Brain cholinergic dysfunction and memory in aged rats

Age related alterations in mnemonic ability and in the functional status of muscarinic receptors were evaluated and compared to biochemical measures of pre- and post-synaptic cholinergic functioning. Retention of a single trial passive avoidance task was considerably disturbed as a function of aging. The functional status of muscarinic receptors, as measured by the ability of microiontophoretically applied acetylcholine to stimulate the firing of hippocampal pyramidal cells, was similarly disturbed in aged rats. A small, but significant decrease in muscarinic receptors was detected in the dorsal hippocampi of these same aged rats, while choline acetyltransferase activity did not change. When considered with prior psychopharmacological studies, these data suggest that specific muscarinic receptor impairments may play a critical role in the memory disturbances associated with old age.     

Morphologic alterations of cholinergic processes in the neocortex of aged rats

In the present study we observed enlarged cholinergic processes in the neocortex of aged Fischer 344 rats. These swollen ChAT-positive profiles appeared either as a single axon enlargement or, in many instances, the bulbous processes coalesced to form grape-like clusters of immunoreactivity. The latter structures looked similar to the immunoreactive profiles observed in the cortex of patients with Alzheimer's disease and in the rat septum following fimbria-fornix transection. Together, these data provide evidence that morphologic changes occur within processes of cholinergic neurons in the aged rat. Moreover, the similarity in appearance between the axonal alterations in the aged rat and in patients with Alzheimer's disease suggests a common pathologic process.     

Muscarinic cholinergic receptor subtypes in the hippocampus of aged rats.

In spite of the suggestion of impaired muscarinic function in adult-onset cognitive disorders, data on the expression of muscarinic receptors in the hippocampus as a function of age are inconsistent. One reason may be that the majority of investigations were unable to differentiate the five brain muscarinic receptors subtypes. In this study, using a protocol based on a combination of both kinetic and equilibrium binding approaches, we have assessed the expression and the density of M1-M5 muscarinic cholinergic receptors in the hippocampus of Fisher 344 rats aged 6, 15 and 22 months. An age-related decrease of the density of M1 receptor was found in pyramidal neurons of the CA1 subfield. In this area, other subtypes of muscarinic receptors were unchanged with the exception of a loss of M2 receptor in the radial layer. In the CA3 subfield, receptor changes involved M2, M3 and M5 subtypes, whereas in the dentate gyrus, the main changes affected M1 and M2 receptors of the granular layer and M2 and M3 receptors of the molecular layer. The above findings indicate that analysis of age-related changes of different muscarinic cholinergic receptors might represent a useful contribution to identifying the basis of cholinergic neurotransmission impairment in adult-onset cognitive dysfunction.     

Distinct manifestations of executive dysfunction in aged rats

Different components of executive function such as working memory, attention, and cognitive flexibility can be dissociated behaviorally and mechanistically; however, the within-subject influences of normal aging on different aspects of executive function remain ill-defined. To better define these relationships, young adult and aged male F344 rats were cross-characterized on an attentional set-shifting task that assesses cognitive flexibility and a delayed response task that assesses working memory. Across tasks, aged rats were impaired relative to young; however, there was significant variability in individual performance within the aged cohort. Notably, performance on the set-shifting task and performance at long delays on the delayed response task were inversely related among aged rats. Additional experiments showed no relationship between aged rats' performance on the set-shifting task and performance on a hippocampal-dependent spatial reference memory task. These data indicate that normal aging can produce distinct manifestations of executive dysfunction, and support the need to better understand the unique mechanisms contributing to different forms of prefrontal cortical-supported executive decline across the lifespan.     

植物雌激素对去卵巢大鼠基底前脑胆碱能神经元的影响

目的：观察植物雌激素对去卵巢大鼠基底前脑胆碱能神经元表达的影响，探讨植物雌激素在中枢神经系统的保护作用及机制。方法：采用乙酰胆碱转移酶(ChAT)免疫组织化学ABC法，观察去卵巢大鼠5w后各组基底前脑内侧隔核(MS)，斜角带垂直支(VDB)胆碱能神经元的数目。结果：与去卵巢对照组相比，植物雌激素用药组、雌激素用药组的内侧隔核，斜角带垂直支胆碱能神经元数目明显升高(P＜0．05)，与假手术组差别不明显。结论：本研究提示植物雌激素能明显增加去卵巢大鼠基底前脑胆碱能神经元的表达，从而对中枢神经系统退行性病变起保护作用，并有望预防和治疗老年性痴呆。     

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.     

