The impact of flavonoids on spatial memory in rodents: from behaviour to underlying hippocampal mechanisms

Emerging evidence suggests that a group of dietary-derived phytochemicals known as flavonoids are able to induce improvements in memory, learning and cognition. Flavonoids have been shown to modulate critical neuronal signalling pathways involved in processes of memory, and therefore are likely to affect synaptic plasticity and long-term potentiation mechanisms, widely considered to provide a basis for memory. Animal dietary supplementation studies have further shown that flavonoid-rich foods are able to reverse age-related spatial memory and spatial learning impairments. A more accurate understanding of how a particular spatial memory task works and of which aspects of memory and learning can be assessed in each case, are necessary for a correct interpretation of data relating to diet-cognition experiments. Further understanding of how specific behavioural tasks relate to the functioning of hippocampal circuitry during learning processes might be also elucidative of the specific observed memory improvements. The overall goal of this review is to give an overview of how the hippocampal circuitry operates as a memory system during behavioural tasks, which we believe will provide a new insight into the underlying mechanisms of the action of flavonoids on cognition.     

SNAP-25 in hippocampal CA3 region is required for long-term memory formation

SNAP-25 is a synaptosomal protein of 25 kDa, a key component of synaptic vesicle-docking/fusion machinery, and plays a critical role in exocytosis and neurotransmitter release. We previously reported that SNAP-25 in the hippocampal CA1 region is involved in consolidation of contextual fear memory and water-maze spatial memory (Hou et al. European J Neuroscience, 20: 1593-1603, 2004). SNAP-25 is expressed not only in the CA1 region, but also in the CA3 region, and the SNAP-25 mRNA level in the CA3 region is higher than in the CA1 region. Here, we provide evidence that SNAP-25 in the CA3 region is also involved in learning/memory. Intra-CA3 infusion of SNAP-25 antisense oligonucleotide impaired both long-term contextual fear memory and water-maze spatial memory, with short-term memory intact. Furthermore, the SNAP-25 antisense oligonucleotide suppressed the long-term potentiation (LTP) of field excitatory post-synaptic potential (fEPSP) in the mossy-fiber pathway (DG-CA3 pathway), with no effect on paired-pulse facilitation of the fEPSP. These results are consistent with the notion that SNAP-25 in the hippocampal CA3 region is required for long-term memory formation.     

Spontaneous alternation, motor activity, and spatial learning in hot-foot mutant mice

Hot-foot mutant mice, characterized by defective innervation of Purkinje cells and an ataxic gait, were less active than normal mice in a T-maze. In spontaneous alternation testing with either single or multiple trials, hot-foot mutants, contrary to normal mice, did not alternate above chance. Moreover, the mutants had a higher number of errors and higher escape latencies in a water-filled Z-maze. These results indicate that in addition to motor coordination deficits, these cerebellar mutants have deficits in spatial learning and perseverate choices of maze arms.     

Assessment of estradiol influence on spatial tasks and hippocampal CA1 spines: evidence that the duration of hormone deprivation after ovariectomy compromises 17beta-estradiol effectiveness in altering CA1 spines

Two pulses of 17β-estradiol (10 µg) are commonly used to increase hippocampal CA1 apical dendritic spine density and alter spatial performance in ovariectomized (OVX) female rats, but rarely are the measures combined. The goal of this study was to use this two-pulse injection protocol repeatedly with intervening wash-out periods in the same rats to: 1) measure spatial ability using different tasks that require hippocampal function and 2) determine whether ovarian hormone depletion for an extended 10-week period reduces 17β-estradiol's effectiveness in elevating CA1 apical dendritic spine density. Results showed that two injections of 10 µg 17β-estradiol (72 and 48 h prior to testing and timed to maximize CA1 apical spine density at behavioral assessment) corresponded to improved spatial memory performance on object placement. In contrast, two injections of 5 µg 17β-estradiol facilitated spatial learning on the water maze compared to rats given two injections of 10 µg 17β-estradiol or the sesame oil vehicle. Neither 17β-estradiol dose altered Y-maze performance. As expected, the intermittent two-pulse injection protocol increased CA1 apical spine density, but 10 weeks of OVX without estradiol treatment decreased the effectiveness of 10 µg 17β-estradiol to increase CA1 apical spine density. Moreover, two pulses of 5 µg 17β-estradiol injected intermittently failed to alter CA1 apical spine density and decreased basal spine density. These results demonstrate that extended time without ovarian hormones reduces 17β-estradiol's effectiveness to increase CA1 apical spine density. Collectively, these findings highlight the complex interactions among estradiol, CA1 spine density/morphology, and task requirements, all of which contribute to behavioral outcomes.     

