Neuroglial alterations in rats submitted to the okadaic acid-induced model of dementia

Several types of animal models have been developed to investigate Alzheimer's disease (AD). Okadaic acid (OA), a potent inhibitor of phosphatases 1 and 2A, induces characteristics that resemble AD-like pathology. Memory impairment induced by intra-hippocampal injection of OA has been reported, accompanied by remarkable neuropathological changes including hippocampal neurodegeneration, a paired helical filament-like phosphorylation of tau protein, and formation of β-amyloid containing plaque-like structures. Rats were submitted to bilateral intrahippocampal okadaic acid-injection (100 ng) and, 12 days after the surgery, behavioral and biochemical tests were performed. Using this model, we evaluated spatial cognitive deficit and neuroglial alterations, particularly astroglial protein markers such as glial fibrillary acidic protein (GFAP) and S100B, metabolism of glutamate, oxidative parameters and alterations in MAPKs. Our results indicate significant hippocampal changes, including increased GFAP, protein oxidation, and phosphorylation of p38^MAPK; and decreases in glutathione content, transporter EAAT2/GLT-1, and glutamine synthetase activity as well as a decrease in cerebrospinal fluid S100B. No alterations were observed in glutamate uptake activity and S100B content. In conclusion, the OA-induced model of dementia caused spatial cognitive deficit and oxidative stress in this model and, for the first time to our knowledge, specific astroglial alterations. Findings contribute to understanding diseases accompanied by cognitive deficits and the neural damage induced by AO administration.     

Effects of chronic infusion of nerve growth factor (NGF) in rats with nucleus basalis magnocellularis lesion

We attempted to evaluate the effects of bilateral injection of ibotenic acid (IA) into the nucleus basalis magnocellularis (nbm) of rats as well as the potential recovery mediated by the infusion of nerve growth factor (NGF). The lesion caused an impairment of learning and memory processes. Also, a severe depletion of choline acetyl transferase activity was detected in cortical areas. After the NGF administration, a significant reversion of these functional changes was observed. Thus, IA-lesioned rats might serve as a model for the evaluation of neurotrophic factors actions on basal forebrain damaged neurons.     

Decrease of somatostatin receptor binding in the rat cerebral cortex after ibotenic acid lesion of the nucleus basalis magnocellularis: a quantitative autoradiographic study

The specific binding of¹²⁵I-Tyr¹¹-somatostatin-14 (¹²⁵I-Tyr¹¹-SS-14) was measured in different cortical regions after unilateral ibotenic acid lesion of the rat nucleus basalis magnocellularis (NBM). A marked loss of acetylcholinesterase-positive fibers was observed in the frontal, parietal, temporal and occipital cortices ipsilateral to the lesion. The loss of cholinergic cell bodies in the NBM was further investigated with cholineacetyltransferase (ChAT) immunohistochemistry which indeed demonstrated a loss of ChAT-positive magnocellular perikarya. Autoradiographic analyses of specific binding of¹²⁵I-Tyr¹¹-SS-14 demonstrated a significant reduction in binding density in the denervated parts of the neocortex. The decrease in specific binding was most pronounced (40–50%) in the superficial layers (I–III) of the frontal, parietal and temporal cortices 2 and 4 weeks after lesion. A significant loss in¹²⁵I-Tyr¹¹-SS-14 binding in the deeper layers was only observed in the frontal cortex after 2 and 4 weeks. In the occipital cortex a significant decrease was measured in the superficial layers only after 4 weeks. The specific binding in all cortical regions returned to normal after 6 weeks. The results suggested that¹²⁵I-Tyr¹¹-SS-14 binding sites are localized on cholinergic afferents in the rat neocortex and that an up-regulation of number of binding sites, alternatively an increased binding affinity occured with time after lesion.     

