Enlarged cholinergic forebrain neurons and improved spatial learning in p75 knockout mice

The p75 low affinity neurotrophin receptor (p75) can induce apoptosis in various neuronal and glial cell types. Because p75 is expressed in the cholinergic neurons of the basal forebrain, p75 knockout mice may be expected to show an increased number of neurons in this region. Previous studies, however, have produced conflicting results, suggesting that genetic background and choice of control mice are critical. To try to clarify the conflicting results from previous reports, we undertook a further study of the basal forebrain in p75 knockout mice, paying particular attention to the use of genetically valid controls. The genetic backgrounds of p75 knockout and control mice used in this study were identical at 95% of loci. There was a small decrease in the number of cholinergic basal forebrain neurons in p75 knockout mice at four months of age compared with controls. This difference was no longer apparent at 15 months due to a reduction in numbers in control mice between the ages of 4 and 15 months. Cholinergic cell size in the basal forebrain was markedly increased in p75 knockout mice compared with controls. Spatial learning performance was consistently better in p75 knockout mice than in controls, and did not show any deterioration with age. The results indicate that p75 exerts a negative influence on the size of cholinergic forebrain neurons, but little effect on neuronal numbers. The markedly better spatial learning suggests that the function, as well as the size, of cholinergic neurons is negatively modulated by p75.     

额叶皮质损伤对大鼠基底前脑胆碱能神经元的影响

本实验探讨了前额叶皮质局限性损伤对大鼠学习、记忆功能及基底前脑胆碱能神经元的影响。用外科手术造成大鼠一侧前额叶皮质局限性损伤后不同时间、用Ｙ型迷宫检测学习、记忆功能、用组织化学技术检测基底前脑含乙酰胆碱酸酶（ＡChE)活性神经元。实验观察到前额叶皮质损伤后１周,动物学习,记忆功能有所障碍,损伤同侧的基底前脑胆碱能神经元有所减少,但均无统计学意义,损伤后２,３,４周,动物学习、记忆障碍明显,损伤同侧基底前脑胆碱能神经元明显减少（Ｐ＜0.05),且两者变化相平行。结果表明单侧前额叶皮质局限性损伤不仅可引起动物学习、记忆功能障碍,且可引起同侧基底前脑胆碱能神经元丢失,且两者发展相平行,提示基底前脑胆碱能神经元逆行性变性在动物额叶皮质损伤引起的学习、记忆障碍中起作用。     

The cholinergic system and spatial learning

Acetlylcholine (ACh) in the central nervous system is critical for a multitude of functions. Here, we concentrate on declarative memory in humans, and its equivalent episodic-like memory in rodents and highlight current understanding of cholinergic system in these processes. Spatial memory formation represents a simple form of episodic-like memory in rodents that engages the basal forebrain cholinergic system and its target structures. In these, ACh exerts numerous functions.

(1)

During spatial acquisition learning, ACh efflux into the extracellular space is immediate in hippocampus and cortex; during consolidation of spatial reference memory, ACh levels are low. These requirements explain why ACh receptor blockade during acquisition blocks memory formation, and it is also consonant with the notion that an unspecific enhancement of cholinergic activity during consolidation is detrimental to memory formation.

(2)

Working and short-term memory for spatial locations engages the nucleus basalis - prefrontal cortex ACh system. ACh activity is trial related and maintained for some time post-training.

(3)

Striatal cholinergic activity is increased during stimulus-response learning and behavioural flexibility (reversal learning, extinction) providing a possible switch between different behavioural strategies.

(4)

At present, there is no clear difference between muscarinic and nicotinergic systems with respect to spatial learning. Antagonists of the respective receptors impair memory formation, agonists can reverse these deficits or may, under specific conditions act more like a general cognitive enhancers by way of improving attention.

(5)

Data reviewed here do not provide conclusive evidence for muscarinic or nicotinic receptors presenting as novel therapeutic targets, and there is no clear indication for ACh derived novel biomarkers for translational medicine.

Unresolved and contradictory results are highlighted and discussed.     

