Systemic injection of an H4 receptor agonist induces a decrease in CREB and pCREB levels in the cerebellar vermis and prefrontal cortex in mice.

Studies have shown that an injection with the histamine H4 receptor agonist VUF-8430 modulates emotional memory processes. In the present study, the aim was to verify if intraperitoneal (ip) injection of VUF-8430 (500 ng/kg) in mice affects the synthesis of proteins required for memory consolidation processes by activating the phosphorylation of CREB (pCREB) in classical structures linked to emotional memory (prefrontal cortex, amygdala, and hippocampus) and the cerebellar vermis, a structure that has also been recently implicated in emotional memory. The results obtained using western blot analysis demonstrated that VUF-8430 induced a decrease in CREB and pCREB levels in the cerebellar vermis and prefrontal cortex, suggesting that this dose impaired the activation of cell signaling pathways in these structures. There was no change in protein expression in the amygdala and hippocampus. Our results are preliminary, and further investigations are needed to investigate the role of the H4 receptors in the central nervous system.

Systemic injection of an H4 receptor agonist induces a decrease in CREB and pCREB levels in the cerebellar vermis and prefrontal cortex in mice

Studies have shown that an injection with the histamine H4 receptor agonist VUF-8430 modulates emotional memory processes. In the present study, the aim was to verify if intraperitoneal (ip) injection of VUF-8430 (500 ng/kg) in mice affects the synthesis of proteins required for memory consolidation processes by activating the phosphorylation of CREB (pCREB) in classical structures linked to emotional memory (prefrontal cortex, amygdala, and hippocampus) and the cerebellar vermis, a structure that has also been recently implicated in emotional memory. The results obtained using western blot analysis demonstrated that VUF-8430 induced a decrease in CREB and pCREB levels in the cerebellar vermis and prefrontal cortex, suggesting that this dose impaired the activation of cell signaling pathways in these structures. There was no change in protein expression in the amygdala and hippocampus. Our results are preliminary, and further investigations are needed to investigate the role of the H4 receptors in the central nervous system.

Intra-vermis H4 receptor agonist impairs performance in anxiety- and fear-mediated models.

The neural histaminergic system modulates cognitive performance in various animal models. However, little is known about the effects of the H4 histaminergic receptor in the central nervous system. The purpose of this study was to investigate the effect of histaminergic H4 agonist VUF-8430 microinjection into the cerebellar vermis on the consolidation of emotional memory in mice subjected to the elevated plus maze (EPM) and inhibitory avoidance task (IAT). All experiments were performed on two consecutive days: exposure (T1 and D1) and 24h after, which we called re-exposure (T2 and D2). The animals received saline (SAL) or VUF (0.15 nmol; 0.49 nmol; 1.48 nmol/0.1µl) administered post-exposure. Experiment 1 was conducted in the EPM, and the animals were free to explore the maze for 5min. In T1, immediately after exposure, the pharmacological treatment was given; in T2, there was only re-exposure to the EPM. Experiment 2 involved the IAT, and the pharmacological treatment was provided post-D1; in D2, the animals were only re-exposed to the IAT. In Experiment 1, increased open arm exploration (% open arm entries and% open arms time) for 0.49 and 1.48nmol of VUF were recorded in T2 compared to T1. In Experiment 2, a significant decrease in consolidation latency was recorded for the group that received 1.48nmol of VUF compared to the SAL group in D2. These results indicate that a 1.48nmol VUF microinjection into the cerebellar vermis impaired performance in both models, even though one model was anxiety-mediated (EPM) and the other was fear-mediated (IAT).

Cholinergic agonist reverses H1-induced memory deficit in mice.

This study investigated the effects of bilateral intraamygdalar microinjections of PNU-282987, a nicotinic cholinergic agonist, on anxiety and the reversal of amnesia induced by chlorpheniramine (CPA), an H1 histaminergic antagonist, in mice subjected to the elevated plusmaze (EPM). Two experiments were performed with seventy-nine adult male Swiss mice. The isolated microinjections of PNU-282987 did not produce effects on emotional memory; however, the combined microinjections of PNU-282987 and CPA were able to reverse the deficit in memory induced by CPA (ANOVA, p<0.05). Taken together, these results suggest that intraamygdalar injections of PNU-282987 did not induce effects on anxiety and emotional memory per se; however, concurrent microinjections of PNU-282987 and CPA-reverse amnesia induced-CPA which is suggestive of an interaction between the histaminergic and cholinergic systems in the modulation of emotion memory acquisition in mice.

Intra-amygdala microinjections of chlorpheniramine impair memory formation or memory retrieval in anxiety- and fear-mediated models.

