Huang YW et al / Acta Pharmacol Sin 2003 Dec; 24 (12): 1270-1276
HUANG Yu-Wen, CHEN Zhong2 , HU Wei-Wei, ZHANG Li-San, WU Wei , YING Li-Yang, WEI Er-Qing
Department of Neurobiology and Pharmacology, School of Medicine, Zhejiang University, Hangzhou 310031, China
1 Project supported by the National Natural Science Foundation of China (No 30000019); Zhejiang Provincial Natural Science Foundation of China (No 300062); partly supported by the Scientific Research Foundations for the Returned Overseas Chinese Scholars.
2 Correspondence to Prof CHEN Zhong, Ph D. Phn 86-571-8721-7150. Fax 86-571-8891-2665. E-mail chenzfl@hotmail.com
Received 2003-01-27 Accepted 2003-06-18
KEY WORDS histamine; maze learning; dizocilpine; memory disorders
ABSTRACT
AIM: To investigate whether or not histamine is involved in spatial
memory deficits induced by dizocilpine (MK-801) as evaluated by 8-arm radial
maze of rats. METHODS: 8-Arm (4-arm baited) radial maze was used to
measure spatial memory in rats. RESULTS: Bilaterally intrahippocampal
(ih) injection of MK-801 (0.3 µg/site) impaired working memory and reference
memory in rats. Both histamine (50, 100 ng/site, ih) and intraperitoneal (ip)
injection of histidine (100, 200 mg/kg) markedly improved the spatial memory
deficits induced by MK-801. On the other hand, the ameliorating effect of histidine
(100 mg/kg, ip) was completely antagonized by 
-fluoromethylhistidine
(-FMH, 5 µg/site, ih), a
potent and selective histidine decarboxylase (HDC) inhibitor, and H1-antagonist
pyrilamine (1 µg/site, ih), but not by H2-antagonist cimetidine,
even at a high dose (2.5 µg/site, ih). CONCLUSION: The hippocampal
histamine plays an important role in the ameliorating effect on MK-801-induced
spatial memory deficits, and its action is mediated through postsynaptic H1-receptor.
INTRODUCTION
Histamine neurons are exclusively located in the tuberomammillary nucleus (TM)
of the posterior hypothalamus receiving input mainly from the limbic system
and project efferent nerve fibers to almost all parts of the brain[1,2].
Histamine plays an important role as a neurotransmitter or neuromodulator in
the mammalian
central neuron system (CNS)[1-3]. The histaminergic neuron system
seems to be involved in various physiological and behavioral functions through
H1-, H2-, and H3-receptors including sleep-wake
cycles, emotion, appetite control, locomotor activity, stress behavior, neuroendocrine,
and epilepsy[2-4]. So far, a role for brain histamine in mechanisms
regulating learning and memory has been well documented[3-7]. For
example, both histamine and its precursor, L-histidine, facilitate memory
retrieval deficits induced by aging, hippocampal lesions and scopolamine as
determined with passive and active avoidance task and 8-arm radial maze in rats[5-7].
-Fluoromethylhistidine (-FMH),
a selective and potent histidine decarboxylase (HDC) inhibitor, has been de-monstrated
to induce significant memory deficits in active avoidance task and 8-arm radial
maze in rats[8]. However, several reports showed conflicting findings
as follows: chronic treatment with -FMH
resulted in facilitation of memory acquisition as evaluated by 8-arm radial
maze of rats[4], and a lesion in TM region showed an ameliorating
effect in aged rats[9]. The mechanisms of these differences seem
to be very complex, which may be due to the different experimental methods,
the species of animals, and drug treated doses and routes etc.
On the other hand, it has been demonstrated that N-methyl-D-aspartate (NMDA) receptors play an important role in learning and memory[10,11]. NMDA receptor antagonist dizocilpine (MK-801) is widely used for a good tool to impair various kinds of learning behavior[10,11]. Recently, the interactions between histaminergic system and NMDA receptor become more and more attractive[3,12,13]. The polyamine site of the NMDA receptor has been suggested to be a binding site for histamine[3]. Bekkers et al and Vorobjev et al discovered in cultured hippocampal pyramidal cells that histamine could dramatically enhance NMDA receptor-mediated synaptic transmission and facilitate the induction of long-term potentiation[12,13]. However, few behavioral studies were made on the interaction of histamine and NMDA receptors in cognition process. D-cyclo-serine, a partial agonist of NMDA receptor, facilitates pyrilamine-induced memory deficits in a three-panel runway task[14]. We have previously reported histamine reversed memory deficits of rats induced by MK-801 in 8-arm radial maze performance[11]. However, little is known about the involvement of the hippocampal NMDA receptor in amelioration of histaminergic system and it remains to be unknown whether working memory, a kind of short-term memory, or reference memory, a kind of long-term memory, is prominently related to histamine. 8-Arm (4-arm baited) radial maze has been widely used to determine working memory and reference memory[15].