GABAergic input to cholinergic forebrain neurons: an ultrastructural study using retrograde tracing of HRP and double immunolabeling

Amygdalopetal cholinergic neurons in the ventral pallidum were identified by combining choline acetyltransferase (ChAT) immunohistochemistry with retrograde tracing of horseradish peroxidase (HRP) following injections of the tracer in the basolateral amygdaloid nucleus. Although ChAT-positive terminals were identified in the ventral pallidum, they were never seen in contact with either immunonegative or ChAT-positive amygdalopetal neurons. In material, in which immunostaining against glutamic acid decarboxylase (GAD), the synthesizing enzyme for GABA was combined with retrograde tracing of HRP from the basolateral amygdaloid nucleus, GAD-positive terminals were seen to contact immunonegative amygdalopetal neurons. In addition, when sections of the rostral forebrain were processed, first to preserve and identify the transported HRP, and then were sequentially tested for both ChAT and GAD immunohistochemistry with the immunoperoxidase reaction for both tissue antigens, GAD-immunopositive terminals were seen to make synaptic contacts with cholinergic amygdalopetal neurons. These results suggest that amygdalopetal, presumably cholinergic, neurons receive GAD-positive terminals. In separate experiments using immunoperoxidase for ChAT and ferritin-avidin for GAD labeling, we confirmed the presence of GAD-containing terminals on cholinergic neurons. In addition, cholinergic terminals were seen in synaptic contact with GAD-positive cell bodies. These morphological studies suggest that direct GABAergic-cholinergic and cholinergic-GABAergic interactions take place in the rostral forebrain.     

Basic fibroblast growth factor (bFGF) increases the survival of embryonic and postnatal basal forebrain cholinergic neurons in primary culture

Basic fibroblast growth factor (bFGF) is found in high concentrations in the mammalian central nervous system. It is a mitogen for glia and it influences the development and survival of specific populations of neurons. In this study, we investigated the effect of various concentrations of bFGF on the survival of embryonic and postnatal cholinergic basal forebrain neurons plated at low and high density in the presence and absence of glia. We observed that 50 and 100 ng/ml of bFGF increased the survival of embryonic cholinergic neurons plated at high density. This effect was observed only in the presence of glia. Lower concentrations of 10 and 20 ng/ml had no effect on cholinergic neuronal survival. The number of GFAP (glial fibrillary acidic protein)-positive cells in high-density embryonic cultures was increased by all concentrations of bFGF. In low-density embryonic cultures, an increase in cholinergic neuron survival was observed at concentrations ranging from 20 to 100 ng/ml. The number of GFAP-positive cells in low-density cultures was also increased by all concentrations of bFGF. Similar to low-density embryonic cultures, the survival of cholinergic neurons from postnatal day 2 cultures was significantly increased in the presence of glia at concentrations of 20, 50 and 100 ng/ml of bFGF. Postnatal glia was affected by all concentrations of bFGF, as was observed in embryonic cultures. This study indicates that high concentrations of bFGF can influence cholinergic neuronal survival by stimulating and increasing glia, which may produce factor(s) that are necessary for cholinergic neuron survival.     

Spatial learning and memory, maze running strategies and cholinergic mechanisms in two inbred strains of mice

The acquisition process of the radial maze task was studied in two inbred strains of mice, C57BL/6 and DBA/2. A quantitative and qualitative evaluation of performance was performed and the pretest level of activity was measured. The results showed a significant correlation between activity and performance since the highly active C57BL/6 mice exhibited better performance of the radial maze task than the less active DBA/2 mice. Moreover, for correct trials, strain-dependent maze-running strategies were observed: while both strains displayed about the same percentage of clockwise and spatial strategies, it was observed that among the spatial strategies C57BL/6 used a larger number of different correct solutions. Subsequently, the effect of scopolamine administration on working memory processes was assessed in sequential and discrete trials. A different reactivity of each strain to anti-cholinergic treatment was found in discrete trials since only DBA/2 mice were impaired. The effect of scopolamine is discussed in relation to the different models of information processing involved in learning and memorizing the experimental rule.     

