Amyloid precursor protein processing is stimulated by metabotropic glutamate receptors.

Stimulation of muscarinic m1 or m3 receptors can, by generating diacylglycerol and activating protein kinase C, accelerate the breakdown of the amyloid precursor protein (APP) to form soluble, nonamyloidogenic derivatives (APPs), as previously shown. This relationship has been demonstrated in human glioma and neuroblastoma cells, as well as in transfected human embryonic kidney 293 cells and PC-12 cells. We now provide evidence that stimulation of metabotropic glutamate receptors (mGluRs), which also are coupled to phosphatidylinositol 4,5-bisphosphate hydrolysis, similarly accelerates processing of APP into nonamyloidogenic APPs. This process is demonstrated both in hippocampal neurons derived from fetal rats and in human embryonic kidney 293 cells transfected with cDNA expression constructs encoding the mGluR 1 alpha subtype. In hippocampal neurons, both an mGluR antagonist, L-(+)-2-amino-3-phosphonopropionic acid, and an inhibitor of protein kinase C, GF 109203X, blocked the APPs release evoked by glutamate receptor stimulation. Ionotropic glutamate agonists, N-methyl-D-aspartate or S(-)-5-fluorowillardiine, failed to affect APPs release. These data show that selective mGluR agonists that initiate signal-transduction events can regulate APP processing in bona fide primary neurons and transfected cells. As glutamatergic neurons in the cortex and hippocampus are damaged in Alzheimer disease, amyloid production in these regions may be enhanced by deficits in glutamatergic neurotransmission.     

Age-related changes of muscarinic cholinergic receptor subtypes in the striatum of Fisher 344 rats

Striatum expresses a cholinergic system involved in the regulation of its activity and changes in striatal cholinergic receptors may be related to cognitive impairment. This study has investigated muscarinic cholinergic M1-M5 receptor subtype expression in striatum of Fischer 344 rats aged 6 (young), 15 (adult) and 22 months (senescent) to assess the contribution of different muscarinic cholinergic receptor subtypes in age-related changes of striatal cholinergic neurotransmission. Western blot analysis revealed the expression of the M1-M5 muscarinic receptor subtytpes in the striatum of rats of the three age groups investigated. Both radioligand binding assay and light microscope autoradiography showed in young rats a M4>M1>M2>M3>M5 rank order of receptor density. With the exception of M1 receptor, the density of which is similar in the dorsal (motor) and ventral (limbic) striatum, other receptor subtypes were more abundant in ventral than in dorsal striatum. M1 receptor expression was unchanged between young and adult rats and decreased in senescent animals both in dorsal and ventral striatum. In dorsal striatum M2 and M5 receptor expression did not show age-related changes, whereas in ventral striatum it was slightly decreased in adult rats compared to young or senescent cohorts. M3 receptor expression did not show age-related modifications, whereas a progressive age-related decrease of M4 receptor was found, both in dorsal and ventral striatum. These data indicate a heterogeneous response to age of different muscarinic receptor subtypes. Striatal cholinergic markers are thought to correlate with cognitive impairment in aged rats. In view of this, the identification of age-related changes of striatal muscarinic receptor subtypes may contribute to develop cholinergic strategies to counter cholinergic neurotransmission changes occurring with aging.     

老年大鼠脑胆碱能M受体分布的放射自显影研究

目的 观察自然衰老大鼠不同脑区胆碱能Ｍ受体密度的变化和滋阴药知母或其中的知母皂甙元（ＺＭＳ）的调整作用。 方法 采用放射自显影技术测定脑切片胆碱能受体。 结果 所得脑切片自显影像灰度层次清晰，主要分布在皮质、海马、纹状体等部位，非特异结合灰度很低。图像分析仪分别给出不同脑区的平均灰度值，老年鼠皮质、海马、纹状体的灰度显著低于青年鼠。皮质降低近２０％，海马和纹状体的降低约为皮质的一半。在大鼠２０月龄     

Comparison of the impact of melatonin on chronic ethanol-induced learning and memory impairment between young and aged rats

Chronic alcohol exposure causes functional and structural changes in nervous system which have all been associated with learning and memory impairments. Furthermore, alcohol consumption has been shown to alter the pattern of neural cell adhesion molecules (NCAM) which are involved in memory processes. In the current work, we investigated the effects of melatonin on learning and memory deficits induced by alcohol exposure in young and aged rats. A group of young rats (3 months old) were administered ethanol for 45 days and half of them were co-treated with melatonin. Similar treatments were performed in the aged (19 months old) rats. Morris water maze test and passive avoidance task were used to assess cognitive performance. Lipid peroxidation (LPO) and glutathione (GSH) levels were determined to characterize the level of oxidative stress in the hippocampus and cortex. NCAM levels were determined by Western blotting in the hippocampal homogenates. There was a significant elevation in LPO levels and a reduction in GSH levels in aged and alcohol-exposed rats. Furthermore, both young and aged rats displayed some cognitive impairment when given with alcohol for 45 days. Co-administration of melatonin with ethanol significantly reduced LPO and elevated GSH levels while improving the learning and memory deficits induced by ethanol; the aged rats exhibited a greater response to melatonin supplementation. Moreover, melatonin modulated NCAM expression in hippocampus. Present findings indicate that exposure to ethanol induces learning and memory deficits probably by generating reactive oxygen species and downregulating NCAM 180 in hippocampus of aged rats. Melatonin improves learning and memory deficits and the behavioral responses of rats to melatonin supplementation are age dependent.     

