Role of the serotonergic system in the forced swimming test.

The effect of manipulations aimed at modifying the function of the 5-HT system has been reviewed. 5-HT uptake inhibitors are devoid of any activity in rats and induce an anti-immobility effect in mice. The so-called 5-HT1A agonists reduce the immobility time with some differences in mice and rats, mice being less sensitive. None of the procedure aimed at reducing 5-HT function reduced immobility time. Therefore, the 5-HT system does not play a tonic role in animals performing the forced swimming test. The involvement of possible brain regions mediating the anti-immobility effects of 5-HT mimetic drugs has been discussed.

DAU 6215, a novel 5-HT3 receptor antagonist, improves performance in the aged rat in the Morris water maze task.

The effects of the new 5-HT3 receptor antagonist, DAU 6215, on aged rats' cognition were assessed in the Morris water maze task. Task performance of aged animals that received acutely the dose of 10 micrograms/kg IP was not different than that of their aged controls treated with the vehicle. Conversely, a repeated IP administration of 10 micrograms/kg DAU 6215 for 3 weeks significantly improved task performance of the aged animals as compared to that displayed by the old rats treated with the vehicle.

The inhibitory effect of 8-OH-DPAT on the firing activity of dorsal raphe serotoninergic neurons in rats is attenuated by lesion of the frontal cortex.

The dose-response inhibitory effect of 8-OH-DPAT on the firing rate of dorsal raphe serotoninergic neurons was shifted 10-fold to the right after acute fronto-cortical deafferentation. This finding suggests that the inhibitory effect of 8-OH-DPAT on the dorsal raphe firing rate might be mediated indirectly by the frontal cortex.

New dibenzothiadiazepine derivatives with antidepressant activities.

A new series of 11-[(aminoalkyl)carbonyl] derivatives of 6,11-dihydrodibenzo[c,f][1,2,5]thiadiazepine 5,5-dioxide (10-39) were synthesized and evaluated for potential antidepressant activity in the apomorphine-induced hypothermia (Apo 16) test. Effects on reserpine-induced hypothermia and toxicity for the most potent antagonists of Apo 16 hypothermia were also studied. Structure-activity relationships are reported. Anticholinergic effects were evaluated for compound 12, identified as the most potent and least toxic in this series, by assessing physostigmine lethality. Compound 12 was also subjected to X-ray analysis.

Effect of antipsychotic drugs and selective dopaminergic antagonists on dopamine-induced facilitatory activity in prelimbic cortical pyramidal neurons. An in vitro study.

Intracellular recordings were obtained from 119 pyramidal neurons localized in prelimbic cortex, five in the dorsal cingulate cortex, one in the infralimbic cortex, one in the border of prelimbic and cingulate cortex and two in the border of prelimbic and infralimbic cortex. The passive membrane properties of these pyramidal neurons (i.e. resting membrane potential, input membrane resistance, shape of the tetrodotoxin-sensitive action potentials, spike frequency adaptation with a prominent postspike afterhyperpolarization, tetrodotoxin-sensitive inward rectification in the depolarizing direction and the absence of bursting) suggested that they resembled regular spiking or intrinsically bursting pyramidal neurons. Bath application of dopamine (EC50 of 1.8 microM) produced a reversible facilitatory effect on all 119 pyramidal neurons localized in the middle layer of the prelimbic cortex. No consistent change in membrane potential was detected during the application of dopamine. No effect of dopamine was noted on the nine pyramidal neurons that were not localized in the prelimbic cortex. The facilitatory effect of dopamine in prelimbic cortex was concentration dependently antagonized by haloperidol, risperidone, quetiapine, clozapine and by the selective D4 dopaminergic receptor antagonist L-745,870, but not by the selective D2/D3 dopaminergic receptor antagonist (-)-sulpiride. (+)-SCH 23390, which is a selective D1/D5 dopamine receptor antagonist, produced, similarly to dopamine, a facilitatory effect per se, and an additive effect when co-administered with dopamine. These results provide evidence that dopamine has a facilitatory effect specifically on pyramidal neurons localized in the middle layer of prelimbic cortex. Antipsychotic drugs and L-745,870 block this effect of dopamine.

