Nerve growth factor prevents cell death and induces hypertrophy of basal forebrain cholinergic neurons in rats withdrawn from prolonged ethanol intake

We have previously reported that the hippocampal cholinergic fiber network is severely damaged in animals withdrawn from ethanol, and that a remarkable recovery in fiber density occurs following hippocampal grafting, a finding that we suggested to be underpinned by the graft production of neurotrophic factors, which are known to be decreased after ethanol exposure. It is widely accepted that nerve growth factor (NGF) signals the neurons of the brain cholinergic system, including those of the medial septum/vertical limb of the diagonal band of Broca (MS/VDB) nuclei, from which the septohippocampal projection arises. Because neurons in these nuclei are vulnerable to ethanol consumption and withdrawal we thought of interest to investigate, in withdrawn rats previously submitted to a prolonged period of ethanol intake, the effects of intraventricular delivery of NGF upon the MS/VDB cholinergic neurons. Stereological methods were applied to estimate neuron numbers and neuronal volumes in choline acetyltransferase (ChAT)-immunostained and Nissl-stained material. We have found that in ethanol-fed rats there was a significant reduction in the total number of Nissl-stained and cholinergic neurons in the MS/VDB, and that the suppression of ethanol intake further decreased neuron numbers. In addition, the somatic size of ChAT-IR neurons was reduced by ethanol intake, and withdrawal further aggravated neuronal atrophy. NGF treatment prevented the withdrawal-associated loss, and induced hypertrophy, of cholinergic neurons.These findings show that exogenous NGF protects the phenotype and prevents the withdrawal-induced degeneration of cholinergic neurons in the MS/VDB. These effects might be due to the trophic action of NGF upon the basal forebrain cholinergic neurons, including the hippocampal fiber network that conveys this neurotrophin retrogradely to the MS/VDB, and/or upon their targets, that is, the hippocampal formation neurons.     

The effects of nerve growth factor upon the neuropeptide content of the suprachiasmatic nucleus of rats withdrawn from ethanol are mediated by the nucleus basalis magnocellularis

It has been previously shown that withdrawal from alcohol decreases the synthesis and expression of vasopressin (VP) and vasoactive intestinal polypeptide (VIP) in the suprachiasmatic nucleus (SCN), and that the infusion of NGF over 1 month completely restores these changes. Because SCN neurons do not express TrkA, NGF might have exerted its effects either through direct signalling of the neurons via p75^NTR or by enhancing the activity of the cholinergic afferents to the SCN, which arise from the nucleus basalis magnocellularis (NBM). The observation that the infusion of NT-3 to withdrawn rats does not elicit any change in neuropeptide expression in the SCN suggests that ACh might be implicated in this process, a hypothesis that we have attempted to clarify in this study. For this purpose we destroyed, with quinolinic acid, the NBM of rats withdrawn from ethanol and later infused them with NGF over a period of 13 days. The total number and the somatic volume of SCN neurons immunoreactive for VP and VIP were stereologically estimated. No differences were found in the total number of neurons between quinolinic-injected NGF-treated withdrawn animals and intact withdrawn rats. However, the somatic volume of SCN neurons from quinolinic-injected animals was significantly reduced relative to control and withdrawn rats. The present results unequivocally demonstrate that the trophic effects exerted by NGF upon SCN neurons do not depend on direct neuronal signalling. Instead, they are indirect and, according to our results, NBM neurons, whose axons give rise to a cholinergic projection to the SCN, seem to be essential for eliciting those effects.     

Behavioral and neuroanatomical consequences of chronic ethanol intake and withdrawal.