Therefore, the objectives of our investigations are to use the radial maze task with 4-arms baited to further elucidate the roles of hippocampal NMDA receptor and histaminergic system in the regulation of spatial memory of rats.
MATERIALS AND METHODS
Animals All experiments were carried out in
accordance with the National Institutes of Health Guide
for the Care and Use of Laboratory Animal. The
animals used in this study were male Sprague-Dawley rats
(
, 220-300 g, n=90, Grade II, Certificate
No 22-9601018, Experimental Animal Center, Zhejiang
University), maintained in an air-conditioned room with
controlled temperature (22-26 ºC) and humidity (40
%-70 %), housed in individual cages with a 12-h
light-dark cycle (lights on from 8:00-20:00). Animals were
given free access to water and kept at 80 %-85 % of
their free feeding body weight throughout the experiment. Experiments were carried out each day
between 10:00-17:00.
Surgical procedure The rats were anesthetized with sodium pentobarbital (35 mg/kg, ip), and fixed on a stereotaxic apparatus (Narishige, SR-5, Tokyo, Japan). A guide cannula made of stainless steel tubing 700 µm in outer diameter, was implanted into the bilateral dorsal hippocampus according to the following coordinates measured from bregma: AP: -4.3 mm, L: ±1.9 mm, H: 3.8 mm from the skull. At least 10 d were allowed for recovery from the surgery. After the behavioral tests, Evans blue with a volume of 1 µL was injected bilaterally into the dorsal hippocampus and the rats were sacrificed by decapitation. The accuracy of the injection site was carefully determined.
Radial-arm maze training The apparatus used was described in our previous report[11]. To make rats familiar with the radial maze, prior to training, they received one daily habituation for 2 d. Food pellets (45 mg each, Bio-Serv, Frenchtown, NJ, USA) were scattered over the entire maze surface, and three or four rats were simultaneously placed in the radial maze and allowed to explore 10 min to take food freely. After adaptation, all rats were trained with 1 trial per day. In each trial, only 4 arms (1, 2, 4, and 7) were baited, and the sequence was never changed throughout the experiment. The rat was placed on the center platform that was closed off by a door. After 15 s, the door was opened and the rat was allowed to make arm choice to obtain food pellets until all 4 pellets had been eaten or 5 min had elapsed. The number of entries into the unbaited arms was regarded as the total error (TE). The number of entries into the never-baited arm was regarded as a reference memory error (RME), while re-entry into the arms where the pellet had already been eaten was regarded as a working memory error (WME). Rats were trained continually until reaching a criterion of at most 1 error per trial for 5 consecutive training trials. Fourteen of ninety rats which were not able to solve the standard radial maze performance or just turn around the maze arm side by side, or lose their guide cannula before drug treatment, were excluded from the following drug test.
Drugs During the drug test, (+) MK-801
hydrogen maleate (Sigma, St Louis, MO, USA), histamine
dihydrochloride (Sigma, St Louis, MO, USA), cimetidine
dimaleate (Sigma, St Louis, MO, USA), pyrilamine dihydrochloride (Sigma, St Louis, MO, USA), and
-fluoromethylhistidine (Merck Sharp & Dohme
Research Lab, Rahway, NJ) were dissolved in saline
and injected into dorsal hippocampus in a fixed 1
µL/site over a period of 120 s at a constant speed with
a continuous infusion pump (KN-201, Natsume, Tokyo,
Japan).
L-histidine monohydrochloride (Sigma, St Louis, MO,
USA) was dissolved in saline and injected ip. Studies
for drug effect were carried out once a week, on
Tuesday or Friday.
Statistical analysis All results were expressed as mean±SD. Differences were analyzed by One-way analysis of variance (ANOVA) with Dunnett's test using computer software (Sigmastat 1.01 for Windows 95, 1992, Jandel Corp, USA). P<0.05 was considered statistically significant.
