Azair Canto-de-Souza

Anxiolytic-like effect of way-100635 microinfusions into the median (but not dorsal) raphe nucleus in mice exposed to the plus-maze: influence of prior test experience

Studies in several laboratories have confirmed the anxiolytic potential of a wide range of 5-HT(1A) receptor antagonists in rats and mice, with recent evidence pointing to a postsynaptic site of action in the ventral hippocampus. It would, therefore, be predicted that blockade of 5-HT(1A) somatodendritic autoreceptors in the midbrain raphe nuclei should produce anxiogenic-like effects. To test this hypothesis, we investigated the effects of WAY-100635 microinfusions (0, 1.0 or 3.0 microg in 0.1 microl) into the dorsal (DRN) or median (MRN) raphe nuclei on behaviours displayed by male Swiss-Webster mice in the elevated plus-maze. As this test is sensitive to prior experience, the effects of intra-raphe infusions were examined both in maze-naive and maze-experienced subjects. Sessions were videotaped and subsequently scored for conventional indices of anxiety (open arm avoidance) and locomotor activity (closed arm entries), as well as a range of ethological measures (e.g. risk assessment). In maze-naive mice, intra-MRN (but not intra-DRN) infusions of WAY-100635 (3.0 microg) increased open arm exploration and reduced risk assessment. Importantly, these effects could not be attributed to a general reduction in locomotor activity. A similar, though somewhat weaker, pattern of behavioural change was observed in maze-experienced animals. This unexpected anxiolytic effect of 5-HT(1A) autoreceptor blockade in the MRN cannot be accounted for by a disinhibition of 5-HT release in forebrain targets (e.g. hippocampus and amygdala), where stimulation of postsynaptic 5-HT(1A) receptors enhances anxiety-like responses. However, as the MRN also projects to the periaqueductal gray matter (PAG), an area known to be sensitive to the anti-aversive effects of 5-HT, it is argued that present results may reflect increased 5-HT release at this crucial midbrain locus within the neural circuitry of defense.

Effects of intra-hippocampal infusion of WAY-100635 on plus-maze behavior in mice. Influence of site of injection and prior test experience.

The positive profile of systemically-administered 5-HT(1A) receptor antagonists in several rodent models of anxiolytic activity suggests an important role for postsynaptic 5-HT(1A) receptor mechanisms in anxiety. To test this hypothesis, we investigated the effects of WAY-100635 microinfusions (0, 0.1, 1.0 or 3.0 microg in 0.2 microl) into the dorsal (DH) or ventral (VH) hippocampus on behaviours displayed by male Swiss-Webster mice in the elevated plus-maze. As prior experience is known to modify pharmacological responses in this test, the effects of intra-hippocampal infusions were examined both in maze-naïve and maze-experienced subjects. Test videotapes were scored for conventional indices of anxiety (% open arm entries/time) and locomotor activity (closed arm entries), as well as a range of ethological measures (e.g. risk assessment). In maze-naïve mice, intra-VH (but not intra-DH) infusions of WAY-100635 (3.0 microg but not lower doses) increased open arm exploration and reduced risk assessment. These effects were observed in the absence of significant changes in locomotor activity. In contrast, neither intra-VH nor intra-DH infusions of WAY-100635 altered the behaviour of maze-experienced mice. These findings suggest that postsynaptic 5-HT(1A) receptors in the ventral (but not dorsal) hippocampus play a significant role both in the mediation of plus-maze anxiety in mice and in experientially-induced alterations in responses to this test.

5-HT2 receptor activation in the midbrain periaqueductal grey (PAG) reduces anxiety-like behaviour in mice

It is widely acknowledged that the indoleamine neurotransmitter serotonin (5-HT) plays a dual role in the regulation of anxiety, a role that in part depends upon neuroanatomical locus of action. Thus, whereas stimulation of 5-HT 1A or 5-HT2 receptors in the limbic forebrain (amygdala, hippocampus) enhances anxiety-like responding in rodents, activation of corresponding receptor populations in the midbrain periaqueductal grey (PAG) more often than not reduce anxiety-like behaviour. The present study specifically concerns the anxiety-modulating influence of 5-HT2 receptors within the mouse PAG. Experiment 1 assessed the effects of intra-PAG infusions of the 5-HT2B/2C receptor agonist mCPP (0, 0.03, 0.1 or 0.3 nmol/0.1 microl) on the behaviour of mice exposed to the elevated plus-maze. As mCPP acts preferentially at 5-HT2B and 5-HT2C receptors, Experiment 2 investigated its effects in animals pretreated with ketanserin, a preferential 5-HT2A/2C receptor antagonist. In both cases, test sessions were videotaped and subsequently, scored for anxiety-like behaviour (e.g., percentage of open arm entries and percentage of open arm time) as well as general locomotor activity (closed arm entries). The results of Experiment 1 showed that mCPP microinfusions (0.03 and 0.1 nmol) into the PAG of mice decreased behavioural indices of anxiety without significantly altering general activity measures. In Experiment 2, the anxiolytic-like profile of intra-PAG mCPP (0.03 nmol) was substantially attenuated by intra-PAG pretreatment with an intrinsically inactive dose of the preferential 5-HT2A/2C receptor antagonist, ketanserin (10 nmol/0.1mul). Together, these data suggest that 5HT2C receptor populations within the midbrain PAG play an inhibitory role in plus-maze anxiety in mice.