We have examined if long-term (13 months) alcohol consumption and the same treatment followed by a 6-week withdrawal period cause different neuropathological changes in rats. Spatial reference and working memory of alcohol-consuming and withdrawn rats were evaluated by comparison of their performance with age-matched controls in the Morris water maze. In the reference memory task we did not observe significant cognitive deficits in rats continuously exposed to ethanol, whereas withdrawn animals showed an obvious impairment of their overall performance. The reference memory deficit in withdrawn rats was evident in the spatial probe trial; these animals required significantly longer swimming distances to approach the former position of the platform when compared with controls and alcohol-consuming animals. In contrast, working memory was not significantly altered in either experimental group. Stereological methods were applied to compare the neurodegenerative changes produced by alcohol intake and withdrawal in the hippocampal formation. In the alcohol-consuming animals there was a significant cell loss in CA1 (18%) and CA3 (19%) hippocampal regions. Moreover, in withdrawn rats there was a further decay in the total number of pyramidal neurons, which amounted to 15% relative to nonwithdrawn animals. In the granular layer of the dentate gyrus there was a trend in the same direction, but it did not reach significance. Thus, our findings indicate that withdrawn rats are cognitively impaired relative to animals submitted to continuous alcohol consumption and to age-matched controls, which fits the morphological data showing that withdrawal aggravates ethanol-induced degenerative processes in the hippocampal formation.     

NGF treatment promotes development of basal forebrain tissue grafts in the anterior chamber of the eye

Summary The effects of nerve growth factor (NGF) on developing central cholinergic neurons were studied using intraocular grafts of rat fetal (E17) basal forebrain tissue. Prior to grafting, grafts were incubated in NGF or saline. Transplants were allowed to mature for six weeks, receiving weekly intraocular injections of NGF or saline. Measurements of NGF levels in oculo after one single injection showed that NGF slowly decreases in the anterior chamber fluid, and after one week, low but significant levels were still present in the eye. Following pretreatment with diisopropylfluorophosphate (DFP), the cholinergic neurons in the grafts were analyzed using three morphological markers: antibodies to cholineacetyltransferase (ChAT), antibodies to acetylcholinesterase (AChE Ab) and acetylcholinesterase histochemistry (AChE). The transplants grew well and became vascularized within the first week. The growth of the NGF-treated basal forebrain grafts was significantly enhanced as compared to the growth of the saline-treated grafts evaluated with repeated stereomicroscopical observations directly through the cornea of the etheranaesthetized hosts. The NGF-treated grafts contained almost twice as many cholinergic neurons seen with all the cholinergic markers used, as the salinetreated grafts. However, there was no difference in cholinergic cell density between the two groups. The morphology and size of an individual cholinergic neuron was similar in the two groups. The fiber density as evaluated with AChE-immunohistochemistry did not change after NGF-treatment. The DFP-treatment did not seem to affect the AChE-immunoreactivity since an extensive fiber network was found, whereas almost no fibers were seen using conventional AChE histochemistry. We have demonstrated that in oculo transplantation of basal forebrain is a useful model for examining in vivo effects of NGF on central cholinergic function. The marked volume increase of NGF-treated grafts and the unchanged density of cholinergic cells and terminals suggests, that NGF increases the survival of not only developing cholinergic neurons, but possibly other non-cholinergic neurons and non-neuronal cells as well. These results support the notion that NGF acts as a neurotrophic factor on cholinergic and possibly non-cholinergic cells in the central nervous system     

The length of hippocampal cholinergic fibers is reduced in the aging brain

Cholinergic deficits occur in the aged hippocampus and they are significant in Alzheimer's disease. Using stereological and biochemical approaches, we characterized the cholinergic septohippocampal pathway in old (24 months) and young adult (3 months) rats. The total length of choline acetyltransferase (ChAT)-positive fibers in the dorsal hippocampus was significantly decreased by 32% with aging (F((1,9))=20.94, p=0.0014), along with the levels of synaptophysin, a presynaptic marker. No significant changes were detected in ChAT activity or in the amounts of ChAT protein, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tropomyosin related kinase receptor (Trk) A, TrkB, or p75 neurotrophin receptor (p75(NTR)) in the aged dorsal hippocampus. The number and size of ChAT-positive neurons and the levels of ChAT activity, NGF and BDNF were not statistically different in the septum of aged and young adult rats. This study suggests that substantial synaptic loss and cholinergic axonal degeneration occurs during aging and reinforces the importance of therapies that can protect axons and promote their growth in order to restore cholinergic neurotransmission.     