RESULTS
Effect of MK-801 on memory retrieval as evaluated by 8-arm radial maze performance in rats Bilaterally intrahippocampal (ih) injection of MK-801 resulted in a dose-dependent increase in the number of errors in memory retrieval process. At a dose of 0.3 µg/site, MK-801-treated rats showed significant increase in the number of TE, WME, and RME (P<0.05, Fig 1).
Fig 1. Effect of bilaterally intrahippocampal (ih) injection of MK-801 on spatial memory of rats in 8-arm radial maze. MK-801 was injected 10 min before radial maze test. n =15-18 rats. Mean±SD. bP<0.05 vs the saline-treated group. TE: total errors; RME: reference memory errors; WME: working memory errors.
Effects of histamine and histidine on MK-801-induced memory deficits in rats Injection (ih) of histamine antagonized the effect of MK-801 in a dose-dependent manner, no significant effect was observed at a dose of 25 ng/site, while at doses of 50 and 100 ng/site significant effects were observed in the number of TE, WME, and RME (P<0.05, Tab 1). Histamine at a dose of 100 ng/site created a bell-shaped inhibition in the number of TE and WME. In addition, ip injection of histidine also produced a dose-dependent and significant inhibition against MK-801-induced memory deficits, at doses of 100 and 200 mg/kg (P<0.05, Tab 1).
Tab 1. Effects of histamine and histidine on memory deficits induced by intrahippocampal (ih) injection of MK-801 in 8-arm radial maze performance. MK-801 was injected 10 min before radial maze test. Histidine was injected 2 h before radial maze test. Histamine was injected 15 min before radial maze test. Mean±SD. bP<0.05 vs saline-treated group. eP<0.05 vs MK-801-treated group. TE: total errors; WME: working memory errors; RME: reference memory errors.
Effects of pyrilamine and cimetidine on memory amelioration of histidine on MK-801-induced memory deficits Injection (ih) of pyrilamine, a representative central H1-antagonist, at a dose of 1 µg/site, significantly antagonized the action of histidine-induced ameliorating effect in the TE, WME, and RME (P<0.05, Fig 2). On the other hand, cimetidine, an H2-antagonist, had no appreciable effect on facilitation of histidine even at a high dose of 2.5 µg/site.
Fig 2. Effects of bilaterally intrahippocampal (ih) injection of pyrilamine and cimetidine on the amelioration of histidine (100 mg/kg, ip) on MK-801-induced memory deficits in rats. MK-801 was injected 10 min before radial maze test. Pyrilamine and cimetidine were injected 5 min before MK-801 treatment. Histidine was injected 2 h before radial maze test. n=15-18 rats. Mean±SD. bP<0.05 vs the saline-treated group. eP<0.05 vs the MK-801-treated group. hP<0.05 vs the histidine+MK-801-treated group. TE: total errors; RME: reference memory errors; WME: working memory errors.
Effect of -FMH on amelioration of
histidine on MK-801-induced memory deficits in rats
-FMH, at a dose of 1 and 2.5 µg/site (ih) showed
a tendency to reverse histidine-induced amelioration on
memory deficits induced by MK-801, but no significant effect was obtained. At a dose of 5 µg/site,
-FMH created a significant increase in the number of
TE, WME, and RME (P<0.05, Tab 2).
Tab 2. Effect of bilaterally intrahippocampal (ih) injection of -fluoromethylhistidine
(a-FMH) on amelioration of histidine (100 mg/kg, ip) on MK-801-induced memory
deficits in rats. MK-801 was injected 10 min before radial maze test. -FMH
was injected 1 h before MK-801 treatment. Histidine was injected 2h before radial
maze test. Mean±SD. bP<0.05 vs saline-treated
group. eP<0.05 vs MK-801-treated group. hP<0.05
vs histidine+MK-801-treated group. TE: total errors; WME: working memory
errors; RME: reference memory errors.
DISCUSSION
In the present study, bilateral ih injection of MK-801 induced serious spatial memory deficits in working memory and reference memory as evaluated by 8-arm (4-arm baited) radial maze in rats. This result was consistent with the previous studies, in which both working memory and reference memory were impaired by ip injection of MK-801 as assessed with 8-arm radial maze and water maze tasks[10,11]. In addition, there appeared no appreciable changes of the running time per choice (locomotor activity) and other behavioral symptoms, such as ataxia (data not shown), which usually occurring in cases of ip or intracerebroven-tricular (icv) treatment of MK-801[10]. Therefore, the inhibition of hippocampal NMDA receptor by MK-801 in the present study mainly contributes to the spatial working and reference memory deficits in rats.