H1 receptor histaminergic antagonist, chlorpheniramine (CPA) participates in cognitive performance in various animal models. However, little is known regarding the effects of CPA microinjection into the amygdala on emotional behavior. The purpose of this study was to investigate whether CPA microinjection into the amygdala has the same effect on two models, one anxiety- and the other fear-mediated, in various memory stages using the elevated plus maze (EPM) and the inhibitory avoidance task (IAT) tests. Two experiments were performed with seventy-two adult male Swiss mice. Behavioral testing was performed on two consecutive days, and in both experiments, before each trial, the animals received bilateral microinjections of saline (SAL) or CPA (0.16 nmol). The animals were re-exposed to the EPM or IAT 24h after the first trial. Four experimental groups were tested: SAL-SAL, SAL-CPA, CPA-SAL and CPA-CPA. In experiment 1, a decreased open arm exploration (% open arm entries, %OAE and% open arms time, %OAT) for SAL-SAL and SAL-CPA was showed, while these measures did not decrease for the CPA-SAL and CPA-CPA groups in Trial 2. In experiment 2, an increase of retention latency in relation to training 2 for the groups SAL-SAL and CPA-SAL and a significant decrease in latency for the group SAL-CPA was revealed. These results indicate that chlorpheniramine microinjection into the amygdala impairs emotional memory acquisition and/or consolidation in the EPM and retrieval of IAT.

Cerebellar vermis H2 receptors mediate fear memory consolidation in mice.

Histaminergic fibers are present in the molecular and granular layers of the cerebellum and have a high density in the vermis and flocullus. Evidence supports that the cerebellar histaminergic system is involved in memory consolidation. Our recent study showed that histamine injections facilitate the retention of an inhibitory avoidance task, which was abolished by pretreatment with an H2 receptor antagonist. In the present study, we investigated the effects of intracerebellar post training injections of H1 and H2 receptor antagonists as well as the selective H2 receptor agonist on fear memory consolidation. The cerebellar vermi 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 the following histaminergic drugs: experiment 1, saline or chlorpheniramine (0.016, 0.052 or 0.16 nmol); experiment 2, saline or ranitidine (0.57, 2.85 or 5.07 nmol); and experiment 3, saline or dimaprit (1, 2 or 4 nmol). Twenty-four hours later, a retention test was performed. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's tests. Animals microinjected with chlorpheniramine did not show any behavioral effects at the doses that we used. Intra-cerebellar injection of the H2 receptor antagonist ranitidine inhibited, while the selective H2 receptor agonist dimaprit facilitated, memory consolidation, suggesting that H2 receptors mediate memory consolidation in the inhibitory avoidance task in mice.

Effect of histamine H1 and H2 receptor antagonists, microinjected into cerebellar vermis, on emotional memory consolidation in mice.

This study investigated the effects of histamine H1 or H2 receptor antagonists on emotional memory consolidation in mice submitted to the elevated plus maze (EPM). The cerebellar vermis of male mice (Swiss albino) was implanted using a cannula guide. Three days after recovery, behavioral tests were performed in the EPM on 2 consecutive days (T1 and T2). Immediately after exposure to the EPM (T1), animals received a microinjection of saline (SAL) or the H1 antagonist chlorpheniramine (CPA; 0.016, 0.052, or 0.16 nmol/0.1 µL) in Experiment 1, and SAL or the H2 antagonist ranitidine (RA; 0.57, 2.85, or 5.7 nmol/0.1 µL) in Experiment 2. Twenty-four hours later, mice were reexposed to the EPM (T2) under the same experimental conditions but they did not receive any injection. Data were analyzed using one-way ANOVA and the Duncan test. In Experiment 1, mice microinjected with SAL and with CPA entered the open arms less often (%OAE) and spent less time in the open arms (%OAT) in T2, and there was no difference among groups. The results of Experiment 2 demonstrated that the values of %OAE and %OAT in T2 were lower compared to T1 for the groups that were microinjected with SAL and 2.85 nmol/0.1 µL RA. However, when animals were microinjected with 5.7 nmol/0.1 µL RA, they did not show a reduction in %OAE and %OAT. These results demonstrate that CPA did not affect behavior at the doses used in this study, while 5.7 nmol/0.1 µL RA induced impairment of memory consolidation in the EPM.

Effect of histamine H1 and H2 receptor antagonists, microinjected into cerebellar vermis, on emotional memory consolidation in mice

This study investigated the effects of histamine H1 or H2 receptor antagonists on emotional memory consolidation in mice submitted to the elevated plus maze (EPM). The cerebellar vermis of male mice (Swiss albino) was implanted using a cannula guide. Three days after recovery, behavioral tests were performed in the EPM on 2 consecutive days (T1 and T2). Immediately after exposure to the EPM (T1), animals received a microinjection of saline (SAL) or the H1 antagonist chlorpheniramine (CPA; 0.016, 0.052, or 0.16 nmol/0.1 µL) in Experiment 1, and SAL or the H2 antagonist ranitidine (RA; 0.57, 2.85, or 5.7 nmol/0.1 µL) in Experiment 2. Twenty-four hours later, mice were reexposed to the EPM (T2) under the same experimental conditions but they did not receive any injection. Data were analyzed using one-way ANOVA and the Duncan test. In Experiment 1, mice microinjected with SAL and with CPA entered the open arms less often (%OAE) and spent less time in the open arms (%OAT) in T2, and there was no difference among groups. The results of Experiment 2 demonstrated that the values of %OAE and %OAT in T2 were lower compared to T1 for the groups that were microinjected with SAL and 2.85 nmol/0.1 µL RA. However, when animals were microinjected with 5.7 nmol/0.1 µL RA, they did not show a reduction in %OAE and %OAT. These results demonstrate that CPA did not affect behavior at the doses used in this study, while 5.7 nmol/0.1 µL RA induced impairment of memory consolidation in the EPM.