Experimental approaches to age-related cognitive impairments

Rats exhibit morphological, biochemical, and metabolic changes in their brains, as well as cognitive deficits, with aging. Aged rats were found to be significantly impaired compared to young rats in a water maze task and test of motor coordination, and show reduced locomotor activity and exploration. Although aged rats did exhibit deficits as a group, not all aged rats were impaired. Additionally, the subgroup that was impaired on one task was not necessarily the subgroup that was impaired on another task. The cholinergic projection neurons in the basal forebrain region were significantly atrophied in the aged rodent. The degree of atrophy was highly correlated with the cognitive impairment exhibited on the Morris water maze task. Swollen choline acetyltransferase (ChAT)-positive "plaque-like" structures were observed in the neocortex of the aged but not the young rats. Declines in cholinergic activity in the brain has also been observed during aging. Biochemical measurements of ChAT in the basal forebrain region of aged rats revealed small but consistent decreases in ChAT activity compared to young rats. General metabolic activity, measured by the 2-deoxyglucose method, was also decreased in the hippocampal CA1 and CA3 fields, the dentate gyrus, the medial septal-diagonal band area, and the prefrontal cortex of aged rats. There was a significant correlation between the decrease in glucose utilization and deficits on the Morris water maze. Most aged rats exhibit pathological EEG patterns as reflected by frequent long-duration high voltage neocortical spindles (HVS) during immobility. Bilateral lesions of the nucleus basalis and scopolamine treatment increased the incidence of HVS, thereby mimicking changes in the aged brain. We attempted to ameliorate the cognitive deficits observed in subgroups or impaired rats by either: (1) implanting fetal cells of basal forebrain origin into the hippocampus, or (2) infusing nerve growth factor (NGF) chronically into the lateral ventricle. The grafts appeared to facilitate an improvement in the ability of the impaired aged rats to perform in the Morris water maze. This improved performance was reversed by injections of atropine at doses that did not affect the behavior of young animals that performed well in the same task. These results suggest that enhancement of the cholinergic system could have an effect on the performance of the impaired aged animals. The study of the effects of infusions of NGF clearly demonstrate that the ability of impaired aged rats to remember what they had previously learned was increased after NGF treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

神经生长因子对大鼠心肌梗塞后胆碱能神经的保护作用

目的　观察神经生长因子(NGF)对大鼠心肌梗塞后胆碱能神经的影响,探讨NGF对心肌梗塞的可 能治疗作用。方法　SD大鼠20只制心梗模型,其中10只心梗后给予NGF为实验组,2天后取材,同时取正常心肌 作对照。以Karnovsky Roots法,显示胆碱能神经纤维,应用多功能真彩色病理图像分析系统分析胆碱能神经纤维 密度。结果　心梗左心室心肌梗塞区及梗塞周围区存活心肌中胆碱能神经纤维密度比正常组明显降低(P<0. 01);实验组应用NGF后胆碱能神经纤维密度较心梗组升高。结论　神经生长因子能增加大鼠心肌梗塞后胆碱能 神经纤维的密度,可能对心梗有治疗作用。     

Intraseptal muscarinic ligands and galanin: influence on hippocampal acetylcholine and cognition

The cholinergic neurons in the septohippocampal projection are implicated in hippocampal functions such as spatial learning and memory. The aim of this study was to examine how septohippocampal cholinergic transmission is modulated by muscarinic inputs and by the neuropeptide galanin, co-localized with acetylcholine (ACh) in septohippocampal cholinergic neurons, and how spatial learning assessed by the Morris water maze test is affected. Muscarinic inputs to the septal area are assumed to be excitatory, whereas galanin is hypothesized to inhibit septohippocampal cholinergic function. To test these hypotheses, compounds were microinjected into the medial septum and hippocampal ACh release was assessed by microdialysis probes in the ventral hippocampus of the rat. Blockade of septal muscarinic transmission by intraseptal scopolamine increased hippocampal ACh release suggesting that septal cholinergic neurons are under tonic inhibition. Stimulation of septal muscarinic receptors by carbachol also increased hippocampal ACh release. Despite this increase, both scopolamine and carbachol tended to impair hippocampus-dependent spatial learning. This finding also suggests a revision of the simplistic notion that an increase in hippocampal ACh may be facilitatory for learning and memory. Galanin infused into the medial septum enhanced hippocampal ACh release and facilitated spatial learning, suggesting that septal galanin, contrary to earlier claims, does not inhibit but excites septohippocampal cholinergic neurons. Galanin receptor stimulation combined with muscarinic blockade in the septal area resulted in an excessive increase of hippocampal ACh release combined with an impairment of spatial learning. This finding suggests that the level of muscarinic activity within the septal area may determine the effects of galanin on hippocampal cognitive functions. In summary, a limited range of cholinergic muscarinic transmission may contribute to optimal hippocampal function, a finding that has important implications for therapeutic approaches in the treatment of disorders of memory function.     