Is the forced swimming test a suitable model for revealing antidepressant activity?

The forced swimming test is reviewed. This test appears to be suitable for detecting antidepressant activity in rats but not in mice. Difference in experimental procedure may account for the different sensitivity to drugs of the two animal species.

Further characterisation of potential antidepressant action of flibanserin.

RATIONALE: Flibanserin has shown antidepressant-like properties in some animal models. In order to better define the probability that flibanserin may act as an antidepressant, its effects were tested in additional tests. OBJECTIVES: To assess the activity of flibanserin in the forced swimming test in rats, in the distress call frequency in isolated chicks, in the tail suspension test in mice and in muricidal rats. Flibanserin was also tested in mice performing an operant schedule of a food reinforcement fixed at an interval of 2 min. METHODS: Flibanserin was given intraperitoneally at a dose range between 0.5 and 32 mg/kg, 60 min before the muricidal test, 30 min before the tail suspension test, once (30 min) or three times (24, 5 and 1 h) before the forced swimming test, or just before testing (distress-induced calls in chicks). In the food reinforcement test in mice, flibanserin was given orally 60 min before testing. RESULTS: Flibanserin showed an antidepressant-like effect in the distress-induced calls in chicks (5 mg/kg) and in the muricidal test (16 and 32 mg/kg), but not in the tail suspension test (from 7.5 to 30 mg/kg). Flibanserin (8 and 16 mg/kg) increased immobility in the forced swimming test, either when administered once or for three times. Flibanserin increased the operant responses in the food reinforcement test (40 mg/kg). CONCLUSIONS: Flibanserin showed antidepressant-like effects in two out of four tests, and increased animal drive in the operant paradigm. These findings, together with others already published, may suggest that flibanserin will exert antidepressant activity in humans.

A model to measure anticipatory anxiety in mice?

Among animals from the same cage, mice removed last had a higher temperature compared to those removed first. This phenomenon a) persisted 2 and 24 h later; b) was present regardless of the number of the animals (5, 10, 15 and 20) in each cage, c) was independent of whether the number of animals was reduced or maintained constant in the cage and d) could even be observed by reversing the order of removal of the animals from the cage. In addition, the fewer the animals allocated to a cage the greater the percentage of those which became hyperthermic. This rise in rectal temperature of mice removed last was prevented by diazepam (2.5 and 5 mg/kg PO, 30 min), nitrazepam (2 and 4 mg/kg PO, 30 min) but not by imipramine (15 and 30 mg/kg PO, 60 min) or haloperidol (0.5 and 1 mg/kg PO, 60 min) and was observed in a greater percentage of mice following subcutaneous yohimbine treatment (2 mg/kg, 60 min). This phenomenon does not seem to depend on physical exercise due to an attempt to escape, since no correlation appears to exist between motor activity (open-field) and rise in rectal temperature. These data would seem to indicate that hyperthermia in the last animals may represent a new tool for studying the neurobiology of anticipatory(?) anxiety.

Pharmacological validation of a novel animal model of anticipatory anxiety in mice.