Recovery of choline acetyltransferase activity without sprouting of the residual acetylcholine innervation in adult rat cerebral cortex after lesion of the nucleus basalis

In view of the divergent literature concerning the long-term effects of ibotenic acid lesions of the nucleus basalis of Meynert (NBM) on the choline acetyltransferase (ChAT) activity in adult rat cerebral cortex, we have critically reassessed the issue of an eventual recovery of this enzymatic activity by sprouting of the residual acetylcholine (ACh) innervation. At short (1 week) and long survival time (3 months) after unilateral ibotenic acid lesion, ChAT activity was biochemically measured in the ipsi and contralateral fronto-parietal cortex of several rats in which the extent of ACh neuronal loss in NBM was also estimated by counts of ChAT-immunostained cell bodies on the lesioned vs. non-lesioned side. In other lesioned rats, particular attention was paid to the distribution of the residual cortical ACh (ChAT-immunostained) innervation, and that of immunostained vasoactive intestinal polypeptide (VIP) axon terminals known to belong in part to intrinsic cortical ACh neurons which co-localize this peptide. One week after NBM lesion, profound decreases of ipsilateral cortical ChAT activity were tightly correlated with the extent of ACh cell body loss in the nucleus. A significant recovery of cortical ChAT activity could be documented after 3 months, despite persistence of NBM cell body losses as severe as after 1 week. At both survival times, the number of ChAT-immunostained axons was markedly reduced throughtout the ipsilateral fronto-parietal cortex, demonstrating that most ACh fibers of extrinsic origin had been permanently removed. This result also indicated that the long-term recovery of ChAT activity had occurred without sprouting of the residual ACh innervation. The laminar distribution and number of VIP-immunostained terminals remained the same on the lesioned and intact side and comparable to normal, ruling out an extensive sprouting of intrinsic ACh/VIP or VIP alone fibers. The return to a near normal cortical ChAT activity in severely ACh-denervated cortex suggested that the intrinsic ACh innervation was primarily responsible for this recovery.     

Short- and long-term effects of nucleus basalis magnocellularis lesions on cortical levels of somatostatin and its receptors in the rat

Cognitive and histological alterations in human Alzheimer's disease (AD) are correlated with selective neuronal loss in nucleus basalis of Meynert. In search of an animal model of AD-linked neurochemical deficits, we examined the effects of short- (2 weeks) and long- (3 and 6 months) term lesions of the nucleus basalis magnocellularis (NBM) on somatostatinergic parameters in rat forebrain. NBM lesions were performed by unilateral injection of ibotenic acid into the NBM. Cortical cholineacetyl transferase (ChAT) activity and acetylcholinesterase staining in the NBM remained significantly decreased ipsi- as compared to contralaterally up to 6 months after the placement of the lesion. Somatostatin (SRIF) content was increased by 120% in the ipsilateral frontal cortex 6 months post-lesion but not at shorter time intervals. Levels of neuropeptide Y (which is extensively co-localized with SRIF in the forebrain) were not significantly altered after unilateral NBM lesions at any time point. A 30% decrease in SRIF binding capacity as well as a marked reduction of SRIF inhibition of adenylate cyclase, indicative of a loss of functional SRIF receptors, was observed in ipsilateral versus contralateral frontal cortex on brain tissue homogenates after short-term unilateral NBM lesion. By film radioautography, the loss in SRIF binding sites was localized to both superficial and deep layers of the frontal cortex. This loss persisted up to 3 months but was no longer apparent after 6 months due to a decrease in SRIF binding capacity on the contralateral side.     

Age-related alterations in responses of nucleus basalis magnocellularis neurons to peripheral nociceptive stimuli

The present study investigated the effects of peripheral noxious stimuli on the spontaneous activity of the nucleus basalis magnocellularis (nbM) neurons in young, adult and old rats. Single unit extracellular recordings from the nbM neurons were obtained with glass micropipettes in urethane-anesthetized rats. A total of 104 units were antidromically identified as nbM-cortical neurons. Noxious but not innocuous mechanical stimulation elicited responses in 72% of the 104 neurons. Most of them were excited. The receptive fields were usually very large and bilateral. Most of the neurons also responded to noxious thermal, chemical and electrical stimuli. No marked differences were observed in the incidence of neurons having different spontaneous firing rates, firing patterns and response type among the three age groups. However, the latency of responses evoked by noxious thermal or electrical stimulation and the threshold of excitatory responses evoked by electrical stimulation were increased with aging. The duration and peak-firing rate of excitatory responses evoked by noxious thermal, chemical or electrical stimulation were decreased in old rats. These findings indicate that there might be some functional changes in the nbM neurons and its projection pathway with aging, which impair their responsive ability to peripheral nociceptive stimuli.     