神经生长因子促进老年鼠大脑皮质胆碱能纤维损伤后再生

目的：探讨神经生长因子（nerve growth factor,NGF)对老年鼠大脑皮质胆碱能神经纤维损伤后再生的影响。方法：老年SD大鼠顶皮质切一宽2mm的冠状切口,侧脑室注射NGF,AChE纤维染色结合体视系分析正常组,损伤对照组和NGF用药组切口嘴,尾侧区的胆碱能纤维的再生情况。结果：损伤对照组损伤1个月后,嘴侧AChE阳性纤维增生至正常111．44％,尾侧纤维减少至正常67．14％;NGF用药组嘴侧纤维密度比损伤对照组增多,为正常145．93％,尾侧纤维为正常127．95％,结论：提示NGF对老年乳类皮质胆碱能纤维后损伤再生具有一定促进作用。     

老年性学习记忆减退大鼠新皮质和海马结构胆碱能纤维的定量研究

用Ｍｏｒｒｉｓ水迷宫行为检测方法以青年组平均逃避潜伏期９５％和９９％正常值范围上限值为界将老年大鼠分为学习记忆正常组（老年正常组）和学习记忆减退组（老年减退组）,以Ｈｅｄｒｅｅｎ等推荐的ＡＣｈＥ组织化学染色方法结合形态计量方法对各组大鼠的额、枕、内嗅皮质、海马ＣＡ１区多形层、腔隙分子层和齿状回分子层外带的胆碱能纤维密度进行分析,结果显示老年减退组较老年正常组、青年组各层（除枕叶外）ＡＣｈＥ阳性纤维数量均明显减少（Ｐ０．０５,Ｐ＜０．０１）。老年正常组与青年组相比各层阳性纤维数量有所减少,但除海马ＣＡ１区腔隙分子层差异显著外,其余差异均无显著性。相关分析结果表明大鼠各层ＡＣｈＥ阳性纤维数量与其平均逃避潜伏期呈负相关关系,与原平台象限游泳距离占总距离百分比呈正相关关系。本研究提示老年性学习记忆能力减退与新皮质、海马结构胆碱能纤维溃变密切相关。     

神经生长因子预处理对拟AD模型大鼠脑内神经原纤维缠结形成和ChAT表达的影响

为了探讨神经生长因子(NGF)预处理对拟Alzheimer病(AD)模型大鼠海马CA1区和顶叶皮层内神经原纤维缠结(NFT)和胆碱乙酰基转移酶(ChAT)表达的影响,本研究将动物分为拟AD组和NGF预处理组。将冈田酸(OA)微量注射至拟AD组大鼠海马CA1区(0.4mmol/L,0.5ml/次;隔天1次,共7次)建立拟AD大鼠模型,NGF预处理组于制模前10d将NGF(0.1mg/ml,5ml/次;隔天1次,共10次)注射至侧脑室。通过Morris水迷宫观察上述大鼠的行为学变化,分别用改进的Bielschowsky染色和免疫组化法观察海马及顶叶皮层内NFT和ChAT表达的变化。结果显示:拟AD模型组大鼠出现认知、学习记忆能力减退,海马CA1区及顶叶皮层出现较多的NFT,而ChAT表达减少;NGF预处理组大鼠的上述症状明显改善。这些结果提示OA的神经毒性可以导致拟AD模型大鼠的学习记忆能力降低,并且出现胆碱能神经元损伤和功能低下;NGF预处理可以显著改善拟AD模型大鼠的学习记忆能力,抑制海马CA1区和顶叶皮层内NFT的形成,减轻对胆碱能神经元的损伤。     