Age-dependent loss of cholinergic neurons in learning and memory-related brain regions and impaired learning in SAMP8 mice with trigeminal nerve damage

The tooth belongs to the trigeminal sensory pathway. Dental damage has been associated with impairments in the central nervous system that may be mediated by injury to the trigeminal nerve. In the present study, we investigated the effects of damage to the inferior alveolar nerve, an important peripheral nerve in the trigeminal sensory pathway, on learning and memory be-haviors and structural changes in related brain regions, in a mouse model of Alzheimer’s disease. Inferior alveolar nerve transection or sham surgery was performed in middle-aged （4-month-old） or elderly （7-month-old） senescence-accelerated mouse prone 8 （SAMP8） mice. When the middle-aged mice reached 8 months （middle-aged group 1） or 11 months （middle-aged group 2）, and the elderly group reached 11 months, step-down passive avoidance and Y-maze tests of learn-ing and memory were performed, and the cholinergic system was examined in the hippocampus （Nissl staining and acetylcholinesterase histochemistry） and basal forebrain （choline acetyltrans-ferase immunohistochemistry）. In the elderly group, animals that underwent nerve transection had fewer pyramidal neurons in the hippocampal CA1 and CA3 regions, fewer cholinergic ifbers in the CA1 and dentate gyrus, and fewer cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band, compared with sham-operated animals, as well as showing impairments in learning and memory. Conversely, no signiifcant differences in histology or be-havior were observed between middle-aged group 1 or group 2 transected mice and age-matched sham-operated mice. The present ifndings suggest that trigeminal nerve damage in old age, but not middle age, can induce degeneration of the septal-hippocampal cholinergic system and loss of hippocampal pyramidal neurons, and ultimately impair learning ability. Our results highlight the importance of active treatment of trigeminal nerve damage in elderly patients and those with Alzheimer’s disease, and indicate that tooth extract     

Loss of cholinergic phenotype in basal forebrain coincides with cognitive decline in a mouse model of Down's syndrome

Mice with segmental trisomy of chromosome 16 (Ts65Dn) have been used as a model for Down's syndrome. These mice are born with a normal density of basal forebrain cholinergic neurons but, like patients with Down's syndrome, undergo a significant deterioration of these neurons later in life. The time course for this degeneration of cholinergic neurons has not been studied, nor is it known if it correlates with the progressive memory and learning deficits described. Ts65Dn mice that were 4, 6, 8, and 10 months old were sacrificed for evaluation of basal forebrain morphology. Separate groups of mice were tested on visual or spatial discrimination learning and reversal. We found no alterations in cholinergic markers in 4-month-old Ts65Dn mice, but thereafter a progressive decline in density of cholinergic neurons, as well as significant shrinkage of cell body size, was seen. A parallel loss of staining for the high-affinity nerve growth factor receptor, trkA, was observed at all time points, suggesting a biological mechanism for the cell loss involving this growth factor. Other than transient difficulty in learning the task requirements, there was no impairment of trisomic mice on visual discrimination learning and reversal, whereas spatial learning and reversal showed significant deficits, particularly in the mice over 6 months of age. Thus, the loss of ChAT-immunoreactive neurons in the basal forebrain was coupled with simultaneous deficits in behavioral flexibility on a spatial task occurring for the first time around 6 months of age. These findings suggest that the loss of cholinergic function and the simultaneous decrease in trkA immunoreactivity in basal forebrain may directly correlate with cognitive impairment in the Ts65Dn mouse Copyright 2000 Academic Press.     

Strain-dependent variations in the number of forebrain cholinergic neurons

The morphological organization of putatively cholinergic neurons was studied in the forebrain of two inbred mouse strains (C57BL/6 and DBA/2) by means of acetylcholinesterase pharmacohistochemistry. In both strains, putatively cholinergic perikarya were seen in the caudato-putamen, medial septum, diagonal band, and basal nucleus of Meynert: in all these regions, their distribution was similar in both strains, but their density was significantly higher (from 20 to 32%) in DBA/2 mice. The present data demonstrate the existence of genetically determined differences in the organization of forebrain cholinergic systems.     

Topographical projection of cholinergic neurons in the basal forebrain to the cingulate cortex in the rat

The cholinergic innervation of the rat's posterior cingulate cortex (Brodmann's area 29) was studied using acetylcholinesterase (AChE) histochemistry. Electrolytic lesion of the ipsilateral medial septum and diagonal band region (MS-DB) reduced the diffuse AChE staining in layers I, II, III and V of the cingulate cortex. Kainic acid lesion of the ipsilateral globus pallidus and substantia innominata area (GP-SI) abolished the dense band of AChE stain in layer IV, with small reductions of AChE stain in other layers. The results indicate that the medial cholinergic pathway from MS-DB terminates diffusely in layers I, II, III and V while the lateral cholinergic pathway from the GP-SI predominantly ends in layer IV of the posterior cingulate cortex.     