The current study investigates the action of anxiolytics, antidepressants, neuroleptics, antipyretics, muscle relaxants, antihypertensives and naloxone in a novel animal model of anxiety, based on the evidence that mice removed last from their cage develop hyperthermia (stress-induced hyperthermia, SIH) when compared to those removed first. Alprazolam (0.15-0.6 mg/kg), chlordiazepoxide (25 mg/kg), estazolam (1 mg/kg), phenobarbital (20 mg/kg), ethanol (2 and 4 g/kg), buspirone (5 and 10 mg/kg) and prazosin (1 and 2 mg/kg), as well as repeatedly administered diazepam (5 mg/kg), inhibited SIH. In contrast, tofisopam (12.5-200 mg/kg), desipramine (15 and 30 mg/kg), amitriptyline (10 mg/kg), fluoxetine (10 and 20 mg/kg), tranylcypromine (5 and 10 mg/kg), chlorpromazine (1 and 2 mg/kg), clozapine (2 and 4 mg/kg), pimozide (0.5 and 1 mg/kg), l-sulpiride (15 and 30 mg/kg), l-propranolol (5 and 10 mg/kg), acetyl salicylic acid (200 and 400 mg/kg), indomethacin (2.5 and 5 mg/kg), verapamil (2.5 and 5 mg/kg), captopril (25 and 50 mg/kg), dantrolene (10 and 20 mg/kg), mephenesin (300 and 600 mg/kg), d-amphetamine (1 and 4 mg/kg) and naloxone (2.5 and 15 mg/kg) were inactive, as were 10 mg/kg imipramine, amitriptyline and fluoxetine injected every day for 21 days. Reserpine at high doses (1.25 and 2.5 mg/kg) but not at a lower dose (0.62 mg/kg) prevented SIH, but in this case animals showed a behavioural syndrome which could have interfered with the occurrence of the hyperthermia.

Lack of relationship between effect of desipramine on forced swimming test and brain levels of desipramine or its demethylated metabolite in rats.

Animals were injected with 20 mg/kg desipramine (DMI) 1 h (acute) or 24, 5 and 1 h (subchronic) or once daily for 7 consecutive days (chronic) before the forced swimming test (FST). DMI was also injected at a dose of 40 mg/kg acutely. Animals were killed immediately after test for evaluation of brain concentrations of DMI and its demethylated metabolite desmethyldesipramine (DDMI). Acute and chronic DMI 20 mg/kg gave rise to similar brain concentrations but only chronic DMI was active on FST. Acute DMI 20 mg/kg and 40 mg/kg gave rise to different brain concentrations but similar effects on FST. DDMI concentrations were similar after the various DMI treatments. Results seem to indicate that no relationship exists between effect of DMI on FST and brain concentrations of either DMI or DDMI.

Discovery of antidepressant activity by forced swimming test may depend on pre-exposure of rats to a stressful situation.

Antidepressant-induced anti-immobility effects have been assessed in animals exposed or not to a pretest session using the forced swimming test. Desipramine, maprotiline, mianserine (15 and 30 mg/kg), nomifensine (2.5 and 5 mg/kg), d-amphetamine (1 and 2 mg/kg) and muscimol (1 and 2 mg/kg), unlike imipramine (15 and 30 mg/kg), LY-171555 (0.1 and 0.2 mg/kg) and scopolamine (0.5 and 0.1 mg/kg), did not reduce immobility time in rats which had not received the pretest session. On the other hand, all of the drugs tested reduced immobility time in rats exposed to a pretest session. In addition, the degree of antiimmobility effects of desipramine (20 mg/kg) and nomifensine (5 mg/kg) increased proportionally with the level of water (0, 4, 15 and 30 cm) to which animals were exposed at the time of pretest. Furthermore, desipramine reduced immobility time in rats pre-exposed to types of stress different from forced swimming, cold, restraint or foot-shock. All drugs were injected intraperitoneally three times, 24, 5 and 1 h before testing. The present findings suggest that a stressful pretest session may reveal pharmacological properties of antidepressants in the forced swimming test. This is also substantiated by the fact that diazepam (2.5 and 5 mg/kg) administered 30 min before the swimming pretest antagonized the anti-immobility effect of 15 mg/kg desipramine.

BIMT 17: a putative antidepressant with a fast onset of action?