Learning and memory deficits after lesions of the nucleus basalis magnocellularis: reversal by physostigmine

The role of the cholinergic nucleus basalis magnocellularis in spatial learning and memory was studied in the rat. Animals received bilateral injections of ibotenic acid (5 micrograms/microliters) into the region of the nucleus basalis magnocellularis. Six weeks postoperatively they were deprived of food and trained for 5 weeks in a 16-arm radial maze in which 9 of the arms were baited with food. The nucleus basalis magnocellularis-lesioned animals showed significant deficits in the acquisition of the task. Further analysis of the data indicated that this was due primarily to a deficit in reference (long-term) as opposed to working (short-term) memory. After the 5-week training period the nucleus basalis magnocellularis-lesioned animals received intraperitoneal injections of physostigmine sulphate (0.5 mg/kg) 30 min before each daily trial for 1 week. This treatment resulted in a significant improvement in the performance of the spatial memory task on all three measures. The ibotenate lesions reduced the activity of choline acetyltransferase by about 40% in the anterior cortex and by 15% in the posterior cortex. Hippocampal choline acetyltransferase activity was not affected, indicating that the septohippocampal cholinergic projection was spared by the lesions. The activity of glutamate decarboxylase was not affected in any of these regions. These results suggest that the cholinergic projections of the nucleus basalis magnocellularis play an important role in the acquisition of a spatial memory task.     

Age-related working memory deficits in the allocentric place discrimination task: possible involvement in cholinergic dysfunction

It is well known that learning and memory ability declines with aging. Age-related long-term changes in learning and memory ability in rats were investigated with the place navigation task and the allocentric place discrimination task (APDT) in a water maze using the same animals for each task. In a working memory place navigation task, aged animals could learn the location of the platform as well as when they were young, although strategy shifts were observed. In contrast, accuracy in the APDT significantly declined from 90% to 65% with aging. This impairment was ameliorated by an acetylcholine esterase inhibitor physostigmine at 22–23 months old. No amelioration was, however, detected in the same animals tested when they further aged to 26–27 months old. These results suggest that the APDT performance is sensitive to age-related memory deficits and that this may be due to the cholinergic dysfunction.     

Cholinergic medial septum neurons do not degenerate in aged 129/Sv control or p75(NGFR)-/-mice

Cholinergic medial septum neurons express TrkA and p75 nerve growth factor receptor (p75(NGFR)) and interactions between TrkA and p75(NGFR) are necessary for high-affinity binding and signaling of nerve growth factor (NGF) through TrkA. In adult p75(NGFR)-deficient (-/-) mice, retrograde transport of NGF and other neurotrophins by these neurons is greatly reduced, however, these neurons maintain their cholinergic phenotype and size. Reduced transport of NGF has been proposed to play a role in Alzheimer's disease. Here, we investigated whether chronic and long-term absence of p75(NGFR) (and possibly reduced NGF transport and TrkA binding) would affect the cholinergic septohippocampal system during aging in mice. In young (6-8 months), middle aged (12-18 months), and aged (19-23 months) 129/Sv control mice the total number of choline acetyltransferase-positive medial septum neurons and the mean diameter and cross sectional area of the cholinergic cell bodies were similar. The cholinergic hippocampal innervation, as measured by the density of acetylcholinesterase-positive fibers in the outer molecular layer of the dentate gyrus was also similar across all ages. These parameters also did not change during aging in p75(NGFR) -/- mice and the number and size of the choline acetyltransferase-positive neurons and the cholinergic innervation density were largely similar as in control mice at all ages. These results suggest that p75(NGFR) does not play a major role in the maintenance of the number or morphology of the cholinergic basal forebrain neurons during aging of these mice. Alternatively, p75(NGFR) -/- mice may have developed compensatory mechanisms in response to the absence of p75(NGFR).     