Microinjection of histamine into the cerebellar vermis impairs emotional memory consolidation in mice.

The biogenic amine histamine is an important neurotransmitter in the central nervous system that has been implicated in learning and memory processes. Experimental evidence indicates that the role of the cerebellum may be more complex than the simple regulation of motor responses, and recent studies have demonstrated significant involvement of the cerebellum in emotional memory consolidation. This study investigated the effect of histamine microinjected into the cerebellar vermis on emotional memory consolidation in mice in the elevated plus-maze (EPM). The cerebellar vermis of male mice (Swiss Albino) were implanted with guide cannulae. The mice weighed between 25 and 30 g. After three days of recovery, behavioral tests in the EPM were performed on two consecutive days; the testing periods were called, Trial 1 and Trial 2. Immediately after Trial 1, the animals received microinjections of histamine in the cerebellar vermis (0.54, 1.36, 2.72, and 4.07 nmol/0.1 μl). On both days, the test sessions were recorded to enable analysis of behavioral measures. The decrease in open arm exploration (% entries and % time spent in the open arms) in Trial 2 relative to Trial 1 was used as a measure of learning and memory. The data were analyzed using One-way Analysis of Variance (ANOVA) and Duncans tests. The percentage of open arm entries (%OAE) and the percentage of time spent in the open arms (%OAT) were reduced in Trial 2 relative to Trial 1 for the control group; the same was true for the group that was microinjected with histamine at doses of 0.54 (%OAE and %OAT) and 1.36 nmol (%OAT). However, when the animals received histamine at doses of 2.72 and 4.07 nmol, their open arm exploration did not decrease. No significant changes were observed in the number of enclosed arm entries (EAE), an EPM index of general exploratory activity. These results suggest that there is a dose-dependent inhibitory effect of histamine microinjected into the cerebellar vermis on emotional memory consolidation.

Emotional memory consolidation impairment induced by histamine is mediated by H1 but not H2 receptors

Histaminergic fibers are present in the molecular and granular layers of the cerebellum and have high density in the vermis and flocculus. Evidence indicates that the cerebellar vermis is involved in memory consolidation. Recently, we demonstrated that when histamine is microinjected into the cerebellar vermis it results in impaired emotional memory consolidation in mice that are submitted to the elevated plus maze (EPM). This study investigated whether histamine impairment was mediated by the H(1) or H(2) receptors. The cerebellar vermis of male mice (Swiss Albino) were implanted using a guide cannula. Three days after recovery, behavioral tests were performed in the EPM on two consecutive days (Trial 1 and Trial 2). Immediately after exposure to the EPM (Trial 1), animals received a microinjection of histaminergic drugs. In Experiment 1, saline (SAL) or histamine (HA, 4.07 nmol/0.1 μl) was microinjected 5 min after pretreatment with the H(1) antagonist chlorpheniramine (CPA, 0.16 nmol/0.1μl) or SAL. In Experiment 2, SAL or HA was microinjected into the mice 5 min after pretreatment with the H(2) antagonist ranitidine (RA, 2.85 nmol/0.1 μl) or SAL. Twenty-four hours later, the mice were re-exposed to the EPM (Trial 2) under the same experimental conditions but did not receive an injection. On both days, the test sessions were recorded to enable analysis of the behavioral measures. The decrease in open arm exploration (% entries and % time spent in the open arms) in Trial 2 relative to Trial 1 was used as a measure of learning and memory. The data were analyzed using the two-way analysis of variance (ANOVA) and Duncans tests. In Experiment 1, the Duncans test indicated that the mice entered the open arms less often (%OAE) and spent less time in the open arms (%OAT) in Trial 2 after being microinjected with SAL+SAL, SAL+CPA and CPA+HA. However, the animals that received SAL+HA did not enter the open arms less frequently or spend less time in them, which was significantly different from the CPA+HA group. The results of Experiment 2 demonstrated that the %OAE and %OAT in Trial 2 were different from Trial 1 for the groups that were microinjected with SAL+SAL and SAL+RA. The animals that were microinjected with RA+HA or with SAL+HA did not show a reduction in %OAE. These results demonstrate that the animals treated with HA did not avoid the open arms less on retesting and indicated that CPA did not alter the behavior parameters but did revert the histamine-induced impairment of memory consolidation. Furthermore, the H(2) antagonist RA, at the dose used in this study, did not affect memory consolidation and failed to revert histamine-induced impairment.

