1A receptor antagonist WAY 100635 were made in order to determine the behavioural and neural specificity of the effects of 8-OH-DPAT. Results: A dose of 100 µg/kg, but not lower doses, significantly reduced choice accuracy and increased errors of omission, latencies to respond correctly and to collect food reward and premature responses. All these effects were completely blocked by WAY 100635, injected SC 5 min before 8-OH-DPAT at doses from 10–100 µg/kg. WAY 100635 by itself had no effect in the task. Dimming the visual stimuli to one-third of the usual brightness did not modify the effect of 8-OH-DPAT on choice accuracy. Prolonging the stimuli from 0.5 to 1.0 s reversed 8-OH-DPAT’s effect on choice accuracy but did not modify the other effects on rats’ performance. An ICV injection of 150 µg 5,7-DHT, which depleted forebrain serotonin by 90%, reversed 8-OH-DPAT’s effect on choice accuracy but did not modify the effects on errors of omission and latency to make correct responses. Similar effects were found by infusing 1.0 µg/0.5 µl WAY 100635 in the dorsal raphe 5 min before 8-OH-DPAT. 8-OH-DPAT increased the latency to collect the reinforcement; this effect was attenuated by ICV 5,7-DHT and completely antagonized by WAY 100635 in the dorsal raphe. Rats treated with 5,7-DHT or 8-OH-DPAT showed more premature responses and these effects were markedly reduced by the combined treatment. Conclusions: The results suggest that stimulation of presynaptic 5-HT1A receptors is involved in the ability of 8-OH-DPAT to cause attentional dysfunction and enhance impulsivity while slowing of responding and increase in errors of omission mainly depend on stimulation of postsynaptic 5-HT1A receptors.

In behavioral and receptor binding studies, 5-(4-methylpiperazin-1-yl)-8-chloro-pyridol[2,3b] [1,5]benzoxazepine (JL13) shows an atypical antipsychotic profile. We used microdialysis in awake rats to study the effects of various intraperitoneal doses of JL13 on extracellular concentrations of dopamine in the prefrontal cortex, nucleus accumbens and striatum. JL13 at 20 mg/kg and 40 mg/kg dose-dependently raised extracellular dopamine (234% and 434% of basal levels at peak, respectively) in the prefrontal cortex whereas lower doses (5 mg/kg and 10 mg/kg) had no effect. Extracellular concentrations of dihydroxyphenylacetic acid and homovanillic acid were also significantly increased in the prefrontal cortex of rats given 40 mg/kg JL13 (310% and 230% of basal levels, respectively). At 20 mg/kg and 40 mg/kg JL13 did not affect the extracellular concentrations of dopamine and its metabolites in the striatum and nucleus accumbens. The mechanisms by which JL13 increases cortical dopamine release and the significance for potential antipsychotic efficacy are discussed.

The expression of S100beta and glial fibrillary acidic protein (GFAP) was analyzed following bilateral injection of kainic acid (KA), a glutamate derivative, into the CA3 region of the adult rat hippocampus. This treatment produces a progressive degeneration of the pyramidal neurons of the hippocampus while sparing the granule cells of the dentate gyrus which undergo sprouting of their axons in the supragranular layer. Messenger RNA and protein levels were measured, by Northern blot and ELISA, in the hippocampus of lesioned and sham-operated rats 1, 7, and 30 days after KA injection. A significant increase of GFAP and its mRNA was demonstrated at each time point, whereas S100beta mRNA levels were significantly enhanced only 30 days after the KA injection and the levels of S100beta protein remained unchanged at all time points. However, when analyzed by immunohistochemistry the S100beta showed clear changes in its expression and distribution depending on the region considered. One month after KA injection, S100beta immunoreactivity was considerably reduced in the stratum radiatum of CA3 region, but there was increased S100beta immunoreactivity in the stratum moleculare. In particular, a notable band of S100beta positive, hypertrophic astrocytes appeared in the supragranular layer of the dentate gyrus where the sprouting of mossy fiber collaterals was detected by Timm's staining. These data show for the first time that an increase in S100beta expression in subpopulations of reactive astrocytes may be involved in the structural reorganization of the hippocampus following KA-induced neurodegeneration.

The effect of reboxetine, a novel antidepressant drug that potently and selectively inhibits neuronal noradrenaline (NA) uptake, on brain extracellular monoamines was studied by microdialysis. Fifteen mg kg−1 i.p. reboxetine raised extracellular NA in the frontal cortex (by 242%) and dorsal hippocampus (by 240%). Idazoxan (1 mg kg−1 s.c.), given 60 min after 15 mg kg−1 reboxetine, markedly potentiated the effect on extracellular NA in the frontal cortex (by 1580%) and dorsal hippocampus (by 1360%), but had no effect by itself. Twenty-four hours after the last injection of a chronic schedule (15 mg kg−1 i.p. once daily for 14 days) reboxetine had no effect on basal extracellular concentrations of NA in the dorsal hippocampus and a challenge dose of reboxetine (15 mg kg−1) raised extracellular NA similarly in rats treated chronically with reboxetine (by 353%) and saline (by 425%). Ten and 20 μg kg−1 i.p. clonidine dose-dependently reduced hippocampal extracellular NA similarly in rats given chronic reboxetine (by 32% and 57%) and saline (by 42% and 56%). Extracellular concentrations of dopamine and 5-HT in the striatum were similar in rats treated chronically with reboxetine and saline. A challenge dose of reboxetine (15 mg kg−1) had no effect on striatal extracellular dopamine and slightly increased striatal extracellular 5-HT to a similar extent in rats treated chronically with reboxetine (by 137%) and saline (by 142%). The results suggest that combining reboxetine with an α2-adrenoceptor antagonist may facilitate its antidepressant activity. Repeated treatment confirmed that reboxetine is fairly selective for the noradrenergic system but provided no evidence of adaptive changes in that system that could facilitate its effect on extracellular NA. British Journal of Pharmacology (1999) 128, 1332–1338; doi:10.1038/sj.bjp.0702926