Low-dose thioperamide injected into the cerebellar vermis of mice immediately after exposure to the elevated plus-maze impairs their avoidance behavior on re-exposure to the apparatus.

The present study investigated the effect of thioperamide (THIO), an H3 histaminergic receptor antagonist, microinjected into the cerebellar vermis on emotional memory consolidation in male Swiss albino mice re-exposed to the elevated plus-maze (EPM). We implanted a guide cannula into the cerebellar vermis using stereotactic surgery. On the third day after surgery, we performed behavioral tests for two consecutive days. On the first day (exposure), the mice (n=10/group) were exposed to the EPM and received THIO (0.06, 0.3, or 1.5 ng/0.1 µL) immediately after the end of the session. Twenty-four hours later, the mice were re-exposed to the EPM under the same experimental conditions, but without drug injection. A reduction in the exploration of the open arms upon re-exposure to the EPM (percentage of number of entries and time spent in open arms) compared with the initial exposure was used as an indicator of learning and memory. One-way analysis of variance (ANOVA) followed by the Duncan post hoc test was used to analyze the data. Upon re-exposure, exploratory activity in the open arms was reduced in the control group, and with the two highest THIO doses: 0.3 and 1.5 ng/0.1 µL. No reduction was seen with the lowest THIO dose (0.06 ng/0.1 µL), indicating inhibition of the consolidation of emotional memory. None of the doses interfered with the animals' locomotor activity. We conclude that THIO at the lowest dose (0.06 ng/0.1 µL) microinjected into the cerebellum impaired emotional memory consolidation in mice.

Low-dose thioperamide injected into the cerebellar vermis of mice immediately after exposure to the elevated plus-maze impairs their avoidance behavior on re-exposure to the apparatus

The present study investigated the effect of thioperamide (THIO), an H3 histaminergic receptor antagonist, microinjected into the cerebellar vermis on emotional memory consolidation in male Swiss albino mice re-exposed to the elevated plus-maze (EPM). We implanted a guide cannula into the cerebellar vermis using stereotactic surgery. On the third day after surgery, we performed behavioral tests for two consecutive days. On the first day (exposure), the mice (n=10/group) were exposed to the EPM and received THIO (0.06, 0.3, or 1.5 ng/0.1 µL) immediately after the end of the session. Twenty-four hours later, the mice were re-exposed to the EPM under the same experimental conditions, but without drug injection. A reduction in the exploration of the open arms upon re-exposure to the EPM (percentage of number of entries and time spent in open arms) compared with the initial exposure was used as an indicator of learning and memory. One-way analysis of variance (ANOVA) followed by the Duncan post hoc test was used to analyze the data. Upon re-exposure, exploratory activity in the open arms was reduced in the control group, and with the two highest THIO doses: 0.3 and 1.5 ng/0.1 µL. No reduction was seen with the lowest THIO dose (0.06 ng/0.1 µL), indicating inhibition of the consolidation of emotional memory. None of the doses interfered with the animals' locomotor activity. We conclude that THIO at the lowest dose (0.06 ng/0.1 µL) microinjected into the cerebellum impaired emotional memory consolidation 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 Duncan's 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.

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 Duncan's tests. In Experiment 1, the Duncan's 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.

H1-histamine receptors in the amygdala are involved in emotional memory but do not mediate anxiety-related behaviors in mice submitted to EPM testing.

This study investigated the role of amygdala H(1) receptors in state-dependent memory deficits induced by l-histidine (LH). Tests using an elevated plus-maze (EPM) were performed on two consecutive days: Trial 1 (T1) and Trial 2 (T2). Before each trial, mice were intraperitoneally (IP) injected with LH (500mg/kg). Two hours later, they were microinjected with the H(1) receptor antagonist, chlorpheniramine (CPA 0.016, 0.052, or 0.16 nmol/0.1µl), or saline (SAL) into the amygdala and submitted to the EPM. LH-CPA did not affect trial 1 performances in the EPM, which indicated that these drugs did not affect anxiety. Emotional memory, as revealed by a reduction in open arm exploration between both trials, was present in the SAL-SAL groups as well as in the SAL-CPA groups for the lower doses of CPA (0.016 and 0.052nmol). On the contrary, neither the LH-SAL group nor the LH-CPA groups exhibited this decrease in open arm activity between both trials, which reveals that LH impaired emotional memory. While intra-amygdalar CPA did not interact with LH effect, it impaired per se the emotional memory performances at the highest dose (0.16nmol). No significant changes were observed in the number of enclosed arm entries (EAE), an EPM index of general exploratory activity. These results may be attributed to a combined effect in the different nucleus of the amygdala. Taken together, these results suggest that the H(1) receptors in the amygdala are not implicated in anxiety-like behaviors but are involved in emotional states induced by the T1/T2 EPM protocol 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 Duncan's 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.