Effects of pregnancy on spatial cognition in female Hooded Long-Evans rats

Studies examining the roles of estrogens and progestins on spatial cognition have been highly contradictory. To determine if the hormonal environment of pregnancy affects spatial cognition, pregnant (n = 7) and virgin (n = 7) Hooded Long-Evans rats were tested in a Morris water maze throughout the 3 weeks of pregnancy and the second week postpartum. Latency to platform, path length, swim velocity, and time in quadrant were compared over trial-days. To compare water maze performance with changes in hormone levels, serum concentrations of estradiol and progesterone were measured on the first, third, and fifth days of testing during the third week of pregnancy. Subjects learned to find the platform as indicated by decreased time and distance to platform over each trial-week and increased time spent in the quadrant where the platform had been located the previous week. However, there were no differences between treatment groups on time or distance to platform over trial-days. Swim velocity did not differ between or within groups over the 4 weeks of testing. Although primigravid and virgin females were similar in their abilities to learn the novel location of a submerged platform and return to it over time, pregnant animals demonstrated less perseveration to previously learned information and were quicker to locate the platform when it moved to a new location. Thus, reproductive status did not affect reference memory but enhanced working memory in the Morris water maze.     

Acute pretreatment with estradiol protects against CA1 cell loss and spatial learning impairments resulting from transient global ischemia

Estradiol can act to protect against hippocampal damage resulting from transient global ischemia, but little is known about the functional consequences of such neuroprotection. The present study examines whether acute estradiol administered prior to the induction of transient global ischemia protects against hippocampal cell death and deficits in performance on a spatial learning task. Ovariectomized female rats were primed with estradiol benzoate or oil vehicle 48 and 24 h prior to experiencing one of three durations of 4-vessel occlusion (0, 5, or 10 min). Performance on the cued and hidden platform versions of the Morris water maze was assessed 1 week following ischemia. On the cued platform task, neither hormone treatment nor ischemia significantly influenced acquisition. When tested on the hidden platform task, however, oil-treated rats exhibited impairments in spatial learning after either 5 or 10 min of ischemia while estradiol-treated rats showed no impairments after 5 min of ischemia and only mild impairments after 10 min of ischemia. Immediately following behavioral testing, rats were perfused and survival of CA1 pyramidal cells was assessed. Ischemia was associated with the loss of CA1 pyramidal cells but rats that received estradiol prior to ischemia showed less severe damage. Furthermore, the extent of cell loss was correlated with degree of spatial bias expressed on a probe trial following hidden platform training. These findings indicate that acute exposure to estradiol prior to ischemia is both neuroprotective and functionally protective.     

Human chorionic gonadotropin (a luteinizing hormone homologue) decreases spatial memory and increases brain amyloid-beta levels in female rats

Numerous studies have suggested that estradiol (E) improves spatial memory as female rats with E perform better than those without E. However there is an inverse relationship between E and luteinizing hormone (LH) levels and LH could play a role. We examined whether treatment with the LH homologue human chorionic gonadotropin (hCG), would impair spatial memory of adult E-treated female rats. In the object location memory task, ovariectomized (ovxed) rats treated with E and either a single high dose (400 IU/kg) or a lower repeated dose of hCG (75 IU/kg hourly for 8 h) showed spatial memory disruption compared to ovxed rats treated with estradiol alone. Impairment was attributed to memory disruption as performance improved with shortened delay between task exposure and testing. Tests on another spatial memory task, the Barnes maze, confirmed that hCG (400 IU/kg) can impair memory: although E + veh treated animals made significantly fewer hole errors across time, E + hCG-treated did not. In humans, high LH levels have been correlated with Alzheimer's disease (AD). Because brain amyloid-beta (Aβ) species have been implicated as a toxic factor thought to cause memory loss in AD, we analyzed whether hCG-treated animals had increased Aβ levels. Levels of Aβ from whole brains or hippocampi were assessed by Western blot. hCG treatment to E-implanted females significantly increased soluble Aβ40 and Aβ42 levels. These results indicate that high levels of LH/hCG can impair spatial memory, and an increase in brain Aβ species may account for the memory impairment in hCG-treated rats.     