The effect of S 15535 (4-benzodioxan-5-yl)1-(indan-2-yl)piperazine), an agonist at presynaptic and antagonist at postsynaptic 5-HT1A receptors, on the impairment of spatial learning caused by intrahippocampal scopolamine in a two-platform spatial discrimination task was studied. Scopolamine (4.0 μg μl−1), injected bilaterally into the CA1 region of the dorsal hippocampus 10 min before each training session, impaired choice accuracy with no effect on choice latency and errors of omission. Administered subcutaneously 30 min before each training session, S 15535 1.0 (but not 0.3) mg kg−1 did not modify choice accuracy but prevented its impairment by intrahippocampal scopolamine. WAY 100635, a 5-HT1A receptor antagonist, injected into the dorsal raphe at 1.0 μg 0.5 μl−1 5 min before scopolamine, had no effect on choice accuracy and latency or errors of omission and did not modify the effect of scopolamine but completely antagonized the effect of S 15535 (1.0 mg kg−1) on scopolamine-induced impairment of choice accuracy. The results confirm a previous report (Carli et al., 1998) that stimulation of presynaptic 5-HT1A receptors in the dorsal raphe counteracts the deficit caused by intrahippocampal scopolamine, probably by facilitating the transfer of facilitatory information from the entorhinal cortex to the hippocampus. Drugs that stimulate action on presynaptic 5-HT1A receptors, such as S 15535 and other partial 5-HT1A receptors agonists, may be useful in the symptomatic treatment of human memory disturbances associated with loss of cholinergic innervation to the hippocampus. British Journal of Pharmacology (1999) 128, 1207–1214; doi:10.1038/sj.bjp.0702915

We studied the effect of WAY 100635, a 5-HT1A receptor antagonist, on the impairment of spatial learning caused by intrahippocampal injection of 3-((R)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a competitive NMDA receptor antagonist, in a two-platform spatial discrimination task. CPP, 3 and 10 ng/microl, administered bilaterally into the CA1 region of the dorsal hippocampus 10 min before each training session, dose-dependently reduced choice accuracy in the two-platform spatial discrimination task with little or no effect on choice latency and errors of omission. A volume of 10 ng/microl intrahippocampal CPP did not affect choice accuracy or latency of a non-spatial visual discrimination task. Subcutaneous doses of 0.3 and 1 mg/kg WAY 100635 did not modify the choice accuracy, but prevented the impairment caused by 10 ng/microl intrahippocampal CPP. A dose of 20 ng/microl WAY 100635 into the dorsal hippocampus prevented the deficit caused by 10 ng/microl CPP administered in the same region. The results suggest that blockade of 5-HT1A receptors can compensate the loss of NMDA-mediated excitatory input to pyramidal cells in the hippocampus. These findings may have clinical relevance for the symptomatic treatment of memory disorders associated with reduced glutamate transmission mediated by NMDA receptors.

The effect of the 5-hydroxytryptamine (5-HT) and noradrenaline (NA) reuptake inhibitor sibutramine was studied in food deprived, neuropeptide Y (NPY)- or muscimol-injected rats. Sibutramine dose-dependently reduced feeding caused by food-deprivation (ED50=5.1±0.8 mg kg−1) or by NPY injection into the paraventricular nucleus of the hypothalamus (ED50=6.0±0.5 mg kg−1). The increase in food intake caused by muscimol injected into the dorsal raphe was not modified by sibutramine (1–10 mg kg−1). The hypophagic effect of 5.1 mg kg−1 sibutramine in food-deprived rats was studied in rats pretreated with different serotonin receptor antagonists. Metergoline (non-selective, 0.3 and 1.0 mg kg−1), ritanserin (5-HT2A/2C, 0.5 and 1.0 mg kg−1) and GR127935 (5-HT1B/1D, 0.5 and 1.0 mg kg−1) did not modify the hypophagic effect of sibutramine, while SB206553 (5-HT2B/2C, 5 and 10 mg kg−1) slightly but significantly reduced it (Fint(2.53)=3.4; P

Seizures increase the synthesis of brain-derived neurotrophic factor in forebrain areas, suggesting this neurotrophin has biological actions in epileptic tissue. The understanding of these actions requires information on the sites and extent of brain-derived neurotrophic factor production in areas involved in seizures onset and their spread. In this study, we investigated by immunocytochemistry the changes in brain-derived neurotrophic factor in the hippocampus, entorhinal and perirhinal cortices of rats at increasing times after acute seizures eventually leading to spontaneous convulsions. We also tested the hypothesis that seizure-induced changes in brain-derived neurotrophic factor induce later modifications in neuropeptide Y expression by comparing, in each instance, their immunoreactive patterns. As early as 100 min after seizure induction, brain-derived neurotrophic factor immunoreactivity increased in CA1 pyramidal and granule neurons and in cells of layers II-III of the entorhinal cortex. At later times, immunoreactivity progressively decreased in somata while increasing in fibres in the hippocampus, the subicular complex and in specific layers of the entorhinal and perirhinal cortices. Changes in neuropeptide Y immunoreactivity were superimposed upon and closely followed those of brain-derived neurotrophic factor. One week after seizure induction, brain-derived neurotrophic factor and neuropeptide Y immunoreactivities were similar to controls in 50% of rats. In rats experiencing spontaneous convulsions, brain-derived neurotrophic factor and neuropeptide Y immunoreactivity was strongly enhanced in fibres in the hippocampus/parahippocampal gyrus and in the temporal cortex. In the dentate gyrus, changes in immunoreactivity depended on sprouting of mossy fibres as assessed by growth-associated protein-43-immunoreactivity. These modifications were inhibited by repeated anticonvulsant treatment with phenobarbital. The dynamic and temporally-linked alterations in brain-derived neurotrophic factor and neuropeptide Y in brain regions critically involved in epileptogenesis suggest a functional link between these two substances in the regulation of network excitability.

The selective 5-hydroxytryptamine reuptake inhibitor citalopram (10 and 20 mg kg−1, i.p.) significantly reduced food intake in male rats (CD-COBS) habituated to eat their daily food during a 4-h period. The 5-HT1A receptor antagonist WAY100635 (0.3 mg kg−1) administered systemically did not modify feeding but significantly potentiated the reduction in food intake caused by 10 mg kg−1 i.p. citalopram. The dose of 5 mg kg−1 i.p. citalopram was not active in animals pretreated with vehicle but significantly reduced feeding in animals pretreated with WAY100635. WAY100635 (0.1 μg 0.5 μl−1) injected into the dorsal raphe significantly potentiated the hypophagic effect of 10 mg kg−1 citalopram. WAY100635 (1.0 μg 0.5 μl−1) injected into the median raphe did not modify feeding or the hypophagic effect of 10 mg kg−1 citalopram. The 5-HT2B/2C receptor antagonist SB206553 (10 mg kg−1, p.o.) slightly reduced feeding by itself but partially antagonized the effect of WAY100635 administered systemically (0.3 mg kg−1, s.c.) or into the dorsal raphe (0.1 μg 0.5 μl−1) in combination with 10 mg kg−1 i.p. citalopram. The hypophagic effect of 10 mg kg−1 i.p. citalopram alone was not significantly modified by SB206553. Brain concentrations of citalopram and its metabolite desmethylcitalopram in rats pretreated with SB206553, WAY100635 and their combination were comparable to those of vehicle-pretreated rats, 90 min after citalopram injection. The hypophagic effect of citalopram was potentiated by blocking 5-HT1A receptors. Only the effect of the WAY100635/citalopram combination seemed to be partially mediated by central 5-HT2C receptors. British Journal of Pharmacology (1998) 124, 1781–1787; doi:10.1038/sj.bjp.0702028