Genistein对去卵巢大鼠基底前脑胆碱能神经元影响的体内研究

本文旨在研究染料木素(genistein)对去卵巢大鼠基底前脑胆碱能神经元的影响。雌性大鼠双侧卵巢切除2周后用genistein和雌激素替代治疗1周。称子宫重量以确定手术是否成功及雌二醇(E2)的治疗是否有效。用免疫组化染色、RT-PCR和Westernblot等方法对胆碱能神经元数量、ChAT基因和蛋白的表达量进行检测。结果显示:去卵巢3周后子宫变轻,雌激素替代治疗能增加去卵巢子宫的重量,而genistein替代治疗对去卵巢子宫的重量影响不明显;去卵巢3周后,内侧隔核(MS)和斜角带垂直臂核(VDB)内的胆碱能神经元数量、ChAT基因和蛋白的表达量均明显减少,雌激素和genistein替代治疗后能显著增加去卵巢大鼠MS和VDB内的胆碱能神经元数量、ChAT基因和蛋白的表达量。本研究结果提示:genistein对去卵巢大鼠基底前脑胆碱能神经元具有类似雌激素样神经保护作用,而对子宫影响不明显。     

Effect of chronic inflammation on dorsal horn nociceptive neurons in aged rats

To elucidate the effect of chronic inflammation on spinal nociceptive neurons in the elderly, we compared nocifensive behavior, peripheral inflammatory responses, and spinal dorsal horn neuronal activities between the aged (29-34 mo) and adult (7-12 mo) male rats after injection of complete Freund's adjuvant (CFA) into the hind paw. Aged rats exhibited a significantly lower mechanical paw withdrawal threshold before inflammation. However, after CFA injection mechanical allodynia developed in both adult and aged rats after CFA injection. The changes of foot temperature and thickness after CFA injection were greater and lasted longer in aged than in adult rats. Sets of 124 wide dynamic range (WDR) neurons (aged: 59, adult: 65) and 26 nociceptive specific (NS) neurons (aged: 13, adult: 13) were recorded from the lumber spinal dorsal horn. NS neurons from the inflamed adult rats showed significantly higher responses to noxious mechanical stimulation than those in aged rats, whereas WDR neurons from inflamed adult and aged rats were similar. Background activity of WDR neurons from the adult rats increased after CFA, whereas WDR neurons of aged rats and NS neurons from either group were not. The afterdischarge followed by noxious mechanical stimulation was significantly greater for WDR neurons in both adult and aged rats, whereas no significant differences were observed in NS neurons. Two days after CFA injection, Fos expression increased similarly in aged and adult rats. Thus the aged rats showed enhanced peripheral inflammatory responses to CFA injection with only a slight change in dorsal horn neuronal activity. Together with our previous finding that nociceptive neurons in aged rats exhibit hyperexcitability, these results suggest that the dorsal horn nociceptive system becomes sensitized with advancing age and its excitability cannot be further increased by inflammation.     

Effect of nitric oxide on auditory cortical neurons of aged rats

Age-related changes in the effects of nitric oxide (NO) on neurons of the auditory cortex have not been determined. We therefore evaluated the anatomical changes and neurophysiological characteristics of these neurons in rats as a function of age. The numbers of cresyl violet stained cells, the numbers and areas of NADPH-d-positive neuronal cell bodies, and their optical density, were measured in Sprague-Dawley rats aged 24 months (aged group) and 4 months (control group). The modulatory effects of NO on K⁺ currents of acutely isolated rat auditory cortical neurons were also assessed. There were no between-group differences in the distribution patterns of glial cells and neurons, or in the numbers and areas of NADPH-d-positive neuronal cell bodies. However, the optical density of NADPH-d-positive neuronal cell bodies was significantly greater in the aged group than in the control group. In addition, voltage-gated K⁺ currents of rat auditory cortical neurons were activated by increased levels of NO. As activation of the K⁺ current likely suppresses neuronal excitability, age-associated increases in NO production can hinder the function of the acoustic center by inhibiting neuron excitability.     

NGF restores decrease in catalase and increases glutathione peroxidase activity in the brain of aged rats.