Aversive Learning under Different Training Conditions: Effects of NMDA Receptor Blockade in Area CA1 of the Hippocampus

Adult male Wistar rats were given either a single training trial or one training trial per day during 3 days followed by a retention test trial in an inhibitory avoidance (IA) task. In animals given a single training trial, pretraining, but not pretest bilateral infusion of the NMDA glutamate receptor antagonist d,l-2-amino-5-phosphonopentanoic acid (AP5) (5.0 μg) into the CA1 hippocampal area blocked IA retention. In animals given three training trials, infusions of AP5 given prior to each of the three training trials severely impaired, but did not block retention. The results indicate that NMDA receptors in the hippocampus are involved in the formation, but not in expression, of aversive memory. In addition, rats given repeated training were able to show a mild improvement of performance across training trials, possibly through mechanisms that do not depend on NMDA receptor activation in the dorsal hippocampus.     

Gas7在大鼠海马和齿状回发育过程中的动态表达

目的研究生长休止蛋白7（Gas7）在大鼠海马和齿状回不同发育阶段的表达。方法采用免疫组织化学方法观察Gas7在SD大鼠胚胎第18d（E18）、新生（P0）、生后第7d（P7）、P14、P21和成年海马和齿状回中的表达和分布。结果在大鼠脑海马和齿状回部位的冠状切片上,Gas7免疫反应阳性产物主要表达在海马的锥体细胞、齿状回的颗粒细胞和门区的多形层细胞。随着发育的进程,在海马,Gas7较早表达在CA3区,其次是CA2和CA1区;在齿状回,Gas7在外臂的表达早于内臂,在颗粒细胞层的表达是按先外层后内层的顺序。在围生期,Gas7在海马和齿状回各区的表达逐渐增强,至P14达到高峰,后逐渐降低,至P21其表达强度和分布趋于恒定至成年水平。结论 Gas7在大鼠海马和齿状回发育过程中的动态表达具有时间和空间上的特异性,提示Gas7可能参与了海马和齿状回形态形成和功能成熟的调控。     

白藜芦醇抑制大鼠海马 CA1区神经元放电

应用细胞外记录单位放电技术,在大鼠海马脑片上观察了白藜芦醇(resveratrol)对海马CAI区神经元放电的影响。实验结果如下：(1)在52个CAI区神经元放电单位给予白藜芦醇(0．05、0．5、5μmol／L)2min,有46个放电单位(88.5％)放电频率明显降低,且呈剂量依赖性;(2)预先用0．2mmol／L的L-glutamate灌流海码腑片,8个放电单位放电频率明显增加,表现为癫痫样放电,在此基础上灌流白藜芦醇(5μmol／L)2min,其癫痫样放电被抑制;(3)预先用L型钙通道开放剂Bay K8644灌流7个海马5脑片,有6个单位(85.7％)放电增加,在此基础上灌流白藜芦醇(5μmol／L)2min,其放电被抑制;(4)9个放电单位灌流一氧化氮合酶抑制剂L-NAME(N^0-nitro-L-arginine methylester)50μmol／L,有7个单位(77．8％)放电明显增加,在此基础上灌流白藜芦醇(5μmol／L)2min,放电被抑制;(5)10个放电单位灌流人电导钙激活性钾通道阻断剂TEA(tetraethylarnmonium chloride)1mmol/L后,有9个单位(90％)放电增加,在此基础上灌流白藜芦醇(5μmol／L)2min,8个放电单位(88,9％)放电频率明显减低。以上结果提示：白藜芦醇能抑制海马神经元自发放电以及由L-glutamate、L-NAME、Bay K8644和TEA诱发的放电,可能与白藜芦醇抑制L型钙通道,减少钙内流有关;似乎与大电导钙激活性钾通道无关。     