Various hippocampal lesions induced by multi-fractional ibotenic acid injections and amygdala kindling in rats

Hippocampal degenerative lesions were made bilaterally by means of multiple ibotenic acid injections and development of amygdala kindling was studies. In groups with lesions in either bilateral dorsal or ventral hippocampus, stimulations required for kindling were almost the same as those of controls. In the group with lesion of the entire hippocampus, kindling development was remarkably slow especially in the early stage of the kindling process. However, kindling effect was finally estabilished in all groups.     

Behavioral and neurochemical consequences of ibotenic acid lesion in the subthalamic nucleus of the common marmoset

Five marmosets were unilaterally lesioned within the subthalamic nucleus (STN) by injection of 10 μg ibotenic acid. Seven marmosets served as saline injected controls. The lesioned marmosets showed an increased locomotor activity, occasional tongue protrusions, posture asymmetry, and abnormal movements of the contralateral legs and arms. The animals were sacrificed 21 days after the lbotenic acid injection and markers of γ-aminobutyric acid (GABA), dopamine (DA), and acetylcholine were studied in a variety brain regions. There was a bilateral increase in the activity of glutamic acid decarboxylase (GAD) in the caudate, putamen, globus pallidus, superior colliculus, and the ventral anterior/ventral lateral (VANL) thalamus, whereas GABA concentrations were only increased ipsilaterally in the ventral posterior medial/centromedial/parafasciculus (VPM/CM/Pf) complex of the thalamus. Tyrosine hydroxylase (TH) activity was bilaterally increased in the medial segment of globus pallidus and nucleus accumbens. However, there were also changes restricted to the side contralateral to the lesion. TH activity and DA concentrations were increased contralateral to the lesion in the putamen. Choline acetyltransferase (CAT) activity was bilaterally increased in the medial segment of globus pallidus and hypothalamus. The lbotenic acid induced STN-lesion in the marmoset, thus, seemed to cause a widespread bilateral activation of neurons within the basal ganglia.     

胶质细胞谷氨酸转运体在大鼠点燃效应中的作用研究

目的 研究点燃形成过程中和点燃后大鼠海马中氨酸天门氨酸转运体（GLAST）和谷氨酸转运体1（GLT－1）的变化,进一步探讨癫痫的形成机制。方法 将78只雄性成年Wistar大鼠随机分为对照组（I组）和戊四氮（PTZ）组（Ⅱ组）。Ⅱ组腹腔注射阈下剂量的PTZ（335mg/kg）,每日1次,直到达到点燃标准;I组腹腔注射等量生理盐水。采有RT－PCR方法检测海马区GLAST和GLT－1mRNA的表达。结果 PTZ组点燃后,GLASTmRNA的表达下降,60天时恢复至对照组;与对照组比较,PTZ组GLT－1mRNA的表达,在给药后15天时开始上升,点燃后0小时和48小时时显著升高,此后呈下降趋势。60天时,两组比较无明显差异。结论 海马区胶质细胞谷氨酸转运体的下降可能与癫痫敏感性的形成有关。     

Glial response to neuronal activity: GFAP-mRNA and protein levels are transiently increased in the hippocampus after seizures