The effects of subchronic administration of nerve growth factor (NGF) into the lateral ventricle on catalase and selenium-dependent glutathione-peroxidase (GSH-Px) activity in several areas of the brain in 3-, 12- and 24-month-old rats were studied. NGF given daily (1 microgram for 28 consecutive days) produced in all brain areas studied a significant increase in catalase activity in 12- and 24-month-old rats. The most important finding was a complete restoration in 12- and 24-month-old rats of catalase activity to levels similar to those occurring in young (3-month-old) rats. In addition, NGF produced in comparison to 3-month-old rats and to same age vehicle-treated rats a significant increase in selenium-dependent GSH-Px in all the brain areas studied in 12- and 24-month-old animals, whereas selenium-independent GSH-Px was unaffected. In conclusion, the present results show that long-term administration of NGF into the lateral ventricle significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of hydrogen peroxide.     

Septal cholinergic neurons suppress seizure development in hippocampal kindling in rats: comparison with noradrenergic neurons

Widespread lesions of forebrain cholinergic or noradrenergic projections by intraventricular administration of 192 IgG-saporin or 6-hydroxydopamine, respectively, accelerate kindling epileptogenesis. Here we demonstrate both quantitative and qualitative differences between the two lesions in their effects on hippocampal kindling in rats. Epileptogenesis was significantly faster after noradrenergic as compared to cholinergic denervation, and when both lesions were combined, kindling development resembled that in animals with 6-hydroxydopamine lesion alone. Furthermore, whereas the 192 IgG-saporin lesion promoted the development only of the early stages of kindling, administration of 6-hydroxydopamine or both neurotoxins accelerated the late stages also. To investigate the contribution of different subparts of the basal forebrain cholinergic system to its seizure-suppressant action in hippocampal kindling, 192 IgG-saporin was injected into medial septum/vertical limb of the diagonal band of Broca or nucleus basalis magnocellularis, leading to selective hippocampal or cortical cholinergic deafferentation, respectively. The denervation of the hippocampus facilitated kindling similar to the extensive lesion caused by intraventricular 192 IgG-saporin, whereas the cortical lesion had no effect. These results indicate that although both noradrenergic and cholinergic projections to the forebrain exert powerful inhibitory effects on hippocampal kindling epileptogenesis, the action of the cholinergic system is less pronounced and occurs specifically prior to seizure generalization. In contrast, noradrenergic neurons inhibit the development of both focal and generalized seizures. The septo-hippocampal neurons are responsible for the antiepileptogenic effect of the cholinergic system in hippocampal kindling, whereas the cortical projection is not significantly involved. Conversely, we have previously shown [Ferencz I. et al. (2000) Eur. J. Neurosci., 12, 2107-2116] that seizure-suppression in amygdala kindling is exerted through the cortical and not the hippocampal cholinergic projection. This shows that, depending on the location of the primary epileptic focus, i.e. the site of stimulation, basal forebrain cholinergic neurons operate through different subsystems to counteract seizure development in kindling.     

Resistance training improves femoral artery endothelial dysfunction in aged rats

Although endurance exercise improves age-associated endothelial dysfunction, few studies have examined the effects of resistance training and the potential molecular mechanisms involved in altering vascular reactivity with age. Young (9 months) and aged (20 months) male, Fisher 344 rats were divided into four groups: Young Sedentary (YS, n = 14), Young Trained (YT, n = 10), Aged Sedentary (AS, n = 12), and Aged Trained (AT, n = 10). Resistance training consisted of climbing a 1 m wire ladder, at an 85° angle, 3 days/week for 6 weeks with increasing weight added to the tail. Endothelial function in femoral arteries was determined by constructing acetylcholine dose–response curves on a wire myograph. Femoral artery phospho-Ser1179-eNOS, eNOS and Hsp90 expression were evaluated by Western blot. Acetylcholine-induced vasorelaxation was significantly (P < 0.05) impaired in AS compared to YS and YT but not AT compared to YS and YT. Phospho-Ser1179-eNOS and eNOS were elevated (P < 0.05) in aged animals but not changed with resistance training. Resistance training increased Hsp90 levels in both young and old animals. Therefore, resistance training improves age-associated endothelial dysfunction in femoral arteries without changes in eNOS phosphorylation and expression. Increased Hsp90 expression, a regulator of eNOS activity and coupling, suggests a potential mechanism for this improvement.     