This study investigated the effect of stimulating 5-HT1A receptors in the dorsal raphe on the impairment of learning caused by 4 microg/microL scopolamine injected in the CA1 region of the dorsal hippocampus in rats performing a two-platform spatial discrimination task. At 1 (but not 0.2) microg/0.5 microL administered in the dorsal raphe on each acquisition training day 5 min before bilateral intrahippocampal injection of 4 microg/microL scopolamine, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, had no effect on choice accuracy and latency or errors of omission but completely antagonized the impairment of choice accuracy by intrahippocampal scopolamine. Administered into the dorsal raphe at 0.2 and 1 microg/0.5 microL, WAY 100635, a 5-HT1A receptor antagonist, had no effect on rats' performance or on the impairment caused by intrahippocampal scopolamine but dose-dependently antagonized the effect of 1 microg/0.5 microL 8-OH-DPAT on the scopolamine-induced deficit. The results show that stimulation of presynaptic 5-HT1A receptors in the dorsal raphe reverses the deficit caused by intrahippocampal scopolamine, probably by facilitating the transfer of facilitatory information from the entorhinal cortex to the hippocampus. Together with a previous study showing that blockade of postsynaptic hippocampal 5-HT1A receptors antagonized the effect of intrahippocampal scopolamine in the two-platform spatial discrimination task (Carli et al., 1995b), the results suggest that drugs with presynaptic stimulatory and postsynaptic blocking actions on 5-HT1A receptors, such as partial agonists at these receptors, may be useful in the symptomatic treatment of human memory disturbances associated with loss of cholinergic innervation to the hippocampus.

Using a balanced conditioned place preference (CPP) paradigm, we studied the role of protein kinases A (PKA) and C (PKC) on the acquisition, consolidation and expression of cocaine place conditioning. H7, a non-selective inhibitor of protein kinases, was administered intracerebroventricularly at 1 and 10 micrograms/10 microliters. The higher dose significantly reduced the time spent by rats in the cocaine compartment when given immediately after each conditioning session (consolidation), whereas it had no effect when administered before cocaine during the training phase (acquisition) or before testing for place preference in the absence of cocaine (expression). The same effect was found on administering immediately after each training session 3 micrograms/10 microliters chelerythrine, a selective PKC inhibitor, or 10 micrograms/10 microliters H89, a selective PKA inhibitor, suggesting that both kinases contribute to the consolidation of stimulus-reward association which determines rats' behavior in the cocaine CPP. Changes in the activity of PKA and PKC may thus be part of the cascade of events that contribute to enhancing synaptic responses in the consolidation phase of cocaine CPP and determine rats' behavior associated with the memory of the rewarding effect of cocaine during cocaine CPP expression. These findings may have implications for the study of cocaine 'craving' and relapse.

Kainate-induced seizure activity causes persistent changes in the hippocampus that include synaptic reorganization and functional changes in the mossy fibers. Using in situ hybridization histochemistry, the expression of PKC alpha, PKC beta, PKC gamma, PKC delta and PKC epsilon mRNAs was investigated in the hippocampus of adult rats following seizures induced by a s.c. injection of kainic acid. In CA1 and CA3, we found a significant decrease in PKC gamma mRNA 1 day after kainic acid which persisted for a 2nd day in CA1. None of the other PKC isoform mRNAs were altered in CA1 or CA3. In granule cells, a significant up-regulation specific to PKC epsilon mRNA was observed. One week after kainic acid administration, a marked increase in PKC epsilon immunoreactivity was found that persisted 2 months after kainic acid administration. PKC epsilon immunoreactivity was found associated with mossy fibers projecting to the hilus of the dentate gyrus and to the stratum lucidum of the CA3 field and presumably with the newly sprouted mossy fibers projecting to the supragranular layer. These data provide the first evidence for a long-lasting increase of the PKC epsilon in the axons of granule cells caused by kainate-induced seizures and suggest that PKC epsilon may be involved in the functional and/or structural modifications of granule cells that occur after limbic seizures.

Kainic acid-induced seizures, in adult rats produce neurodegeneration in the hippocampus followed by sprouting of the mossy fibres in the inner molecular layer of the dentate gyrus and changes in GAP-43 expression in the granule cells. In the present study we observed that 4 days after kainic acid injection a dense plexus of silver-impregnated degenerating terminals detected by Gallyas's method and a decrease of GAP-43 immunostaining was observed in the inner molecular layer of the dentate gyrus indicating deafferentiation of this region. This was associated with the formation of an intense GAP-43 immunostained band in the supragranular layer. MK-801, a non-competitive inhibitor of the NMDA receptor, which partially inhibited the behavioural seizures induced by KA, also protected from the inner molecular layer deafferentation and markedly reduced the expression of GAP-43 mRNA in the granule cells and the intense GAP-43 immunostained band in the supragranular layer, suggesting a relationship among these events. Two months after kainic acid injection the intense supragranular GAP-43 positive band was no longer evident but the whole inner molecular layer appeared more labelled in association with the formation of the collateral sprouting of the mossy fibres in the inner molecular layer as detected by Timm's staining. These effects were also markedly reduced by the pretreatment with MK-801. Taken together, these experiments indicate for the first time a direct relationship between the increase of GAP-43 immunostaining in the inner molecular layer of the dentate gyrus and the collateral sprouting of mossy fibres in this district in response to kainic acid induced seizures. This further supports the hypothesis that the early induction of GAP-43 in granule cells may be one of the molecular mechanisms required for the synaptic reorganization of the mossy fibres.