L-histidine provokes a state-dependent memory retrieval deficit in mice re-exposed to the elevated plus-maze

The effects of L-histidine (LH) on anxiety and memory retrieval were investigated in adult male Swiss Albino mice (weight 30-35 g) using the elevated plus-maze. The test was performed on two consecutive days: trial 1 (T1) and trial 2 (T2). In T1, mice received an intraperitoneal injection of saline (SAL) or LH before the test and were then injected again and retested 24 h later. LH had no effect on anxiety at the dose of 200 mg/kg since there was no difference between the SAL-SAL and LH-LH groups at T1 regarding open-arm entries (OAE) and open-arm time (OAT) (mean +/- SEM; OAE: 4.0 +/- 0.71, 4.80 +/- 1.05; OAT: 40.55 +/- 9.90, 51.55 +/- 12.10, respectively; P > 0.05, Kruskal-Wallis test), or at the dose of 500 mg/kg (OAE: 5.27 +/- 0.73, 4.87 +/- 0.66; OAT: 63.93 +/- 11.72, 63.58 +/- 10.22; P > 0.05, Fisher LSD test). At T2, LH-LH animals did not reduce open-arm activity (OAE and OAT) at the dose of 200 mg/kg (T1: 4.87 +/- 0.66, T2: 5.47 +/- 1.05; T1: 63.58 +/- 10.22; T2: 49.01 +/- 8.43 for OAE and OAT, respectively; P > 0.05, Wilcoxon test) or at the dose of 500 mg/kg (T1: 4.80 +/- 1.60, T2: 4.70 +/- 1.04; T1: 51.55 +/- 12.10, T2: 43.88 +/- 10.64 for OAE and OAT, respectively; P > 0.05, Fisher LSD test), showing an inability to evoke memory 24 h later. These data suggest that LH does not act on anxiety but does induce a state-dependent memory retrieval deficit in mice.

Intra-cerebellar microinjection of histamine enhances memory consolidation of inhibitory avoidance learning in mice via H2 receptors

Studies have demonstrated the relationship between the histaminergic system and the cerebellum, and we intend to investigate the role of the cerebellar histaminergic system on memory consolidation. This study investigated the effect of intra-cerebellar microinjection of histamine on memory retention of inhibitory avoidance in mice, and the role of H1 and H2 receptors in it. The cerebellar vermis of male mice were implanted with guide cannulae, and after three days of recovery, the inhibitory avoidance test was performed. Immediately after a training session, animals received a microinjection of histaminergic drugs: in the experiment 1, saline (SAL) or histamine (HA 0.54, 1.36, 2.72 or 4.07 nmol); experiment 2, SAL or 1.36 nmol HA 5 min after a pretreatment with 0.16 nmol chlorpheniramine (CPA) or SAL; and experiment 3, SAL or 1.36 nmol HA 5 min after a pretreatment with 2.85 nmol ranitidine (RA) or SAL. Twenty-four hours later, a retention test was performed. The data were analyzed using one-way analysis of variance (ANOVA) and Duncans tests. In experiment 1, animals microinjected with 1.36 nmol HA showed a higher latency to cross to the dark compartment compared to controls and to 2.72 and 4.07 nmol HA groups. In experiment 2, the combined infusions revealed difference between control (SAL+SAL) and SAL+HA and CPA+HA; while in the experiment 3 the analysis indicated differences in retention latency between mice injected with SAL+SAL and SAL+HA. The groups that received the H2 antagonist RA did not show difference compared to control. These results indicate that 1.36 nmol HA enhances memory consolidation of inhibitory avoidance learning in mice and that the pretreatment with H2 antagonist RA was able to prevent this effect.

l-histidine induces state-dependent memory deficit in mice mediated by H1 receptor