Self-Stimulation Rewarding Experience Restores Stress-Induced CA3 Dendritic Atrophy,Spatial Memory Deficits and Alterations in the Levels of Neurotransmitters in the Hippocampus

Chronic restraint stress causes spatial learning and memory deficits, dendritic atrophy of the hippocampal pyramidal neurons and alterations in the levels of neurotransmitters in the hippocampus. In contrast, intracranial self-stimulation (ICSS) rewarding behavioral experience is known to increase dendritic arborization, spine and synaptic density, and increase neurotransmitter levels in the hippocampus. In addition, ICSS facilitates operant and spatial learning, and ameliorates fornix-lesion induced behavioral deficits. Although the effects of stress and ICSS are documented, it is not known whether ICSS following stress would ameliorate the stress-induced deficits. Accordingly, the present study was aimed to evaluate the role of ICSS on stress-induced changes in hippocampal morphology, neurochemistry, and behavioral performance in the T-maze. Experiments were conducted on adult male Wistar rats, which were randomly divided into four groups; normal control, stress (ST), self-stimulation (SS), and stress + self-stimulation (ST + SS). Stress group of rats were subjected to restraint stress for 6 h daily over 21 days, SS group animals were subjected to SS from ventral tegmental area for 10 days and ST + SS rats were subjected to restraint stress for 21 days followed by 10 days of SS. Interestingly, our results show that stress-induced behavioral deficits, dendritic atrophy, and decreased levels of neurotransmitters were completely reversed following 10 days of SS experience. We propose that SS rewarding behavioral experience ameliorates the stress-induced cognitive deficits by inducing structural and biochemical changes in the hippocampus.     

氧化还原对成年大鼠海马CA1区锥体神经元外向整流氯通道的调控作用

采用膜片钳内面向外式记录技术,研究急性分离成年大鼠海马CAl区锥体神经元外向整流氯离子通道的氧化还原调控。发现细胞内侧给予氧化剂DTNB(5,5'-dithiobis-2-nitrobenzoic acid),可显著减弱氯通道的活动,IC50值为(28.05±2.42)μmol/L;还原剂DTT(dithiothreitol)对氯通道没有明显影响,但可逆转DTNB引起的抑制效应。说明DTNB不改变通道电导,其引起的通道活动减弱是由氯通道关闭时间延长而开放时间缩短所致。研究还发现,另一对氧化型和还原型谷胱甘肽具有与DTNB和DTT同样的效应。本研究结果显示,成年大鼠海马CA1区锥体神经元外向整流氯通道可以被细胞内氧化还原剂所调控。     

一氧化氮对谷氨酸诱发的大鼠海马脑片CA1区神经元活动的抑制

用细胞外记录单位放电技术,在大鼠海马脑片上观察了Ｌ－精氨酸（Ｌ－ａｒｇ）、Ｎ－硝基Ｌ－精氨酸（Ｌ－ＮＮＡ）及ＳＩＮ－１对谷氨酸（ｇｌｕｔａｍａｔｅ,Ｇｌｕ）诱导的ＣＡ１区神经元放电的影响。旨在了解Ｌ－精氨酸：ＮＯ通路在谷氨酸诱发的海马放电中的作用及其可能的机制。结果如下：（１）用ＧｌＵ（０．５ｍｍｏｌ／Ｌ）灌流海马脑片１ｍｉｎ,１２个放电单位放电频率明显增加,表现为癫痫样放电;（２）海马脑片２ｍｉ     