雌激素对去卵巢大鼠基底前脑胆碱能神经元及一氧化氮合酶阳性神经元的影响

目的　探讨雌激素补充治疗对去卵巢大鼠胆碱能神经元及一氧化氮合酶 (NOS)阳性神经元表达的影响及剂量效应关系。方法　 2 0只去卵巢SD大鼠分成 4个不同剂量组 :0 μg(对照组 )、2 0 μg(0 0 8mg/kg)、5 0 μg(0 2 0mg/kg)、10 0 μg(0 4 0mg/kg)组 ;1周后 ,用乙酰胆碱转移酶 (ChAT)免疫化学方法及尼克酰胺腺嘌呤二核苷酸黄递酶 (NADPH d)组织化学方法研究。结果　NOS阳性神经元在内侧隔核 (MS) ,其数目在各组间无明显差异 (P >0 0 5 )。在斜角带垂直支 (VDB) ,5 0 μg剂量组与对照组相比有明显差异 (P <0 0 5 ) ;ChAT阳性神经元在内侧隔核 (MS) ,其 2 0 μg、5 0 μg剂量组数目出现剂量递增效应 ,与对照组比较有明显差异 (P <0 0 5 ) ,但 10 0 μg剂量组与对照组比较没有明显差异 (P >0 0 5 ) ,同时 5 0 μg与 10 0 μg剂量组比较有明显差异 (P <0 0 5 )。ChAT阳性神经元在斜角带水平支 (HDB) ,各组间均无明显差异 (P >0 0 5 )。结论　雌激素补充治疗能选择性影响基底前脑各亚区NOS和胆碱能神经元 ,并有可能影响学习和记忆能力。     

Responsiveness of sympathetic and sensory iridial nerves to NGF treatment in young and aged rats

Altered neuronal responses to trophic factors may play a role in neuronal maintenance in adulthood and may also be involved in neuronal atrophy in old age. We have investigated this issue in the rat iris, studying responsiveness of sympathetic and sensory iridial nerves to a range of NGF concentrations in mature and aged rats. We show here that growth responses of sensory nerves to NGF, as measured by quantitative immunohistochemistry and image analysis, were unchanged in old rats. In contrast, there was a small but significant reduction in responsiveness of aged sympathetic neurons. The shapes of the dose-response curves for sensory and sympathetic neurons were different, with a larger response over a narrower range of concentrations in sensory neurons. Lower levels of p75 immunoreactivity were observed in iridial nerves from old compared to young rats. NGF treatment had no effect on receptor staining in young rats but restored 'young' levels of p75 staining in old rats. Our results do not support the hypothesis of a primary role for NGF in maintenance or atrophy of nerves in ageing.     

Profound effects of combining choline and piracetam on memory enhancement and cholinergic function in aged rats

In an attempt to gain some insight into possible approaches to reducing age-related memory disturbances, aged Fischer 344 rats were administered either vehicle, choline, piracetam or a combination of choline or piracetam. Animals in each group were tested behaviorally for retention of a one trial passive avoidance task, and biochemically to determine changes in choline and acetylcholine levels in hippocampus, cortex and striatum. Previous research has shown that rats of this strain suffer severe age-related deficits on this passive avoidance task and that memory disturbances are at least partially responsible. Those subjects given only choline (100 mg/kg) did not differ on the behavioral task from control animals administered vehicle. Rats given piracetam (100 mg/kg) performed slightly better than control rats (p<0.05), but rats given the piracetam/choline combination (100 mg/kg of each) exhibited retention scores several times better than those given piracetam alone. In a second study, it was shown that twice the dose of piracetam (200 mg/kg) or choline (200 mg/kg) alone, still did not enhance retention nearly as well as when piracetam and choline (100 mg/kg of each) were administered together. Further, repeated administration (1 week) of the piracetam/choline combination was superior to acute injections. Regional determinations of choline and acetylcholine revealed interesting differences between treatments and brain area. Although choline administration raised choline content about 50% in striatum and cortex, changes in acetylcholine levels were much more subtle (only 6–10%). No significant changes following choline administration were observed in the hippocampus. However, piracetam alone markedly increased choline content in hippocampus (88%) and tended to decrease acetylcholine levels (19%). No measurable changes in striatum or cortex were observed following piracetam administration. The combination of choline and piracetam did not potentiate the effects seen with either drug alone, and in certain cases the effects were much less pronounced under the drug combination. These data were discussed as they relate to possible effects of choline and piracetam on cholinergic transmission and other neuronal function, and how these effects may reduce specific memory disturbances in aged subjects. The results of these studies demonstrate that the effects of combining choline and piracetam are quite different than those obtained with either drug alone and support the notion that in order to achieve substantial efficacy in aged subjects it may be necessary to reduce multiple, interactive neurochemical dysfunctions in the brain, or affect activity in more than one parameter of a deficient metabolic pathway.     