We have recently demonstrated that electrically induced seizures lead to dramatic increases in mRNA for GFAP in areas in which seizures occur. The present study evaluates the time course of the changes in the GFAP-mRNA levels after seizures and the relationship between these changes and GFAP protein levels to understand the role of neuronal activity in regulating glial gene expression. GFA protein and mRNA levels were measured in hippocampi from rats in which seizures were induced by: (1) 50-Hz stimulus trains delivered 12 times over the course of 1 day via indwelling electrodes implanted chronically in the CA3 region of the hippocampus; and (2) intraperitoneal injections of pentylenetetrazol. In the case of the electrically induced seizures, we also compared the glial response in animals that had never experienced a seizure with the response in animals that previously had been kindled but had not experienced a seizure for 30 days. Electrically induced seizures led to rapid transient increases in GFAP-mRNA levels in the hippocampus ipsi- and contralateral to the stimulation. GFAP-mRNA increased about five-fold 1 day after the end of seizure activity and returned to near-control levels by 4 days. There were no detectable increases in GFA protein at 1 day but by 2 days GFA protein levels had increased about two-fold. GFA protein levels remained elevated until 4 days poststimulation and then began to decrease. The responses were similar when seizures were induced in kindled animals, except that the GFAP protein levels remained elevated for somewhat longer. Pentylenetetrazol-induced seizures also led to increases in GFAP-mRNA and GFA protein levels but the extent of the increases was not as great as after kindled seizures. These results suggest that gene expression in astrocytes in likely to be upregulated in any situation in which seizures occur. These changes may fundamentally alter the homeostatic activities of the affected astrocytes which, in turn, could have important consequences on the development of the epileptic state.     

Loss of hippocampal CA1 neurons and learning impairment in subicular lesioned rats

30-Day-old male Wistar rats were tested for acquisition and retention of operant conditioned behavior after bilateral subicular lesions made either electrolytically or chemically (ibotenic acid). The acquisition of operant learning was carried out in lesioned rats by assessing the number of sessions required to learn the operant task, whereas the retention test was performed after lesions by assessing performance on a previously learnt operant task. The acquisition of pedal press operant learning was significantly delayed in both types of lesioned rats, without any impairment in the retention of the previously learned task after lesioning. In these animals the cell densities were quantified in cresyl violet-stained sections in different subfields of hippocampus. Following the lesion of subiculum, selective degeneration of CA1 cells without the involvement of other hippocampal subfields was observed. This might be due to the loss of target area (subiculum) through which hippocampus is connected with neocortical and subcortical structures. This, in turn, might have resulted in behavioral deficits. The data suggest that the subiculum might be involved in the acquisition of new information rather than in retention.     

红藻氨酸致痫大鼠海马S100B、CGRP蛋白表达及病理改变

目的 研究红藻氨酸(KA)致痫大鼠海马S100B、降钙素基因相关肽(CGRP)的表达及病理改变.方法 雄性SD大鼠按照完全随机数字表法分成对照组(8只)和模型组(40只),模型组再根据处死时间分为造模后6 h、12 h、24 h、72 h、1周5个亚组,每组8只.模型组采用KA建立颞叶癫痫动物模型,对照组用等体积生理盐水代替KA注射.模型组造模后6 h、12 h、24 h、72 h、1周,对照组注射后24 h取大鼠海马组织行Nissl染色、Timm染色和免疫组化染色,观察S100B、CGRP蛋白的表达情况以及海马神经元和胶质细胞的病理变化.结果 Nissl染色结果显示,模型组大鼠1周后CA3区出现大量固缩的坏死神经元,胞体萎缩,尼氏体消失.Timm染色结果显示,模型组大鼠1周后CA3区始层出现条带状分布的棕色颗粒,齿状回内分子层亦可见少量棕色颗粒.免疫组化染色结果显示,模型组大鼠海马CGRP蛋白大量表达,72 h时达到高峰,同时伴随大量神经元丧失及胶质细胞增生.结论 KA致痫大鼠出现S100B、CGRP蛋白高表达,尼氏体消失,苔藓纤维发芽等一系列病理学改变,推测S100B、CGRP蛋白参与了癫痫发生.