胍丁胺对大鼠海马 CA1区神经元放电的影响

应用细胞外记录单位放电技术,在大鼠海马脑片上观察了胍丁胺(agmatine,Agm)对CAl区神经元放电的影响。实验结果如下：(1)在47个海马脑片放电单位上灌流Agm(0．1—1．0μmol／L)2min,有38个单位(80．9％)自发放电频率明显降低,且呈剂量依赖性,9个单位(19．1％)无明显的反应;(2)预先用0．2mmol／L的L-谷氨酸(L-glutamate,L-Glu)灌流12个海马脑片放电单位,有9个单位(75％)放电频率明显增加,表现为癫痫样放电,在此基础上灌流Agm(1．0μmol／L)2min,其癫痫样放电被抑制;(3)在7个海马脑片放电单位上给予L型钙通道激动剂Bay K8644(0．1μmoL／L)时,有6个单位(85．7％)放电频率明显增加,另外1个单位(14．3％)无明显变化,再给予Agm(1．0μmol／L)2min,其放电频率被明显抑制;(4)13个CAl放电单位,灌流50μmoL／L一氧化氮合酶(NOS)抑制剂N^G-nitro-L-arginine methyl ester。(L-NAME)5min后其放电频率明显增加,在此基础上再给予Agm(1．0μmol／L)2min,有11个单位(84．6％)的放电频率被抑制,有2个单位(15．4％)的变化不明显。上述结果提示：胍丁胺能抑制海马CAl区神经元自发放电以及由谷氨酸、BayK8644和L-NAME诱发的放电,这一抑制效应可能与胍丁胺阻断CAl区锥体细胞上的NMDA受体,并减少钙离子内流有关。     

Basic Fibroblast Growth Factor Prevents the Memory Impairment Induced by Gastrin-Releasing Peptide Receptor Antagonism in Area CA1 of the Rat Hippocampus

Increasing evidence indicates that the gastrin-releasing peptide receptor (GRPR) is implicated in regulating synaptic plasticity and memory formation in the hippocampus and other brain areas. However, the molecular mechanisms underlying the memory-impairing effects of GRPR antagonism have remained unclear. Here we report that basic fibroblast growth factor (bFGF/FGF-2) rescues the memory impairment induced by GRPR antagonism in the rat dorsal hippocampus. The GRPR antagonist [D-Tpi⁶, Leu¹³ psi(CH₂NH)-Leu¹⁴] bombesin (6–14) (RC-3095) at 1.0 μg impaired, whereas bFGF at 0.25 μg enhanced, 24 h retention of inhibitory avoidance (IA) when infused immediately after training into the CA1 hippocampal area in male rats. Coinfusion with an otherwise ineffective dose of bFGF blocked the memory-impairing effect of RC-3095. These findings suggest that the memory-impairing effects of GRPR antagonists might be partially mediated by an inhibition in the function and/or expression of neuronal bFGF or diminished activation of intracellular protein kinase pathways associated with bFGF signaling.     

Ultrastructural changes in granule cell somata and mossy fibers of the rat hippocampus during picrotoxin-induced convulsions

Summary Ultrastructural changes in hippocampal granule cells, mossy fibers and mossy fiber boutons were examined following the administration of picrotoxin in adult rats. Generalized seizures occurred within 5–10 min after the intraperitoneal injection of picrotoxin. The electron-microscopic examination of hippocampal tissues from rats that had been perfused with fixative during the seizure revealed that the large dense-core vesicles increased in number and accumulated on the presynaptic membranes of mossy fiber boutons; some of these vesicles appeared to be fused with the membranes, and omega-shaped exocytotic profiles were frequently seen. Furthermore, greatly increased numbers of coated vesicles (60–90 nm in diameter) were observed on the maturing faces of Golgi fields of granule cells. Thus, our study not only indicates an increased incidence of exocytosis of large dense-core vesicles during picrotoxin-induced seizures, but also suggests that these vesicles are replaced in excess from the perikaryon of the granule cell.     