Both histamine and histidine created protective effects against MK-801-induced spatial memory deficits, including working memory and reference memory, which suggested histamine ameliorated both short-term and long-term spatial memory. It has been demonstrated that histamine in the hippocampus plays an important role in memory retrieval process, and ip injection of histidine could result in an increase in hippocampal histamine levels as same as icv injection of histamine[6,16,17]. Moreover, histamine has been recently indicated to facilitate NMDA-mediated excitatory transmission, and to increase glutamate release via H1- and H2-receptors in the hippocampus[12,13,18]. Therefore, our behavioral results provide more evidences that the glutamate released by hippocampal histamine contributes to the ameliorating effect on the MK-801-induced working memory and reference memory impairment in rats. Further biochemical studies are needed to elucidate its mechanism.
Interestingly, we found, histamine at a dose of 100 ng/site created a bell-shaped inhibition in the 8-arm radial maze. We have no more data to explain it. Similar findings were obtained by Sakai et al[19] and Ghi et al[20], who found biphasic effects of histamine on locomotor activity, dependent on doses administered. Kiyono et al also reported dual effects of histamine on waking-sleep from studies on EEG of rats[21]. A lower dose of histamine (45 nmol) treatment in the hippocampus improved memory retrieval in contrast to a higher dose (90 nmol)[17]. In addition, histamine at a dose of 10 µmol was found to inhibit the glutamate release in the hippocampus of rat[22]. These findings suggest that the dual effects of histamine were probably mediated through glutamate systems[12], and memory facilitation of histamine only existed within a certain range of low histamine level in the hippocampus. Recently, a facilitated learning acquisition of radial maze has been reported in histidine-deficient rats[15].
-FMH, which is a specific HDC inhibitor
and created no effects in other neurotransmitters at low
doses used in the study[23] has been widely used as a
powerful tool for elucidating the brain histamine function.
Previous report showed that -FMH induced a
severe impairment in memory retrieval in radial maze,
correlated with a decrease in hippocampal histamine
contents[8]. We found that the memory amelioration of
histamine was antagonized by -FMH, at a dose of
producing no appreciable effect on both working memory and reference memory when administered
alone (data not shown). These results strongly
confirm the ameliorating effect of endogenous histamine in
hippocampus on spatial memory deficits induced by
MK-801.
Postsynaptic H1-receptors play an important role in learning and memory[24,25]. Both clinical and animal studies indicated that H1-receptor antagonists could produce disturbances in arousal, which affected the cognitive function[25,26]. The human positron emission tomography (PET) studies indicated that marked decreases in the content of H1-receptors exited in the frontal and temporal areas of Alzheimer's diseases patients[26]. We found that the ameliorations of both spatial working memory and reference memory induced by histamine were reversed by pyrilamine, a central H1-antagonist. In contrast, no effect was observed with cimetidine. It was previously reported that icv injection of H1-agonist, 2-thiazoly-lethylamine, but not H2-agonist, 4-methylhistamine reversed the working memory deficits induced by MK-801 as evaluated by 8-arm radial maze in rats[11]. These results consisted well with the study by Payne et al[24], who has reported the ability of histamine to facilitate the NMDA receptor-mediated synaptic responses was reduced by histamine H1-receptor antagonists, suggesting that amelioration of histamine is mediated by postsynaptic H1-receptor, but not H2-receptor.
In conclusion, histamine facilitates spatial memory deficits of both spatial working memory and reference memory induced by MK-801, and its action is mediated by postsynaptic H1-receptors.
ACKNOWLEDGEMENT We thank Prof C KAMEI (Okayama University, Japan) for his valuable suggestion of this study.
REFERENCES
-fluorescent
immunohistochemical analysis with histidine decarboxylase as a marker. Brain
Res 1984; 295: 13-25.
-fluoromethyl-histidine
enhances radial maze performance in rats with modulation of brain amino acid
level. Life Sci 1998; 62: 989-94.
-fluoromethylhistidine
on radial maze performance in rats. Pharmacol Biochem Behav 1999; 64: 513-8.
-fluoromethylhistidine
increases feeding-associated locomotor activity in mice with a modulation
of brain amino acid levels. Behav Brain Res 1996; 72: 83-8.
-fluoromethylhistidine on
sleep waking parameters in rats. Physiol Behav 1985; 34: 615-7.
-fluoromethylhistidine, a specific
inhibitor of histidine decarboxylase. Trends Pharmacol Sci 1990; 11: 363-7.