In situ hybridization histochemistry with somatostatin sst1-sst5 receptor messenger RNA-selective oligoprobes and quantitative receptor autoradiographic binding studies using [125I]Tyr3-octreotide, [Leu2,D-Trp22,125I-Tyr25]somatostatin-28 and [125I]CGP 23996 ([125I]c[Asn-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) were performed to determine the level of expression of somatostatin receptor messenger RNA and receptor binding sites in the hippocampal formation, limbic system and cerebral cortex of adult rats electrically kindled in the dorsal hippocampus. In control rats (implanted with electrodes but not electrically stimulated), the somatostatin-1 receptor-selective [125I]Tyr3-octreotide and the non-subtype-selective [Leu3,D-Trp22,125I-Tyr25]somatostatin-28 preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the molecular layer of the dentate gyrus, the subiculum and presubiculum of the hippocampal formation, the inner layer of the frontal cortex, and the lateral and basolateral nuclei of the amygdala. The non-subtype-selective radioligand [125I]CGP 23996 (in 5 mM Mg2+ buffer) preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the subiculum and the basolateral nucleus of the amygdala. Under conditions where primarily somatostatin-2 receptors were labelled, [125I]CGP 23996 (in 120 mM Na+ buffer) showed strong binding in the strata oriens and radiatum of the CA1 subfield of the hippocampus and the frontal cortex, whereas the dentate gyrus, subiculum and amygdala showed only weak signals. During and after kindling, no significant differences were observed between the ipsi- and contralateral sides of the hippocampus. A significant decrease (about 40%) of somatostatin receptor binding sites was observed in the molecular layer of the dentate gyrus with all radioligands (except [125I]CGP 23996 in Na+ buffer, which did not label this area) at stage 2 (pre-convulsive stage) and one week, but not one month, after stage 5 (generalized motor seizures). In contrast to somatostatin receptor binding, no alterations of the messenger RNA levels for sst1-sst5 receptors were found either at stage 2 or at stage 5. Similarly, no changes in receptor binding or messenger RNA levels were observed in the brain of rats which experienced a single afterdischarge. The present study shows a significant and selective decrease of somatostatin-1 receptor binding sites in the dentate gyrus of kindled rats. This is part of the plastic changes induced by kindling and may contribute to the increased sensitivity for the induction of generalized seizures during kindling.

1. We studied whether the stimulation of 5-HT1A receptors by 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a specific 5-HT1A receptor agonist, reduced electroencephalographic (EEG) seizures induced by intrahippocampal injection of 0.04 microgram in 0.5 microliter of the glutamate analogue kainic acid in freely-moving rats. 2. Pretreatment with 8-OH-DPAT 15 min earlier at the same site as kainic acid injection, caused a dose-dependent decrease of kainic acid-induced seizure activity. One and 10 micrograms significantly reduced the total time spent in seizures by 72% on average and the total number of seizures by 58% (P < 0.01) and 43% (P < 0.05) respectively. The latency to onset of the first seizure was increased 2.8 times (P < 0.01) only after 1 microgram 8-OH-DPAT; 0.1 microgram was ineffective on all seizure parameters. 3. Systemic administration of 25, 100 and 1000 micrograms kg-1 8-OH-DPAT significantly reduced the total number of seizures and the total time in seizures induced by intrahippocampal kainic acid by 52% and 74% on average. The latency to onset of the first seizure was delayed 1.8 times by 100 and 1000 micrograms kg-1 (P < 0.05). 4. The anticonvulsant action of 8-OH-DPAT given intrahippocampally or systemically was significantly blocked by 5 micrograms, but not 1 microgram WAY 100635, a selective 5-HT1A receptor antagonist, administered in the hippocampus before the agonist. 5. These results indicate that postsynaptic 5-HT1A receptors in the hippocampus mediate the anticonvulsant action of 8-OH-DPAT and that their stimulation has an inhibitory role in the generation of limbic seizures.

The in vitro release of somatostatin and neuropeptide Y, their tissue concentration and immunocytochemical pattern were examined in the entorhinal cortex of chronically epileptic rats. A systemic administration of 12 mg/kg kainic acid causing generalized tonic-clonic seizures for at least 3 h after injection was used to induce, 60 days later, a chronically enhanced susceptibility to seizures in the rats. The release of both peptides under depolarizing conditions was significantly reduced by 15% on average from slices of the entorhinal cortex two days after kainic acid-induced status epilepticus. At 60 days, the spontaneous and 30 mM KCl-induced release of somatostatin was significantly enhanced by 30% on average. The release induced by 100 mM KCl was raised by 70%. The spontaneous, 30 mM and 100 mM KCl-induced release of neuropeptide Y from the same slices was increased, respectively, by 120%, 76% and 36%. The late changes were associated with an increased tissue concentration of neuropeptide Y but not of somatostatin. This was confirmed by immunocytochemical evidence showing that neuropeptide Y-, but not somatostatin-immunoreactive neurons were increased in the entorhinal cortex of kainic acid-treated rats. These results indicate that neurotransmission mediated by somatostatin and neuropeptide Y, two peptides previously shown to play a role in limbic epileptogenesis, is enhanced in the entorhinal cortex of chronically epileptic rats.

The effect of 5-HT1 and 5-HT2 receptor agonists administered into the paraventricular hypothalamus was studied on the hyperphagia caused by neuropeptide Y (NPY) injected into the same area. The 5-HT2A/2C receptor agonist DOI (10-20 nmol/0.5 microliter) significantly reduced NPY overeating while the 5-HT1A/1B receptor agonist RU 24969 (3.5-14 nmol/0.5 microliter) and the 5-HT1B/2C receptor agonist mCPP (5-20 nmol/0.5 microliter) had no such effect. The 5-HT2A receptor antagonist spiperone (5 microgram/0.5 microliter) and the corticotropin releasing factor antagonist alpha-helical-CRF9-41 (0.5-1 micrograms/0.5 microliter) completely antagonized the effect of 10 nmol DOI.

Drugs with different intrinsic activity at 5-HT1A receptors and antagonists at 5-HT2A/2C and 5-HT3 receptors were studied for their ability to increase the rates of punished operant responding in rats. Like chlordiazepoxide (5 and 10 mg/kg) and diazepam (1.25 and 2.5 mg/kg), 0.125 mg/kg 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, and 5 and 10 mg/kg ipsapirone, a partial agonist at these receptors, increased the rates of punished responding, whereas (S)-WAY 100135, a 5-HT1A receptor antagonist, had no effect at doses from 1 to 10 mg/kg. 8-OH-DPAT and ipsapirone, like benzodiazepines, significantly reduced unpunished responding. The 5-HT2A/2C receptor antagonists ritanserin (2 mg/kg), mianserin (8 mg/kg), and mesulergine (0.1 mg/kg) significantly increased the rates of punished responding, whereas 0.5-2 mg/kg ketanserin, that has higher affinity for 5-HT2A than 5-HT2C receptors, had no effect. Antagonists, at 5-HT3 receptors such as ondansetron (0.001-0.1 mg/kg) and tropisetron (0.001-0.1 mg/kg), had no effect on punished or unpunished responding. The results show that agents acting as full or partial agonists at 5-HT1A receptors and blockers of postsynaptic 5-HT2C receptors have anxiolytic-like effects in a model of punished operant responding, whereas antagonists at 5-HT1A and 5-HT3 receptors have no such effect.