BIMT 17, the only compound reported to be a full 5-HT1A agonist and a 5-HT2A antagonist at the frontal cortex, was assessed in three animal paradigms sensitive to antidepressants in rats: olfactory bulbectomy (OB), differential-reinforcement-of-low rate 72-s (DRL 72-s) and learned helplessness (LH). In the OB rats, BIMT 17, given once daily for 14 consecutive days at an i.p. dose of 10 mg/kg, but not of 20 mg/kg, reduced the increase in ambulation of OB rats, 24 h after the last administration. In the DRL 72-s test, BIMT 17 had a different profile than imipramine. A single i.p. injection of 5, 10, 15 or 20 mg/kg BIMT 17, in contrast to the same doses of imipramine, did not affect response and reinforcement rate in DRL 72-s 1 h after the administration. On the other hand, BIMT 17 slightly shifted the peak of the interresponse time (IRT) distribution towards shorter IRT duration, while imipramine shifted the peak of the IRT distribution towards longer IRT duration. In the LH test, acute oral doses (36, 48 or 60 mg/kg) of BIMT 17, given 30 min before testing, reduced the number of escape failures in LH without altering the intertrial crossings. This effect was also induced by a repeated, but not single, administration with 8 or 16 mg/kg imipramine. The plasma levels following i.p. 10 or oral 48 mg/kg BIMT 17 were in the same range. These results indicate that BIMT 17 does not behave like imipramine in all the tests, and suggest that BIMT 17 acts through different mechanisms of action than imipramine. Only clinical trials will tell whether these mechanisms will be relevant, but if so, BIMT 17 might induce a faster onset of therapeutic activity.

Further studies on the mechanism of serotonin-dependent anorexia in rats.

4-(3-Indolyl-2-ethyl) piperidine (LM 5008), 2-(1-piperazinyl) quinoline (quipazine), and metachlorophenylpiperazine (mCPP) were studied for their ability to affect serotonergic mechanisms in vitro. Their relative potency in inhibiting serotonin (5-HT) uptake in vivo and reducing food intake in rats was also examined. mCPP was very potent in displacing 3H-5-HT bound to brain membranes (IC50, 6.2 X 10(-7) M), followed by quipazine, which showed an IC50 of 3.8 X 10(-6) M. LM 5008 was the least effective with an IC50 of 3.6 X 10(-5) M. mCPP and quipazine were less potent than d-fenfluramine in releasing 14C-5-HT from brain synaptosomes, while LM 5008 caused no significant effects at a concentration of 10(-5) M. Conversely, both in vitro and in vivo studies on 5-HT uptake showed that LM 5008 was the most potent compound in inhibiting 5-HT uptake and mCPP the least potent. Since a 50% reduction of food intake was not reached even with a dose of LM 5008 27-times higher than the ED50 for inhibiting 5-HT uptake in vivo, it is suggested that even marked inhibition of 5-HT uptake at central synapses is not sufficient per se to trigger serotonin-dependent anorexia in the rat. Increased release and/or direct stimulation of post-synaptic receptors may be necessary to obtain this effect. This could be of interest for developing new agents which can cause anorexia by interacting with brain serotonin.

Further characterisation of [3H]8-hydroxy-2-(di-N-propylamino)tetralin binding sites in human brain postmortem.

The saturation parameters and the pharmacological characteristics of the binding of the serotonin 1A (5-HT1A) receptor agonist [3H]8-hydroxy-2-(di-N-propylamino)tetralin ([3H]8-OH-DPAT), as well as the effects of nucleotides and divalent cations (Mg2+, Mn2+) on it, were compared in some human postmortem brain regions: the main cortical areas, hippocampus and striatum. [3H]8-OH-DPAT labelled a single population of recognition sites with the highest maximal capacity (Bmax) in the hippocampus and the lowest affinity in the striatum. Among the various cortical areas, the frontal cortex exhibited the highest Bmax. The pharmacological profile of the [3H]8-OH-DPAT binding sites was consistent with the labelling of the 5-HT1A receptor in the hippocampus and cortex, whereas the striatal site shared strong similarity to the presynaptic serotonin transporter. Modulation of [3H]8-OH-DPAT binding by divalent cations and nucleotides was detectable and stable in autopsy brains. In particular, nucleotide effects were area-dependent: guanosine thiotriphosphate (GTP gamma S) reduced [3H]8-OH-DPAT binding to the same extent in the hippocampus and frontal cortex, while having no effect in the striatum. Divalent cation effects depended also upon the brain area: in the striatum, they inhibited [3H]8-OH-DPAT binding, while stimulating it in the hippocampus and, with less extent, in the frontal cortex. In summary, these findings suggest that the [3H]8-OH-DPAT binding and its modulatory parameters in human brain tissues seem to show similarities but also some differences with respect to those determined in the rat brain. Furthermore, postmortem stability of GTP and divalent cation sensitive 5-HT1A receptors underlines the need for further studies on the regulatory and functional properties of this receptor in the human brain.