神经生长因子预处理阿尔茨海默病大鼠海马神经原磷酸化Tau蛋白的变化

背景：神经生长因子能够促进胆碱能神经元的分化,决定轴突的生长,并可参与损伤神经的再生和功能修复。 目的：进一步验证神经生长因子预处理对拟阿尔茨海默病模型大鼠脑内神经原纤维缠结和磷酸化Tau蛋白表达的影响。 方法：将3-5月龄雄性Wistar大鼠随机分为3组：模型组将冈田酸微量注射至拟大鼠海马CA1区建立拟阿尔茨海默病大鼠模型;预处理组于造模前将神经生长因子注射至侧脑室;对照组用同样方法注入等体积的二甲亚砜作为对照。通过Morris水迷宫观察上述大鼠的行为学变化,分别用改进的Bielschowsky染色观察海马CA1区神经原纤维缠结,用免疫组织化学和免疫印迹法观察海马区磷酸化Tau蛋白表达的变化。 结果与结论：拟阿尔茨海默病模型组大鼠出现认知、学习记忆能力减退;与对照组比较,模型组海马CA1区出现较多神经原纤维缠结, 而且磷酸化的Tau蛋白表达增多;神经生长因子预处理组大鼠的上述症状明显改善。提示神经生长因子预处理可以显著改善拟阿尔茨海默病模型大鼠的学习记忆能力,抑制神经原纤维缠结的形成,减少磷酸化Tau蛋白的表达。     

Histamine innervation and activation of septohippocampal GABAergic neurones: involvement of local ACh release

Recent studies indicate that the histaminergic system, which is critical for wakefulness, also influences learning and memory by interacting with cholinergic systems in the brain. Histamine-containing neurones of the tuberomammillary nucleus densely innervate the cholinergic and GABAergic nucleus of the medial septum/diagonal band of Broca (MSDB) which projects to the hippocampus and sustains hippocampal theta rhythm and associated learning and memory functions. Here we demonstrate that histamine, acting via H₁ and/or H₂ receptor subtypes, utilizes direct and indirect mechanisms to excite septohippocampal GABA-type neurones in a reversible, reproducible and concentration-dependent manner. The indirect mechanism involves local ACh release, is potentiated by acetylcholinesterase inhibitors and blocked by atropine methylbromide and 4-DAMP mustard, an M₃ muscarinic receptor selective antagonist. This indirect effect, presumably, results from a direct histamine-induced activation of septohippocampal cholinergic neurones and a subsequent indirect activation of the septohippocampal GABAergic neurones. In double-immunolabelling studies, histamine fibres were found in the vicinity of both septohippocampal cholinergic and GABAergic cell types. These findings have significance for Alzheimer's disease and other neurodegenerative disorders involving a loss of septohippocampal cholinergic neurones as such a loss would also obtund histamine effects on septohippocampal cholinergic and GABAergic functions and further compromise hippocampal arousal and associated cognitive functions.     

Age-related changes in rostral basal forebrain cholinergic and GABAergic projection neurons: relationship with spatial impairment

Both cholinergic and GABAergic projections from the rostral basal forebrain contribute to hippocampal function and mnemonic abilities. While dysfunction of cholinergic neurons has been heavily implicated in age-related memory decline, significantly less is known regarding how age-related changes in codistributed GABAergic projection neurons contribute to a decline in hippocampal-dependent spatial learning. In the current study, confocal stereology was used to quantify cholinergic (choline acetyltransferase [ChAT] immunopositive) neurons, GABAergic projection (glutamic decarboxylase 67 [GAD67] immunopositive) neurons, and total (neuronal nuclei [NeuN] immunopositive) neurons in the rostral basal forebrain of young and aged rats that were first characterized on a spatial learning task. ChAT immunopositive neurons were significantly but modestly reduced in aged rats. Although ChAT immunopositive neuron number was strongly correlated with spatial learning abilities among young rats, the reduction of ChAT immunopositive neurons was not associated with impaired spatial learning in aged rats. In contrast, the number of GAD67 immunopositive neurons was robustly and selectively elevated in aged rats that exhibited impaired spatial learning. Interestingly, the total number of rostral basal forebrain neurons was comparable in young and aged rats, regardless of their cognitive status. These data demonstrate differential effects of age on phenotypically distinct rostral basal forebrain projection neurons, and implicate dysregulated cholinergic and GABAergic septohippocampal circuitry in age-related mnemonic decline.     