Citalopram, 1 and 10 mg/kg i.p., that in a previous study using identical treatment and dialysis conditions had little or no effect on dialysate serotonin (5-HT) in the frontal cortex, dose-dependently raised the extracellular concentrations of 5-HT in the dorsal hippocampus, by 70% and 205% respectively at the peak. In animals given 10 mg/kg citalopram twice daily for 14 days, intraperitoneal doses of 1 and 10 mg/kg or infusion of 10(-8) - 10(-6) M through the hippocampal probe raised dialysate 5-HT in the dorsal hippocampus similarly in animals treated chronically with citalopram or saline. A dose of 100 micrograms/kg 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), an agonist at 5-HT1A receptors, reduced hippocampal extracellular 5-HT concentrations to the same extent in rats repeatedly given saline or citalopram. Half this dose had no such effect in either group. The effect of citalopram and the sensitivity of autoreceptors controlling 5-HT release in the dorsal hippocampus were unaffected by a chronic treatment known to facilitate the drug's effect on dialysate 5-HT and to reduce the sensitivity of 5-HT1A receptors controlling 5-HT release in the frontal cortex. The effects of acute and chronic treatment with citalopram on dialysate 5-HT in the rat brain thus appear to differ in at least two brain regions: the frontal cortex and the dorsal hippocampus.

The effect of ipsapirone, a partial agonist at 5-HT1A receptors, and of diazepam on punished operant responding was studied in rats injected intracerebroventricularly with 150 microg 5,7-dihydroxytryptamine to deplete brain serotonin or pretreated with (S)-WAY 100135 (N-tert-butyl) 3-4-(2-methoxyphenyl)piperazin-1-yl-2-phenylpropanamide dihydrochloride), an antagonist at 5-HT1A receptors. 5,7-Dihydroxytryptamine markedly depleted brain serotonin and caused a sustained increase in punished responding with no effect on rates of unpunished responding in sham-operated rats but had no effect in animals which had received 5,7-dihydroxytryptamine. At 5 and 10 mg/kg ipsapirone reduced unpunished responding similarly in sham-operated and 5,7-dihydroxytryptamine-treated rats. Diazepam 2.5 mg/kg i.p.significantly increased punished responding and reduced rates of unpunished responding similarly in sham-operated and in 5,7-dihydroxytryptamine-treated animals. At 3 and 10 mg/kg (S)-WAY 100135 did not modify punished or unpunished responding but at 10 mg/kg it completely antagonized the effect of 5 mg/kg/s.c. ipsapirone on unpunished and punished responding. The results suggest that ipsapirone releases behaviour that is suppressed by punishment by stimulating presynaptic 5-HT1A receptors.

d-Fenfluramine (0.63 mg/kg i.p.), a serotonin (5-hydroxytryptamine, 5-HT) releaser and re-uptake inhibitor, reduced the eating caused by neuropeptide Y (235 pmol) injected into the paraventricular nucleus of the hypothalamus. The 5-HT1 and 5-HT2 receptor antagonist metergoline (1.0 and 2.0 mg/kg i.p.) and the 5-HT1A and 5-HT1B receptor antagonist (+/-)-cyanopindolol (3.0 and 8.0 mg/kg s.c.) significantly antagonized the effect of d-fenfluramine. The 5-HT2A and 5-HT2C receptor antagonist mesulergine (0.1 and 0.3 mg/kg s.c.) and the 5-HT2A receptor antagonist ketanserin (2.5 and 5.0 mg/kg i.p.) did not significantly modify the effect, nor did the 5-HT1A and 5-HT1B receptor antagonist (-)-propranolol (20-40 nmol), injected bilaterally into the paraventricular nucleus of the hypothalamus. The results suggest that d-fenfluramine reduces neuropeptide Y's hyperphagia by indirectly stimulating 5-HT1B receptors outside the paraventricular nucleus of the hypothalamus.

The effect of chronic treatment with (3-alpha-tropanyl)1H-benzimidazolone-3-carboxamide chloride (DAU 6215; 15 micrograms/kg s.c. twice daily for 21 days), a serotonin3 receptor antagonist, on the extracellular concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was studied by intracerebral dialysis in the striatum, nucleus accumbens and frontal cortex of conscious rats. Twenty-four hours after the last injection, the basal extracellular concentrations of DA in the nucleus accumbens of rats given DAU 6215 were significantly lower than in saline-treated rats. DA output in the dorsolateral striatum or frontal cortex was not significantly different between the DAU 6215 and saline-treated rats. Chronic DAU 6215 significantly reduced the extracellular concentrations of DOPAC and HVA in the frontal cortex but had no effect in the other brain regions. A subcutaneous challenge dose of DAU 6215 (15 micrograms/kg) did not significantly modify the extracellular concentrations of DA and its metabolites in either DAU 6215 or saline treated rats in any of the brain regions examined. The present investigation is the first on the effect of chronic administration of a 5-HT3 receptor antagonist on basal extracellular DA in the rat brain. The results provide evidence of an association between the electrophysiological and biochemical effects of chronic treatment with a serotonin3 receptor antagonist on the activity of the mesolimbic DA system. In line with the theory that hyperactivity of the mesolimbic dopaminergic system is involved in psychosis, the results suggest that DAU 6215 may be useful in the treatment of psychotic disorders, possibly with limited extrapyramidal effects.

The effect of chronic oral treatment with clozapine (20 mg/kg daily for 21 days) on the extracellular concentrations of dopamine, dihydroxyphenylacetic acid and homovanillic acid in the dorsolateral anterior striatum, nucleus accumbens and frontal cortex was studied by microdialysis in conscious rats. Basal levels of dopamine and its metabolites in the three brain regions of rats treated chronically with clozapine were not significantly different from those of vehicle-treated rats. A subcutaneous challenge dose of clozapine (20 mg/kg) significantly increased the extracellular concentrations of dopamine and its metabolites in the three brain regions, with no differences between chronic vehicle- and clozapine-treated rats.

The levels of neuropeptide Y and somatostatin may change when serotoninergic neurotransmission is altered in different brain regions. To assess whether serotonin regulates the synthesis of these peptides, we measured the levels of preproneuropeptide Y (ppNPY) and preprosomatostatin (ppSOM) mRNA in different brain regions after intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), a selective serotonin neurotoxin. The mRNA of these peptides significantly increased in the striatum but not in hippocampus and frontal cortex. It thus appears that serotonin has an inhibitory effect on the biosynthesis of neuropeptide Y and somatostatin in striatum whereas it probably acts by stimulating the release of these peptides in hippocampus and frontal cortex.