Lack of stereoselectivity of 8-hydroxy-2(di-N-propylamino)tetralin-mediated inhibition of forskolin-stimulated adenylyl cyclase activity in human pre- and post-synaptic brain regions.

The stereoselectivity of the serotonin1A (5-HT1A) receptor compound 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT) on forskolin-stimulated adenylyl cyclase activity was investigated in membranes from human 5-HT pre-synaptic (raphe nuclei) and post-synaptic (hippocampus and prefrontal cortex) regions of autopsy brains. After sample incubation with agonists and antagonists, results showed that both the racemic mixture of 8-OH-DPAT or its (+) and (-) enantiomers behaved as full agonists in the tested brain regions. Enantiomer potency (EC50, nM) and efficacy (percentage of maximal inhibition, %) values were similar in all regions under investigation. However, some inter and intra-region variations in racemic 8-OH-DPAT potency and efficacy have been observed. In particular, the potency of racemic 8-OH-DPAT was higher in the prefrontal cortex and raphe nuclei than in the hippocampus, where it was in fact lower than either single enantiomers. Agonist effects were competitively reversed by 5-HT1A antagonists, although once again a different profile was revealed in the hippocampus. The data underscores the lack of stereospecificity of 8-OH-DPAT-mediated inhibition of adenylyl cyclase activity in either pre- or post-synaptic human brain regions. Moreover, such results have significant implication, as they support the notion that human 5-HT1A receptors might vary from one brain region to the other.

Hyper- and hyposensitivity of central serotonin receptors:[3H]serotonin binding and functional studies in the rat.

The effect of repeated treatment with D-fenfluramine, a serotonin releaser, or methergoline, a serotonin antagonist, on [3H]5-HT binding was studied in various rat brain areas. In animals with the same pretreatments, the anorectic activity of m-chlorophenylpiperazine, a serotonin agonist, was investigated. A 14-day treatment with D-fenfluramine caused a significant decrease in the number of [3H]5-HT binding sites (Bmax) in the diencephalon. A reduction of binding sites was found in the cortex too when D-fenfluramine was administered for 28 days. Methergoline caused no changes of [3H)5-HT binding in any brain area examined when given for 14 days but 28-day treatment led to a significant increase in the striatum, hippocampus and cortex. D-Fenfluramine and methergoline caused, respectively, a decrease and increase in the effect of m-chlorophenylpiperazine on food intake. The data show that central 5-HT receptor numbers and sensitivity may change after repeated treatments with drugs acting on brain serotonin.

The effect of 4-aminopyridine on micturition reflex in normal or capsaicin-desensitized rats.

4-aminopyridine (4-AP) produced a dose-related (0.15-2 mg/kg i.v.) potentiation of the voiding cycle of the urinary bladder and increased frequency of micturition in urethane-anesthetized rats. In bladders containing a subthreshold amount of fluid for eliciting reflex micturition 4-AP (1-3 mg/kg i.v.) activated a series of high-amplitude, hexamethonium-sensitive rhythmic bladder contractions. In rats desensitized to capsaicin as newborns, reflex micturition was almost abolished: in these animals i.v. 4-AP did not affect bladder voiding unless at high doses (1-2 mg/kg), at which a reversal from anesthesia occurred. This was accompanied by a prompt micturition. In unanesthetized rats, neither the 4-AP-induced convulsions nor the behavioral response (assessed in an open field) to 4-AP were affected by neonatal capsaicin desensitization. Daily urine production of capsaicin-pretreated animals did not differ from that of controls. However, when measurements were made during daytime, almost no spontaneous urine emission was found in capsaicin-treated rats. On the rat isolated urinary bladder, 4-AP potentiated the response to field stimulation in preparations from both vehicle- and capsaicin-pretreated animals. These findings indicate that 4-AP has a potent excitatory action on bladder voiding in rats and support the hypothesis that in this species 'conscious' bladder voiding can be initiated through capsaicin-resistant mechanisms.