Abstract：

Objective To investigate the expressions of S100B and calcitonin gene related peptide (CGRP) and the pathologic alterations of the hippocampus in kainic acid (KA)-induced epileptic rats. Methods Male SD rats were randomly divided into control group (n=8) and model group (n=40).Animal models of temporal lobe epilepsy were established by intracerebroventricular injection of KA; the same volume of saline was injected into the rats in the control group. Hippocampal tissues within various phases after seizures (6, 12, 24 and 72 h, and 24 h after the success of model making) were performed Nissl staining, Timm staining and immunohistochemical staining. The expressions of S100B and CGRP were observed, and the pathologic alterations of the hippocampal neurons and glial cells were studied.Results All rat models were successfully induced with epileptic seizures. Nissl staining showed that pyknotic neuronal necrosis appeared in the CA3 area of the hippocampus in the model group with cell body atrophy and disappearance of Nissl bodies 1 week after the injection. Timm staining showed that brown particles showed stripped distribution in the CA3 area of the hippocampus and some brown particles in the molecular layer of fascia dentate. Immunohistochemical staining indicated that significant neurons lost and gliosis appeared after seizures with abundant expressions of S100B and CGRP.Conclusion KA-induced epileptic rats express abundant S100B and CGRP and appear such pathological changes as disappearance of Nissl bodies and mossy fiber sprouting, indicating that both S100B and CGRP participate in the onset of epilepsy.     

饮酒致大鼠海马区GFAP和S-100B蛋白改变及对学习、记忆功能的影响

目的探讨饮酒致大鼠海马区神经胶质细胞内胶质纤维酸性蛋白(GFAP)和S-100B蛋白表达的改变,并观察饮酒对大鼠学习、记忆功能的影响。方法将60只3月龄大鼠随机分为不饮酒组、饮酒1周、2周、4周、6 周、8周组各10只,饮酒组酒精灌胃,喂养过程中进行大鼠跳台实验观察饮酒对大鼠学习记忆功能的影响,喂养结束后取大鼠脑组织,常规制片,作GFAP和S-100B的免疫组化染色,光镜下进行形态学观察及定量分析。结果各饮酒组GFAP和S-100B免疫反应产物阳性细胞计数值均较未饮酒组显著增高且有细胞形态改变。大鼠跳台试验学习成绩显示饮酒组自第4周开始反应期较未饮酒组明显延长,错误次数从第6周开始明显增加;记忆成绩显示饮酒组自第2周开始潜伏期明显缩短,错误次数从第4周开始明显增加。结论饮酒使大鼠海马区GFAP和S-100B的表达增加,增加的程度与饮酒时间呈正相关。随饮酒周数的增加大鼠学习、记忆功能减退。     

Effects of chronic infusion of nerve growth factor (NGF) in rats with nucleus basalis magnocellularis lesion

We attempted to evaluate the effects of bilateral injection of ibotenic acid (IA) into the nucleus basalis magnocellularis (nbm) of rats as well as the potential recovery mediated by the infusion of nerve growth factor (NGF). The lesion caused an impairment of learning and memory processes. Also, a severe depletion of choline acetyl transferase activity was detected in cortical areas. After the NGF administration, a significant reversion of these functional changes was observed. Thus, IA-lesioned rats might serve as a model for the evaluation of neurotrophic factors actions on basal forebrain damaged neurons.     

Transplantation of embryonic ventral forebrain grafts to the neocortex of rats with bilateral lesions of nucleus basalis magnocellularis ameliorates a lesion-induced deficit in spatial memory

Embryonic ventral forebrain grafts containing developing cholinergic cells were transplanted to the neocortex of rats with bilateral quisqualic acid lesions of the nucleus basalis magnocellularis. A lesion-induced deficit on performance of a spatial alternation test of memory was reduced by such transplants. When the same animals were treated with the acetylcholinesterase inhibitor physostigmine (0.05 mg/kg), however, performance on the behavioral task was not further promoted, and therefore, under these conditions, the cholinergic cortical transplants appear not to be subject to modulation by anticholinesterase drugs.     

Effects of tetrahydro-9-aminoacridine on the electrocorticogram of rats with a unilateral lesion of the nucleus basalis magnocellularis.

The effects of tetrahydro-9-aminoacridine (THA) were studied on the electrocortical activity of control rats and of rats with a unilateral lesion of the nucleus basalis magnocellularis (NBM) produced by alpha-amino-3-hydroxy-4-isoxozole propionic acid (AMPA). This lesion almost completely deprived the lesioned hemisphere of its cholinergic innervation. In control rats, THA (10 mg/kg i.p.) increased the amplitude of the slow components of the electrocorticogram (less than 9 Hz). These effects were antagonised by atropine (5 mg/kg i.p.). Lesion of the NBM alone decreased the amplitude of frequencies in the 12-16 Hz frequency band but did not significantly affect the slower frequencies. THA (10 mg/kg) restored the amplitude of the 12-16 Hz activity to the level seen in control rats before THA but did not affect activity in the other frequency bands. The results suggest that THA requires some residual cholinergic innervation in order to exert its effect.     