Studies with dexfenfluramine, an anorectic agent which releases 5-hydroxytryptamine (5-HT) from nerve terminals and inhibits its reuptake, have considerably increased our knowledge of the role of 5-HT in feeding control. 5-HT1B receptors mediate the satiating effect of dexfenfluramine, whereas the mechanism by which 5-HT uptake inhibitors such as fluoxetine and sertraline cause anorexia is not clear. Anorexia induced by (+)-amphetamine, phentermine, diethylpropion and phenylpropanolamine seems to be the result of their ability to increase the release of noradrenaline and/or dopamine from nerve terminals and inhibit their reuptake or, in the case of phenylpropanolamine, to stimulate directly alpha 1-adrenoceptors. It has been suggested that beta- and alpha 1-adrenoceptors and D1 dopamine receptors are involved in their effect on food intake. The difficulties of extrapolation across species limit our knowledge of the mechanism of the anorectic action in humans. Significant advances in the treatment of feeding pathology will be linked to identifying new receptor types and subtypes for neurotransmitters and quantifying and modelling eating disorders such as binge-eating and food craving.

The effects of various doses of tianeptine on extracellular concentrations of dopamine were studied in the frontal cortex and nucleus accumbens of the rat. At 5 and 10 mg/kg intraperitoneally, tianeptine raised extracellular dopamine in the nucleus accumbens but only the higher dose caused a significant increase in the frontal cortex. At 10 mg/kg tianeptine significantly raised the concentrations of dihydroxyphenylacetic acid and homovanillic acid in both brain regions. In another experiment, 10 and 20 mg/kg tianeptine did not modify the extracellular concentrations of noradrenaline in the frontal cortex but dose dependently blocked the increase in extracellular noradrenaline caused by restraint stress.

Administration of 2 and 5 but not 0.4 microgram/microliter 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) into the CA1 region of the dorsal hippocampus 10 min before the acquisition trial in a passive avoidance task significantly reduced retention latencies 24 h later. The effect of 5 micrograms 8-OH-DPAT on retention latencies was completely antagonized by 1 microgram/microliter spiroxatrine, a 5-HT1A receptor antagonist, infused into the dorsal hippocampus 5 min before 8-OH-DPAT. Administered 5 min after the acquisition trial, 5 micrograms/microliters 8-OH-DPAT had no effect on retention latencies 24 h later. Administration of 5 micrograms/microliters 8-OH-DPAT into the dorsal hippocampus did not significantly change the thresholds for responses to the same electrical stimuli used in the passive avoidance task and had no antidepressant-like effect in the forced swimming test. The dose of 5 micrograms/microliters 8-OH-DPAT administered into the dorsal hippocampus caused anxiolytic-like effects assessed by stress-induced deficit in open field locomotor activity. The results suggest that stimulation of 5-HT1A receptors in the dorsal hippocampus impairs rats' performance in a passive avoidance task by interfering with memory processes or by attenuating the emotional impact of the shock through an anxiolytic action.

Ritanserin (0.5 and 1 mg/kg) and ketanserin (2.5 mg/kg), two antagonists with high affinity for 5-HT2 receptors, attenuated restraint stress-induced hypophagia in rats. Two injections of the 5-HT2 receptor antagonist cinanserin ( 30 nmol 0.5 μ l ) in the paraventricular nucleus of the hypothalamus completely reversed the effect of stress on food intake. (±)Cyanopindolol (3 and 8 mg/kg), an antagonist at 5-HT1A and 5-HT1B receptors, had no effect whereas 8-hydroxy-2-di-n-propylamino) tetralin (30–300 μg/kg), an agonist at 5-HT1A receptors, significantly attenuated the hypophagia. The results suggest that restraint stress-induced hypophagia is mediated by 5-HT2 receptors in the paraventricular nucleus of the hypothalamus. The potential utility of this model in anorexia nervosa is discussed.

Intracerebral microdialysis combined with a sensitive and specific radioimmunoassay was used to monitor the neuronal release of somatostatin (somatostatin-like immunoreactivity, SLI) in the dorsal hippocampus of freely moving rats. The sensitivity of the radioimmunoassay was optimized to detect < 1 fmol/ml. The basal concentration of SLI in 20-min dialysate fractions (5 microliters/min) collected 24 h after probe implantation was stable over at least 200 min. The spontaneous efflux dropped by 54 +/- 6.4% (p < 0.05) when Ca2+ was omitted and 1 mM EGTA added to the Krebs-Ringer solution and by 65.5 +/- 3.2% (p < 0.05) in the presence of 1 microM tetrodotoxin. Depolarizing concentrations of the Na+ channel opener veratridine (6.25, 25, 100 microM) induced 11 +/- 2 (p < 0.05), 17 +/- 2 (p < 0.05), and 21 +/- 5 (p < 0.01) fold increase in SLI concentration, respectively, during the first 20 min of perfusion. The effect of 100 microM veratridine was blocked by coperfusion with 5 microM tetrodotoxin (p < 0.01) and reduced by 79% (p < 0.01) in the virtual absence of Ca2+. Neuronal depolarization by 20 min of perfusion with Krebs-Ringer solution containing 25 and 50 mM KCl and proportionally lowered Na+ increased the dialysate SLI 4.4 +/- 1 (p < 0.05) and 17 +/- 3 (p < 0.01) fold baseline, respectively. Ten micromolar ouabain, a blocker of Na+,K(+)-ATPase, increased the dialysate SLI 15-fold baseline, on average (p < 0.05), during 80 min of perfusion. The results demonstrate the suitability of brain microdialysis for monitoring the neuronal release of SLI and for studying its role in synaptic transmission.