Intracerebroventricular microinjection of kaempferol on memory retention of passive avoidance learning in rats: involvement of cholinergic mechanism(s)

Abstract

Purpose of the study: Kaempferol (KM) is a flavonoid found in plant-derived foods and medicinal plants. Recently, it is well established that KM plays a protective role to develop Alzheimer's disease. The current study aimed at evaluating the effect of intracerebroventricular micro-injection of KM on memory retention of passive avoidance learning (MRPAM) and identifying the potentially related cholinergic mechanisms (ChMs) in rats.

Materials and methods: In the current study, male Wistar rats randomly divided into control, vehicle and KM (10, 20 and 40?μg/rat) groups. Moreover, MRPAM was evaluated by shuttle box test. The role of ChM was studied using non-selective and selective acetylcholine antagonists (scopolamine [SCN], 4-DAMP and methoctramine [MN], respectively) as well as pirenzepine (PZ) in combination with KM.

Results: The employment of KM (40?μg/rat) improved the SCN-induced memory impairment in MRPAM. Co-treatment with KM (40?μg/rat) plus 4-DAMP significantly increased the step-through latency (STL, P?<?0.05; 167?±?28?s) and decreased the total dark chamber (TDC, P?<?0.05; 121?±?31?s) compared with those of the 4-DAMP group (STL: 75?±?13?s; TDC: 178?±?46?s). Co-treatment with KM (40?μg/rat) plus PZ attenuated STL, and also increased TDC (P?<?0.01; 220?±?28?s) compared with those of the PZ group. Co-treatment with KM (10 and 20?μg/rat) and MN increased STL (P?<?0.05), and deceased TDC compared with those of the MN group (P?<?0.01).

Conclusions: Totally, the results of the present study showed that cholinergic system may be involved in improving effect of KM on SCN-induced memory impairment.     

Basal forebrain magnocellular neurons stain for nerve growth factor receptor: correlation with cholinergic cell bodies and effects of axotomy

Recent evidence has demonstrated the presence of nerve growth factor (NGF) in areas of the central nervous system characterized by cholinergic innervation. We report that a unique population of rat basal forebrain magnocellular neurons that project to the cortex and hippocampus are immunoreactive to a monoclonal antibody to the NGF receptor. Removal of target contact results in a time-dependent loss or shrinkage of cells in the basal forebrain that stain for NGF receptor and acetylcholinesterase, suggesting that under normal conditions, basal forebrain cholinergic neurons utilize NGF for trophic support.     

Threshold relationship between lesion extent of the cholinergic basal forebrain in the rat and working memory impairment in the radial maze

The cholinergic basal forebrain (CBF) degenerates in Alzheimer's Disease (AD), and the degree of this degeneration correlates with the degree of dementia. In the present study we have modeled this degeneration in the rat by injecting various doses of the highly selective immunotoxin 192 IgG-saporin (192-sap) into the ventricular system. The ability of 192-sap-treated rats to perform in a previously learned radial maze working memory task was then tested. We report here that 192-sap created lesions of the CBF and, to a lesser extent, cerebellar Purkinje cells in a dose-dependent fashion. Furthermore, we found that rats harboring lesions of the entire CBF greater than 75% had impaired spatial working memory in the radial maze. Correlational analysis of working memory impairment and lesion extent of the component parts of the CBF revealed that high-grade lesions of the hippocampal-projecting neurons of the CBF were not sufficient to impair working memory. Only rats with high-grade lesions of the hippocampal and cortical projecting neurons of the CBF had impaired working memory. These data are consistent with other 192-sap reports that found behavioral deficits only with high-grade CBF lesions and indicate that the relationship between CBF lesion extent and working memory impairment is a threshold relationship in which a high degree of neuronal loss can be tolerated without detectable consequences. Additionally, the data suggest that the CBF modulates spatial working memory via its connections to both the hippocampus and cortex.     