5-HT(1B) receptor-mediated regulation of serotonin clearance in rat hippocampus in vivo

The 5-hydroxytryptamine (5-HT; serotonin) transporter (5-HTT) is important in terminating serotonergic neurotransmission and is a primary target for many psychotherapeutic drugs. Study of the regulation of 5-HTT activity is therefore important in understanding the control of serotonergic neurotransmission. Using high-speed chronoamperometry, we have demonstrated that local application of 5-HT(1B) antagonists into the CA3 region of the hippocampus prolongs the clearance of 5-HT from extracellular fluid (ECF). In the present study, we demonstrate that the 5-HT(1B) antagonist cyanopindolol does not produce this effect by increasing release of endogenous 5-HT or by directly binding to the 5-HTT. Dose-response studies showed that the potency of cyanopindolol to inhibit clearance of 5-HT was equivalent to that of the selective 5-HT reuptake inhibitor fluvoxamine. Local application of the 5-HT(1A) antagonist WAY 100635 did not alter 5-HT clearance, suggesting that the effect of cyanopindolol to prolong clearance is not via a mechanism involving 5-HT(1A) receptors. Finally, the effect of low doses of cyanopindolol and fluvoxamine to inhibit clearance of 5-HT from ECF was additive. These data are consistent with the hypothesis that activation of terminal 5-HT(1B) autoreceptors increases 5-HTT activity.     

The spatial and temporal expression of delta-like protein 1 in the rat pituitary gland during development

An analysis of secreted proteins by the signal sequence trap method using a cDNA library of the rat pituitary anlage at embryonic days (E) 13.5 revealed the abundant expression of delta-like protein 1 (Dlk1) in the pituitary gland. Dlk1, an epidermal growth factor-like repeat protein in preadipocytes, functions in maintaining the preadipose state. Expression of Dlk1 mRNA in the pituitary at E13.5 and in the adult pituitary was confirmed by in situ hybridization. The expression pattern of Dlk1 during pituitary development was also studied by immunohistochemistry. Dlk1 protein first appeared in Rathke’s pouch and the infundibulum at E11.5; as development proceeded, expression became restricted to the pars distalis and pars tuberalis (PT). Dlk1 was expressed in most ACTH cells during the embryonic stages, but its expression was limited to only a few ACTH cells in the adult pituitary. It was also expressed in a small population of TSH, GTH, and PRL cells throughout development, whereas it was present in the cytoplasm of most GH cells at all developmental stages. Similarly, Dlk1 was localized in the cytoplasm of PT cells during development. These findings provide new insights into the mechanism of Dlk1 regarding its regulation of pituitary hormone-secreting cells during development.     

Spatial clustering property and its self-similarity in membrane potentials of hippocampal CA1 pyramidal neurons for a spatio-temporal input sequence

To clarify how the information of spatiotemporal sequence of the hippocampal CA3 affects the postsynaptic membrane potentials of single pyramidal cells in the hippocampal CA1, the spatio-temporal stimuli was delivered to Schaffer collaterals of the CA3 through a pair of electrodes and the post-synaptic membrane potentials were recorded using the patch-clamp recording method. The input–output relations were sequentially analyzed by applying two measures; “spatial clustering” and its “self-similarity” index. The membrane potentials were hierarchically clustered in a self-similar manner to the input sequences. The property was significantly observed at two and three time-history steps. In addition, the properties were maintained under two different stimulus conditions, weak and strong current stimulation. The experimental results are discussed in relation to theoretical results of Cantor coding, reported by Tsuda (Behav Brain Sci 24(5):793–847, 2001) and Tsuda and Kuroda (Jpn J Indust Appl Math 18:249–258, 2001; Cortical dynamics, pp 129–139, Springer-Verlag, 2004).     

CCL28 in the mouse hippocampal CA1 area and the dentate gyrus during and after pilocarpine-induced status epilepticus

The present study showed a wide presence of CCL28 in mouse CNS, including cerebral, cerebellum, brain stem and spinal cord. In hippocampus, the expression of CCL28 at both mRNA and protein level was clarified. The CCL28 expression was mainly localized in pyramidal cells of CA area, granular cells of dentate gyrus and some interneurons in CA area and hilus. Double-labelling immunocytochemistry revealed that most of calbindin, calretinin and parvalbumin immunopositive neurons expressed CCL28. During and after pilocarpine induced status epilepticus (SE), a down-regulated expression of CCL28 in hippocampal interneurons in the CA1 area and in the hilus of the dentate gyrus was demonstrated. The present study may, therefore provide evidence that CCL28 may have a novel role in CNS and may be involved in the loss of hippocampal interneurons, and subsequent disinhibition of pyramidal neurons.