This study investigated the role of H(1) receptor in the state-dependent memory deficit induced by l-histidine (LH) in mice using Trial 1/2 protocol in the elevated plus-maze (EPM). The test was performed for two consecutive days: Trial 1 (T1) and Trial 2 (T2). Before both trials, mice received a combined injection i.p. of saline+saline (SAL/SAL), 500 mg/kg L-histidine+saline (LH/SAL), 500 mg/kg L-histidine+16 mg/kg chlorpheniramine (LH/CPA) or saline+16 mg/kg chlorpheniramine (SAL/CPA). The trials were performed in the EPM 10 min after the last injection. Each animal was placed in the center of the maze facing the open arm and had five minutes to explore it. On both days, test sessions were videotaped. The behavioral measures were scored from videotape. Data were analyzed based on Analysis of Variance (ANOVA) and the Fishers LSD test. The data showed no effects on anxiety since there was no difference between the SAL/SAL and the other groups in Trial 1, respectively, open arm entries (OAE), open arm time (OAT) and their percentages (%OAE and %OAT). During Trial 2, OAE, OAT, %OAE and %OAT were reduced in mice treated with SAL/SAL, LH/CPA and SAL/CPA, while the group LH/SAL did not show any difference in these measures. No significant changes were observed in enclosed arm entries (EAE), an EPM index of general exploratory activity. Thus, it can be suggested that LH induces emotional memory deficit and the treatment with chlorpheniramine was able to revert this effect, suggesting this action of LH was mediated by the H(1) receptor.

H1 but not H2 histamine antagonist receptors mediate anxiety-related behaviors and emotional memory deficit in mice subjected to elevated plus-maze testing

This study investigated the role of H1 and H2 receptors in anxiety and the retrieval of emotional memory using a Trial 1/Trial 2 (T1/T2) protocol in an elevated plus-maze (EPM). Tests were performed on 2 consecutive days, designated T1 and T2. Before T1, the mice received intraperitoneal injections of saline (SAL), 20 mg/kg zolantidine (ZOL, an H2 receptor antagonist), or 8.0 or 16 mg/kg chlorpheniramine (CPA, an H1 receptor antagonist). After 40 min, they were subjected to the EPM test. In T2 (24 h later), each group was subdivided into two additional groups, and the animals from each group were re-injected with SAL or one of the drugs. In T1, the Student t-test showed no difference between the SAL and ZOL or 8 mg/kg CPA groups with respect to the percentages of open arm entries (%OAE) and open arm time (%OAT). However, administration of CPA at the highest dose of 16 mg/kg decreased %OAE and %OAT, but not locomotor activity, indicating anxiogenic-like behavior. Emotional memory, as revealed by a reduction in open arm exploration between the two trials, was observed in all experimental groups, indicating that ZOL and 8 mg/kg CPA did not affect emotional memory, whereas CPA at the highest dose affected acquisition and consolidation, but not retrieval of memory. Taken together, these results suggest that H1 receptor, but not H2, is implicated in anxiety-like behavior and in emotional memory acquisition and consolidation deficits in mice subjected to EPM testing.

Involvement of the midbrain periaqueductal gray 5-HT1A receptors in social conflict induced analgesia in mice

Recent results from our laboratory have shown that 30-bites social conflict in mice produces a high-intensity, short-term analgesia which is attenuated by systemically injected 5-HT1A receptor agonists, such as BAY R 1531 (6-methoxy-4-(di-n-propylamino)-1,3,4,5-tetrahydrobenz((c,d)indole hydrochloride) and gepirone. The present study investigated the effects of these drugs, as well as the 5-HT1A receptor antagonist WAY 100135 (N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide ) injected into the midbrain periaqueductal gray matter of mice on 30-bites analgesia. Four to five days after guide-cannula implantation, each mouse received microinjection of gepirone (30 nmol/0.2 microl), BAY R 1531 (10 nmol/0.2 microl), WAY 100135 (10 nmol/0.2 microl), saline (0.9% NaCl) or vehicle (saline + 4% Tween 80) 5 min before either an aggressive (30 bites) or a non-aggressive interaction. Nociception was assessed by the tail-flick test made before as well as 1, 5, 10 and 20 min after social interaction. The full 5-HT1A receptor agonist BAY R 1531 blocked, whereas, WAY 100135 and gepirone intensified 30-bites analgesia. Neither non-aggressive interaction, per se, nor the three compounds given after this type of social interaction significantly changed nociception. These results indicate that 5-HT1A receptors in the periaqueductal gray inhibit analgesia induced by social conflict in mice.