戊四氮点燃癫癎大鼠空间学习记忆改变及海马NR2B表达

目的观察戊四氮点燃癫癎大鼠空间学习记忆功能变化及海马NMDA2型受体(NR2)B亚单位(NR2B)表达,探讨二者的关系及PTZ致癎大鼠认知障碍发生的分子机制。方法采用戊四氮(PTZ)慢性癫癎(CE)模型,Y-迷宫对两组大鼠进行行为学检测,免疫组织化学方法观察两组大鼠海马CA3区NR2B表达的变化,反转录多聚酶链反应(RT-PCR)方法检测大鼠海马NR2B mRNA的表达。结果癫癎组大鼠空间学习记忆能力受损;其海马CA3区NR2B阳性细胞较对照组明显减少(P<0.01),同时伴有海马NR2B mRNA表达下降(P<0.01)。结论戊四氮点燃癫癎大鼠空间学习记忆受损可能与海马神经元NR2B的表达减少有关。     

Effects of estrogen on basal forebrain cholinergic neurons and spatial learning

Estrogen receptors are expressed in several areas of the brain associated with cognition, including the basal forebrain cholinergic nuclei, and numerous reports have described improvements in memory in response to estrogen supplementation. The relationship between estrogen's effects on the basal cholinergic system and improvements in cognitive function, however, are obscure. We therefore undertook a study to determine the effects of estrogen on several parameters of the cholinergic system in ovariectomized rats and measured the concomitant effects on performance in the Barnes maze, a test of spatial memory. Six weeks of estradiol treatment caused an increase in choline acetyltransferase activity throughout the projection fields of the basal forebrain, including the hippocampal formation (14%), olfactory bulb (30%), and cerebral cortex (35%). Estrogen treatment also caused an increase in cell soma size of cholinergic neurons in the horizontal diagonal limb of the band of Broca and in the basal nucleus of Meynert. There was no change in the number of neurons positive for p75(NTR), nor in the level of p75(NTR) expression per neuron. Barnes maze performance was markedly improved after estradiol treatment, reinforcing the view that estrogen has beneficial cognitive effects, particularly on spatial memory. The beneficial cognitive effect was likely mediated in part by stimulation of the basal forebrain cholinergic system, especially in its neocortical projection, but was not associated with changes in the level of p75(NTR) expression.     

Aberrant trafficking of the high-affinity choline transporter in AP-3-deficient mice

The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha . In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.     

Reduced mastication stimulates impairment of spatial memory and degeneration of hippocampal neurons in aged SAMP8 mice

The involvement of reduced mastication in senile dementia was evaluated by examining the effect of cutting off the upper molars (molarless) on spatial memory and numbers of hippocampal neurons in aged SAMP8 mice. Molarless mice showed a decrease in both learning ability in a water maze and neuron density in the hippocampal CA1 region compared with control mice. These changes increased the longer the molarless condition persisted. The data suggest a possible link between reduced mastication and hippocampal neuron loss that may be one risk factor for senile impairment of spatial memory.     

Differential effects of short- and long-term hyperhomocysteinaemia on cholinergic neurons, spatial memory and microbleedings in vivo in rats

Hyperhomocysteinaemia (HHcy) has been identified as a cardiovascular risk factor for neurodegenerative brain diseases. The aim of the present study was to investigate the effects of short (5 months) or long (15 months) HHcy in Sprague–Dawley rats in vivo. Short- and long-term HHcy differentially affected spatial memory as tested in a partially baited eight-arm radial maze. HHcy significantly reduced the number of choline acetyltransferase (ChAT)-positive neurons in the basal nucleus of Meynert and ChAT-positive axons in the cortex only after short-term but not long-term treatment, while acetylcholine levels in the cortex were decreased at both time points. Nerve growth factor (NGF) was significantly enhanced in the cortex only after 15 months of HHcy. HHcy did not affect cortical levels of amyloid precursor protein, beta-amyloid(1-42), tau and phospho-tau181 and several inflammatory markers, as well as vascular RECA-1 and laminin density. However, HHcy induced cortical microbleedings, as illustrated by intensive anti-rat IgG-positive spots in the cortex. In order to study the regulation of the key enzyme ChAT, organotypic rat brain slices were incubated with homocysteine, which induced a decline of ChAT that was counteracted by NGF treatment. In conclusion, our data demonstrate that chronic short- and long-term HHcy differentially caused memory impairment, cholinergic dysfunction, NGF expression and vascular microbleedings.