A five-choice serial reaction time task was used to study the effects of serotonin (5-HT) receptor agonists and antagonists on accuracy of performance and food-motivated behaviour. Lysergic acid diethylamide (LSD), 0.1 mg/kg IP and quipazine, 2.5 mg/kg IP significantly reduced the percentage of correct responses and increased the percentage of omissions with no effect on other measures such as latency to collect the reinforcement or to respond correctly. The effects of LSD and quipazine were reversed by 1–2 mg/kg ritanserin, a potent 5-HT2 and 5-HT1C receptor antagonist. Metachlorophenylpiperazine (mCPP) 2.5 mg/kg IP, an agonist at 5-HT1B and 5-HT1C receptors, andd-fenfluramine (DF) 1.25 mg/kg IP, a releaser of 5-HT from nerve terminals and inhibitor of 5-HT uptake, increased the percentage of omissions and the latency to respond correctly or to collect the reinforcement with no effects on the correct responses. Effects similar to those of mCPP and DF were obtained by 60 min access to food before testing. Haloperidol, 0.1 mg/kg IP, did not affect the percentage of correct responses or the latency to collect the reinforcement, but significantly increased the proportion of errors of omission and the latency to respond correctly. The results show that 5-HT2 receptor agonists cause attentional disturbances at doses that have no marked effect on motivation for food or speed. An increase in the latency to collect the reinforcement was found only with prefeeding and drugs supposed to cause satiety such as mCPP and DF. An increase in latency to respond correctly and in the percentage of omissions seemed related to haloperidol-induced motor retardation and reduced level of arousal. The five-choice serial reaction time task seems useful for separating effects on attentional processes from those on food-motivated behaviour or motor activity.

The effects of d-fenfluramine (1.5 mg/kg) and sertraline (10 mg/kg), administered intraperitoneally once daily for seven days were studied on feeding parameters of rats over various periods. On the first day of treatment both drugs markedly reduced meal size and meal duration during the first hour and, to a lesser extent, the first 4 h. No effects were seen later. The size and duration of eating bouts were also markedly reduced by both drugs in the first hour. There was no significant effect of either drug on meal frequency in any period. Only d-fenfluramine significantly reduced the rate of eating within 4 h from the start of testing. Sertraline, but not d-fenfluramine, markedly increased locomotor activity in the first 4 h after the start of testing. The d-fenfluramine effect on eating rate disappeared by the second day whereas total intake and meal size were still reduced on day five. By days six and seven however the d-fenfluramine-treated rats did not differ from the controls. During the seven-day treatment sertraline always reduced total food eaten and meal size but caused only transient changes of locomotor activity and eating rate. Since the effects of d-fenfluramine and sertraline on meal size and food intake could be separated from the effects on eating rate and arousal, it appears that at appropriate doses these drugs specifically increase the satiating effect of food. Tolerance to this effect appears to develop more rapidly for d-fenfluramine than for sertraline.

Stimulation of glutamate receptors has been reported to modulate the expression of neuropeptides and their receptors in neurons. On the other hand, neuropeptides are known to regulate the presynaptic glutamate release and neuronal responses to excitatory neurotransmission. This evidence indicates a functional interaction between glutamatergic and neuropeptidergic transmission in the central nervous system (CNS). In this report, we provide pharmacologic evidence in experimental models of seizures, suggesting that somatostatin (SRIF) and neuropeptide Y (NPY) are endogenous modulators of glutamate-mediated hyperexcitability in the CNS. Electroencephalographic (EEG) and behavioral seizures were induced in rats by intrahippocampal or systemic injection of kainic acid, a glutamate analog. The number of EEG seizures and their total duration were inhibited significantly by intracerebral application of a SRIF(1) receptor agonist. Similarly, kainate seizures were reduced by N[-2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl-D-arginamide++ +] (BIBP 3226), a NPY Y(1) receptor antagonist. Enhanced seizure susceptibility to pentylentetrazol, ensuing in rats after a systemic administration of kainic acid, was reduced significantly by intracerebral application of RC 160, a SRIF(1) receptor agonist, or NPY 13-36, a Y(2)/Y(5) receptor agonist. This evidence suggests that neuropeptide analogs may be of value for controlling seizures and possibly in other pathologic conditions associated with excessive glutamate function.

The levels of preproneuropeptide Y (ppNPY) mRNA and preprosomatostatin (ppSOM) mRNA were analyzed in different brain regions during the development of hippocampal kindling in rats. ppNPY mRNA levels were markedly elevated in the dorsal hippocampus bilaterally, two days after stage 2 (preconvulsive stage) and stage 5 (full seizure expression). The contents of ppSOM mRNA were slightly, although not significantly. increased in the dorsal hippocampus at stage 2 whereas a significant increase was observed in the ipsilateral hippocampus of fully kindled rats. ppNPY and ppSOM mRNA levels were unchanged in the cortex and striatum at both stages of kindling. These results show that an increased synthesis of somatostatin and neuropeptide Y, with a greater effect for the latter, occurs during hippocampal kindling in rats. The relative role of the two peptides in the development and expression of kindling phenomenon remains to be elucidated.

The effects of 1-20 mg/kg diazepam were studied on the extracellular concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the nucleus accumbens and striatum of conscious rats, using intracerebral microdialysis. Five, but not 1 mg/kg diazepam significantly reduced extracellular DA, DOPAC and HVA in the nucleus accumbens. Twenty mg/kg diazepam significantly reduced extracellular DA, DOPAC and HVA in the striatum. A significant effect on striatal DOPAC, but not on DA and HVA, was seen with 10 mg/kg diazepam, while no changes were found with 5 mg/kg diazepam. The results suggest that diazepam reduces the release and metabolism of DA in the nucleus accumbens more than in the striatum.

In one experiment, the effect of d-fenfluramine (DF) on gastric emptying was studied in rats treated i.p. with metergoline, a non-selective serotonin (5-HT) receptor antagonist, ritanserin, a selective 5-HT2 and 5-HT1C receptor antagonist, and xylamidine, a 5-HT antagonist which has poor access to the brain. Metergoline (1 mg/kg) but not ritanserin (0.5 mg/kg) or xylamidine (3 mg/kg) blocked the effect of 2.5 mg/kg DF studied 2 and 4 h after injection. In a second experiment, we studied the ability of metergoline to antagonise the effect of DF, administered after a meal, on runway performance, food intake and gastric emptying assessed 4 h later. Metergoline at a dose of 1 mg/kg did not antagonise the effect of DF (2.5 mg/kg) on runway performance but completely blocked the effect on gastric emptying. The data clearly show that DF delays gastric emptying by indirectly activating 5-HT1 receptors; this effect is not important for the ability of DF to reduce runway performance and food intake when the drug is injected after a pre-feeding period. While there is evidence that DF hastens the termination of the meal by a 5-HT mechanism, the data suggest that DF may prolong the satiating effect of food during the post-absorptive phase by mechanisms other than 5-HT.