Effects of postmortem delay on serotonin and (+)8-OH-DPAT-mediated inhibition of adenylyl cyclase activity in rat and human brain tissues.

The reproducibility of serotonin (5-HT) and (+)8-OH-DPAT-mediated inhibition of adenylyl cyclase activity was assessed in membranes, stimulated by forskolin, of rat frontal cortex postmortem as well as of human fronto-cortical, hippocampal and dorsal raphe tissues obtained from autopsy brains. The results revealed that differences between basal and forskolin-stimulated enzyme activities were still significant after 48 h postmortem in rat cortex and in all human brain regions up to 46 h after death. However, a decrease of about 17 and 26% in forskolin-stimulated adenylyl cyclase activity was observed at 24 and 48 h, respectively, in rat cortex. 5-HT and the 5-HT1A receptor agonist, (+)8-hydroxy-2(di-N-propylamino)tetraline (8-OH-DPAT), were able to inhibit forskolin-stimulated adenylyl cyclase activity in a dose-dependent manner for 48 h after death in rat and human brain. In rat cortex, both 5-HT and (+)8-OH-DPAT potencies (EC50, nM) and efficacies (percent of maximum inhibition capacity, %) varied significantly with postmortem delay. Conversely, in human tissues, postmortem delay and subject age did not modify agonist potencies and efficacies. Furthermore, a regionality of 5-HT potency and efficacy was revealed in the human brain. 5-HT was equally potent in cortex and raphe nuclei, while being more potent but less effective in hippocampus. (+)8-OH-DPAT was more active in hippocampus and raphe nuclei than in cortex. (+)8-OH-DPAT behaved as an agonist in all areas, as its efficacy was similar or greater than those obtained with 5-HT. The (+)8-OH-DPAT dose-response curve was completely reversed by 5-HT1A receptor antagonists in rat cortex and all human brain areas. In conclusion, we suggest here that differences between rat and human brain might exist at the level of postmortem degradation of 5-HT-sensitive adenylyl cyclase activity. In human brain, 5-HT1A receptor-mediated inhibition of adenylyl cyclase seems to be reproducible, suggesting that reliable experiments can be carried out on postmortem specimens from patients with neuropsychiatric disorders.

Localization and gene expression of serotonin 1A (5HT1A) receptors in human brain postmortem.

We investigated the binding parameters, i.e. the maximum binding capacity (Bmax) and the dissociation constant (Kd), of [3H]8-hydroxy-2-(di-N-propylamino)tetralin ([3H]8-OH-DPAT) labeling the serotonin receptor of the 1A type (5HT1A), and the distribution of the mRNA encoding it in some human brain areas obtained from autoptic samples. The results showed that the Bmax was significantly higher in the hippocampus than in the prefrontal cortex and the striatum, while the Kd had the inverse, although not significant, pattern. The expression study revealed that 5HT1A mRNA distribution in the hippocampus and prefrontal cortex was consistent with the data of the [3H]8-OH-DPAT binding. A different result was obtained in the striatum where no 5HT1A mRNA expression was detected, despite the measurement of specific [3H]8-OH-DPAT binding. These findings underline the different nature of [3H]8-OH-DPAT binding sites in different brain areas and the need for further studies on 5HT receptor gene expression in human brain.

Developmental effects of early postnatal treatment of rats with prochlorperazine.

This study examined effects of neonatal prochlorperazine in rats tested as adults in a variety of behaviors. The drug exposure impaired open field behavior during treatment neonatally. However, no differences in open field behavior were observed in adulthood. Adult females that had received drug were less efficient in obtaining food reinforcement in a conditioned maze performance than were respective controls and showed a slight increase in motor activity during accommodation. d-Amphetamine-induced motor activity was greater in both treated males and females relative to respective controls. Spaced trials in shuttle-box avoidance uncovered an avoidance decrement in the treated females only. This drug treatment appears to cause subtle changes in brain development in the rat.