Amelioration of cholinergic neurons dysfunction in aged rats depends on the continuous supply of NGF

The present study was designed to examine whether NGF-induced improvement in morphology of senile basal forebrain cholinergic neurons persist after discontinuation of NGF treatment. Trophic effect of continuous intraventricular infusion of NGF was tested in the 4- and 28 months old male Wistar rats immediately after cessation of NGF and 3 or 6 weeks after termination of treatment. Immunohistochemical procedure for ChAT, TrkA, and p75(NTR) receptor has been applied to identify cholinergic cells in the basal forebrain structures. Using the quantitative image analyzer, morphometric and densitometric parameters of cholinergic cells were measured. In untreated 28-month-old rats a reduction in the number, size and intensity of staining of cholinergic neurons was observed in all basal forebrain structures. NGF significantly improved morphological parameters of ChAT- and TrkA-positive cells in aged rats. In 28-month-old rats tested within 3 and 6 weeks after discontinuation of infusion a renewed progressive deterioration of cholinergic phenotype of basal forebrain neurons was observed when compared with the NGF-treated immediately tested group. The parallel staining for p75(NTR) revealed normal morphology of the basal forebrain neurons, despite of the age of rats or the NGF treatment. Analysis of Nissl stained sections also showed that 28-month-old rats did not display significant losses of neurons in the basal forebrain when compared with the young animals. These findings demonstrate that senile impairment of cholinergic neurons is induced by a loss of cholinergic phenotype rather than an acute degeneration of cell bodies. NGF may be beneficial in enhancing cholinergic neurochemical parameters, but the protective effects seem to be dependent on the continuous supply of NGF.     

Cholinergic influences on hippocampal glucose metabolism

2-Deoxy-D-[3H]glucose autoradiography was employed to investigate the effects of acute cholinergic manipulations on hippocampal glucose metabolism. In general, manipulations designed to reduce cholinergic activity (medial septal ablation, atropine treatment) reduced hippocampal glucose metabolism. Maximal decrements were found in the terminal fields of the septohippocampal projection after medial septal lesions, while maximal deficits after atropine treatment correlated with muscarinic receptor binding. Electrical stimulation of the medial septum resulted in increased glucose utilization in some terminal fields of the septohippocampal projection and decreased utilization in the terminal fields of the perforant pathway. Our data clearly indicate that acute alterations in cholinergic activity can affect hippocampal glucose metabolism but the distribution, direction and degree of these changes is dependent on the specific treatment.     

Presynaptic cholinergic mechanisms in brain of aged rats with memory impairments

Presynaptic cholinergic mechanisms were investigated in various brain regions of aged Fisher 344 rats with documented 24 hr retention deficits measured in a single-trial passive avoidance task. Sodium-dependent high affinity choline uptake was found to be decreased by 22% in hippocampus of 23–26 month old animals as compared to 6 month old controls. Prior depolarization of hippocampal or cortical synaptosomes with K⁺ resulted in stimulation of choline uptake which was similar in aged rats and young controls. No age-related differences were observed either in hippocampal, cortical, striatal acetylcholine or choline concentrations, or in the activity of choline acetyltransferase in hippocampus. Synthesis of acetylcholine in hippocampal and cortical slices under basal conditions, as well as under K⁺-stimulated conditions, did not differ in the two age groups examined. These neurochemical findings are consistent with an age-related decrease in hippocampal cholinergic neuronal activity without an actual loss in cholinergic neuron number. It is further suggested that this reduction in cholinergic neuronal activity may be related to the deficit in cognitive performance observed in aged Fisher rats.