The effects of d-fenfluramine (DF) and d-norfenfluramine (DNF), administered intracerebroventricularly (i.c.v.) on levels of serotonin (5-HT) in the brain, was assessed in relation to levels of drugs in brain. d-Fenfluramine, as a single injection (500 micrograms/20 microliters), caused no significant changes in 5-HT in whole brain from 15 to 480 min after injection. When infused intraventricularly for 2 hr, DF and DNF at 500 but not at 125 250 micrograms/hr, markedly reduced concentrations of 5-HT in brain 4 hr after the end of the infusion. At this time levels of DNF in brain were similar (between 4 and 5 micrograms/g) with both compounds, whereas levels of DNF after single intraventricular injections of DF were below 2 micrograms/g at all times after injection. Infusion of 500 micrograms/hr of DNF for 2-hr reduced concentrations of 5-HT in various regions of the brain, with the exception of the brainstem, whereas 250 micrograms/hr of DNF significantly lowered levels of 5-HT only in the cortex. The effect of infusion of 500 micrograms/hr of DNF was specific for 5-HT (no effect on dopamine and norepinephrine) and lasted for at least 168 hr. The results suggest that the effect on 5-HT in brain of intraventricular infusion of DF, but not a single injection, was due to the fact that, only in the former condition were adequate levels of DNF, the active metabolite of DF, reached in the brain. These results are relevant to the interpretation of studies in which biochemical changes in the brain after intraventricular administration, are reported without any measurement of the drug or its active metabolites, in plasma and brain.

Following administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.04-5.0 micrograms/0.5 microliter) in the raphe nucleus dorsalis (DR) or medianus (MR), the synthesis of serotonin (5-HT), as assessed by the accumulation of 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition, was measured in various regions of the rat CNS. At all doses, 8-OH-DPAT in the DR significantly reduced 5-HTP accumulation in the striatum, nucleus accumbens, cortex, and prefrontal cortex, whereas even the highest dose had no effect in the hippocampus, hypothalamus, and spinal cord. One microgram of 8-OH-DPAT in the MR significantly reduced 5-HTP accumulation in the nucleus accumbens and prefrontal cortex, and 5 micrograms had an effect in all the areas except the striatum and spinal cord. One and 5 micrograms of 8-OH-DPAT, administered in either the DR or MR, did not significantly modify the accumulation of dihydroxyphenylalanine in the striatum and nucleus accumbens. The results confirm that DR and MR have different sensitivities to 5-HT1A receptor agonists, and that activation of 5-HT1A receptors in these nuclei produces different effects on 5-HT synthesis in different brain regions.

Neurons expressing the tryptophan hydroxylase (TPH) mRNA within the raphe nuclei of control rats showed a distributioin similar to that observed using an antibody for TPH. Numerous packed cells expressing the TPH mRNA were observed in the ventral and dorsal zone of the nucleus raphe dorsalis (NDR) and in the pars dorsalis of the nucleus centralis superior (NCS) whereas fewer and more scattered neurons were found in the pars medialis of NCS. Five days after the intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), which markedly reduced the serotonin (5-HT) content in the hippocampus, caudate putamen and cortex, the hybridization signal had completely disappeared in the dorsal region of the NDR. In the ventromedial region, above and between the medial longitudinal fasciculus (MLF), which includes the pars dorsalis of NCS, there was a partial decrease of cell number and a marked increase of the grain density over spared neurons. No significant change was noted in the number of TPH-positive cells and hybridization signal in individual neurons of the pars medialis of NCS. Consistent with previous evidence of increased TPH activity in the residual 5-HT terminals, the present study shows that synthesis of the TPH mRNA may be augmented in some neurons surviving the lesion.

The effects of sulpiride isomers injected into the nucleus accumbens on the anti-immobility activity of desipramine in rats were studied to clarify the role of dopamine receptors. (-)-Sulpiride 100 ng/0.5 microliters injected bilaterally into the nucleus accumbens 5 min before testing reduced the effect of a 7-day treatment with 10 mg/kg per day desipramine in the swimming test but 5 micrograms/0.5 microliter (+)-sulpiride injected in the same area had no effect. Fluphenazine, 5 micrograms/0.5 microliter, or 6-hydroxydopamine-induced depletion of dopamine in the nucleus accumbens also reduced the anti-immobility effect of desipramine. The data further support the hypothesis that dopamine transmission in the nucleus accumbens has a permissive role in the effect of desipramine in the forced swimming test.

The influence of alpha 2-adrenoceptor blockade on the activity of desipramine in an experimental model of depression was studied by using idazoxan and 1-(pyrimidinyl)piperazine (1-PP). The two drugs antagonists at these receptors, were studied for their ability to modify the effect of repeated treatment with the antidepressant, desipramine in the forced swimming test. Idazoxan (0.03, 0.3 and 3 mg/kg s.c.) and 1-PP (0.3 and 3 mg/kg p.o.) given with the last dose of a 7-day schedule of 10 mg/kg i.p. desipramine significantly reduced the effect of the latter on immobility. On its own neither drug modified the immobility time of rats at any dose. Infusion of various concentrations of idazoxan (1.6, 8 and 40 ng/microliters) in the rat locus coeruleus (LC), dose dependently antagonized the effect of desipramine without causing any appreciable change in motor behavior or immobility. The effect of idazoxan (8 ng/microliters) infusion in the LC was completely prevented by administering 6 micrograms 6-hydroxydopamine in the same region 12 days earlier. It thus appears that alpha 2-adrenoceptor blockade prevents the effect of desipramine in the forced swimming test, presumably by an effect on noradrenaline-containing cells in the LC. The question of how blockade or activation of alpha 2-adrenoceptors, both in the LC and in other sites, could influence antidepressant activity is discussed.

Several lines of evidence indicate that neuropeptide Y (NPY)-mediated neurotransmission in the hippocampus is altered by limbic seizures. The functional consequences of this change are still unresolved and clearly depend on the type of NPY receptors involved. We have investigated the role of NPY Y1 receptor subtypes, which are enriched in the dentate area of the hippocampus, on EEG seizures induced by a local injection of 0.04 microg kainic acid in rats. Intrahippocampal administration of 10 microg BIBP3226 (N2- (diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]D-arginamide), a non-peptide selective antagonist at the NPY Y1 receptors, increased threefold on average (P < 0.01) the time to onset of seizures and reduced the number of seizures and the total time in seizures three- and fourfold, respectively (P < 0.01). Its inactive S-enantiomer BIBP3435 was ineffective on seizure activity. One microgram [Leu31,Pro34]NPY, an agonist at Y1 receptors, did not modify per se the EEG sequelae induced by kainic acid but it antagonized the anticonvulsant effect of BIBP3226. These results indicate that NPY Y1 receptors in the hippocampus are involved in epileptic phenomena and suggest that selective Y1 receptor antagonists may be of value for attenuating limbic seizures.