Quantitative distribution of AChE-positive neurons in the hippocampus of young and aged rats

Summary Acetylcholinesterase (AChE)-positive neurons were counted in the different layers of the rostral (septal) third, the middle third and the caudal (temporal) third of the hippocampus from 3 month (young) and 27 month old rats (aged) using AChE stained cryostat sections. The rats were treated with 3 and 2.5 mg of diisopropylphosphofluoridate/kg body weight, respectively 3 h before sacrifice. The study showed — 1) a high numerical density of AChE-positive neurons (10.9 to 18.9 perikarya/mm²) in the hilus (fascia dentata), the str. oriens/pyramidale of CA1 and the subiculum, a particularly low density (< 0.1 perikarya/mm²) in the str. granulosum and moleculare of the dentate area; — 2) a significant (p < 0.05) linear increase of the numerical density in most of the hippocampal layers from the rostral to the caudal pole; — 3) no significant differences between young and aged animals; — and 4) a higher sensitivity to DFP-treatment in aged than in young animals. The distribution of AChE-positive neurons corresponds with the distribution of somatostatin-immunoreactive neurons described in the literature. A modulatory effect on neurotransmission is discussed as a possible function of the AChE in peptidergic neurons.     

Paradoxical sleep insomnia and decreased cholinergic neurons after myocardial infarction in rats

Study Objectives:

Acute myocardial infarction (MI) is followed, within a few hours, by neuronal loss in the central nervous system (CNS), including the limbic system, the hypothalamus, and the brainstem. Sleep before and after MI was investigated in the first experiment. In a parallel experiment, 2 weeks after MI, we quantified brainstem cholinergic neurons known to control paradoxical sleep (PS).

Measurements and Results:

Data were obtained from 28 adult male Sprague-Dawley rats weighing 350-375 g and maintained under a 12-12 light-dark cycle in 2 experiments on 16 and 12 rats, respectively. The 16 animals in the first experiment were implanted with chronic electroencephalographic (EEG) and electromyographic (EMG) electrodes. A week after surgery, these animals were habituated for 2 days to the recording equipment, and baseline sleep was charted for 24 h. The next morning, MI was induced in 8 rats by occluding the left anterior descending coronary artery for 40 min. The remaining 8 rats served as sham-operated controls. Sleep was recorded again 2 weeks after MI. The number of choline acetyltransferase (ChAT)-positive neurons was counted in the second, parallel experiment on 6 MI and 6 sham rats.Compared to the sham controls, MI rats displayed longer latency to sleep onset, shorter latency to paradoxical sleep (PS), and curtailed PS duration. The number of ChAT-positive neurons in the pedunculopontine tegmentum (PPT) area of MI rats was significantly decreased compared to the sham controls, while the number of laterodorsal tegmentum (LDT) cholinergic neurons was not different.

Conclusion:

Acute MI is accompanied, within 2 weeks, by PS-specific insomnia that can be explained, at least partly, by a specific loss of cholinergic neurons in an area known to control PS.

Citation:

Bah TM; Laplante F; Wann BP; Sullivan R; Rousseau G; Godbout R. Paradoxical sleep insomnia and decreased cholinergic neurons after myocardial infarction in rats. SLEEP 2010;33(12):1703-1710.     

单侧磨牙缺失对老年学习记忆减退大鼠基底前脑胆碱能系统的影响

目的:探讨单侧缺牙对不同学习记忆能力老年大鼠基底前脑胆碱能系统的影响。方法:用Morris水迷宫筛选出老年记忆减退鼠和老年记忆正常鼠,拔除单侧磨牙后2个月,用免疫组化和组织化学染色观察对其斜角带核垂直支胆碱能神经元和海马CA1区、前额皮质胆碱能纤维密度的影响。结果:斜角带核垂直支ChAT阳性细胞数和海马CA1区、前额皮质AChE阳性纤维密度在老年记忆减退鼠中,拔牙组较对照组(未拔牙)明显下降;海马CA1区AChE阳性纤维密度在老年记忆正常鼠中,拔牙组较对照组明显下降。结论:单侧磨牙缺失可加速老年学习记忆减退鼠基底前脑胆碱能系统的损害。