Neurochemical and behavioral recovery after colchicine lesions of the nucleus basalis magnocellularis in rats

Experimentally-induced lesions of the basal forebrain have been used to test the hypothesis that the cholinergic system plays a critical role in learning and memory. In the present study, a basal forebrain infusion of colchicine, a microtubule assembly inhibitor, was used to characterize the relationship between a cholinergic marker and behavioral function. Bilateral infusions were made in the nucleus basalis magnocellularis (NBM) of male Long-Evans rats. At 4 weeks post-lesion, behavioral assessments were made on half of the rats in each group. These rats were sacrificed 1 week later and regional choline acetyltransferase (ChAT) activity was measured. The remaining rats were behaviorally tested 11 weeks post-lesion and sacrificed 12 weeks post-lesion. The brains of additional rats were studied for Nissl-staining, ChAT-, GAD- and metEnk immunoreactivity (IR) and AChE histochemistry. At 5 weeks after colchicine infusion, there was a significant decrease in parietal and frontal cortical ChAT activity, impaired acquisition of a water maze spatial navigation task and decreased passive avoidance cross-over latency. At 12 weeks after colchicine infusion, ChAT activity was decreased in frontal but not parietal cortex; acquisition of the water maze task was not significantly different from vehicle-infused rats, and a significant deficit was observed in passive avoidance latency. ChAT-IR in the NBM showed a significant decrease at both time points, while changes in AChE-stained cortical fibers paralleled the ChAT activity. GAD- and metEnk-IR were decreased but were not different between the two time points. These data show task-specific behavioral recovery associated in time with recovery of regional cholinergic markers.     

Neurochemical recovery in the neocortex after colchicine lesions of the nucleus basalis magnocellularis in rats

Neurochemical recovery was investigated in male, Fischer-344 rats up to 3 months after lesions of the nucleus basalis. Bilateral injections of colchicine (1.0 micrograms/site) into the nucleus basalis magnocellularis (NBM) resulted in a 30% decrease in choline acetyltransferase (ChAT) activity in frontal cortex 4 weeks after surgery, compared to unlesioned controls. ChAT activity in the frontal cortex gradually recovered to control levels by 12 weeks. The loss of ChAT-immunoreactive neurons in the NBM observed 4 weeks after surgery was still evident 12 weeks after surgery. These results suggest that surviving cholinergic neurons in the NBM contribute to recovery of ChAT activity in the neocortex.     

Behavioral impairments after lesions of the nucleus basalis by ibotenic acid and quisqualic acid

Ibotenic acid (IBO) or quisqualic acid (QUIS) was infused into the region of the nucleus basalis magnocellularis (NBm) in F344 rats in order to behaviorally and biochemically characterize the effects of these two neurotoxins. QUIS infusion resulted in a slightly higher depletion of choline acetyltransferase (ChAT) activity in both anterior and posterior regions of cortex than did lesions caused by infusion of IBO. Both QUIS- and IBO-treated rats demonstrated significantly longer latencies than controls to find a hidden platform in a Morris water maze task. In addition, QUIS-treated rats performed significantly better than IBO-treated rats in the water maze. Analysis of swim speed and open field behavior did not show significant differences in general motor activity. Passive avoidance retention was unaffected by either neurotoxin. Cortical amino acid levels, [3H]neurotensin binding, dopamine, norepinephrine, and serotonin levels were unaffected by either neurotoxin. The levels of HVA and 5-HIAA in the IBO and QUIS groups were significantly reduced compared to controls, but were not significantly different from each other. Histological examination showed greater damage to non-NBm structures with IBO than with QUIS, including the basolateral nucleus of the amygdala and the reticular formation of the thalamus. The greater behavioral deficit seen after IBO lesions may be due to damage to other areas rather than differences in the extent of depletion of corticai ChAT, amino acids, catecholamines or indolamines.