Elimination of the vesicular acetylcholine transporter in the forebrain causes hyperactivity and deficits in spatial memory and long-term potentiation

Basal forebrain cholinergic neurons, which innervate the hippocampus and cortex, have been implicated in many forms of cognitive function. Immunolesion-based methods in animal models have been widely used to study the role of acetylcholine (ACh) neurotransmission in these processes, with variable results. Cholinergic neurons have been shown to release both glutamate and ACh, making it difficult to deduce the specific contribution of each neurotransmitter on cognition when neurons are eliminated. Understanding the precise roles of ACh in learning and memory is critical because drugs that preserve ACh are used as treatment for cognitive deficits. It is therefore important to define which cholinergic-dependent behaviors could be improved pharmacologically. Here we investigate the contributions of forebrain ACh on hippocampal synaptic plasticity and cognitive behavior by selective elimination of the vesicular ACh transporter, which interferes with synaptic storage and release of ACh. We show that elimination of vesicular ACh transporter in the hippocampus results in deficits in long-term potentiation and causes selective deficits in spatial memory. Moreover, decreased cholinergic tone in the forebrain is linked to hyperactivity, without changes in anxiety or depression-related behavior. These data uncover the specific contribution of forebrain cholinergic tone for synaptic plasticity and behavior. Moreover, these experiments define specific cognitive functions that could be targeted by cholinergic replacement therapy.     

Effect of treatment with the cholinesterase inhibitor rivastigmine on vesicular acetylcholine transporter and choline acetyltransferase in rat brain

A decline of cholinergic neurotransmission probably contributes to cognitive dysfunction occurring in Alzheimer's disease (AD) and vascular dementia (VaD). Acetylcholinesterase (AChE)/cholinesterase (ChE) inhibitors are the only drugs authorized for symptomatic treatment of AD and are also under investigation for VaD. The present study has investigated the influence of two doses of the AChE inhibitor rivastigmine (0.625 mg/Kg/day and 2.5 mg/Kg/day) on vesicular acetylcholine transporter (VAChT) and on choline acetyltransferase (ChAT) expression in frontal cortex, hippocampus, striatum and cerebellum of normotensive and spontaneously hypertensive rats (SHR). Cholinergic markers were assessed by immunochemical (Western blotting) and immunohistochemical techniques. In frontal cortex and striatum of normotensive rats, treatment with the lower dose (0.625 mg/Kg/day) of rivastigmine had no effect on VAChT immunoreactivity and increased slightly ChAT protein immunoreactivity. The higher dose (2.5 mg/Kg/day) of the compound increased significantly VAChT and ChAT protein immunoreactivity. In hippocampus rivastigmine induced a concentration-dependent increase of VAChT protein expression and no significant changes of ChAT protein expression. A similar pattern of VAChT and ChAT protein expression was observed in control SHR, whereas treatment of SHR with rivastigmine induced a more pronounced increase of VAChT protein immunoreactivity in frontal cortex, hippocampus and striatum compared to normotensive rats. Our data showing an increase of VAChT after treatment with rivastgmine further support the notion of an enhancement of cholinergic neurotransmission by AChE/ChE inhibitors. The observation of a greater expression of this cholinergic marker in SHR suggest that AChE inhibition may provide beneficial effects on cholinergic neurotransmission in an animal model of VaD.     

Long-Term Effects of Chronic Ethanol on Muscarinic Receptor Binding in Rat Brain

Effects of chronic ethanol treatment (CET) on muscarinic acetylcholine receptor (mAChR) binding properties were investigated via quantitative autoradiography in rats maintained on an ethanol-containing liquid diet for 28 weeks and withdrawn from ethanol for 8 weeks before harvesting of tissues. Controls received an identical diet in which sucrose was substituted isocalorically for ethanol. Maximal binding of the radiolabeled mAChR antagonist quinuclidinyl benzilate ([³H]QNB) was not reduced in hippocampal area CA1, dentate gyrus, neocortex, striatum, or thalamus, suggesting that CET results in no significant mAChR loss in these regions. Binding affinities of the cholinergic agonist carbachol to mAChRs were unaffected by CET in each of these regions, as determined by competitive displacement of [³H]QNB labeling. These results suggest that CET-induced functional deficits in brain cholinergic responses are not due to direct effects of CET on mAChR binding properties.     

Effect of Treatment with the Cholinesterase Inhibitor Rivastigmine on Vesicular Acetylcholine Transporter and Choline Acetyltransferase in Rat Brain

A decline of cholinergic neurotransmission probably contributes to cognitive dysfunction occurring in Alzheimer's disease (AD) and vascular dementia (VaD). Acetylcholinesterase (AChE)/cholinesterase (ChE) inhibitors are the only drugs authorized for symptomatic treatment of AD and are also under investigation for VaD. The present study has investigated the influence of two doses of the AChE inhibitor rivastigmine (0.625 mg/Kg/day and 2.5 mg/Kg/day) on vesicular acetylcholine transporter (VAChT) and on choline acetyltransferase (ChAT) expression in frontal cortex, hippocampus, striatum and cerebellum of normotensive and spontaneously hypertensive rats (SHR). Cholinergic markers were assessed by immunochemical (Western blotting) and immunohistochemical techniques. In frontal cortex and striatum of normotensive rats, treatment with the lower dose (0.625 mg/Kg/day) of rivastigmine had no effect on VAChT immunoreactivity and increased slightly ChAT protein immunoreactivity. The higher dose (2.5 mg/Kg/day) of the compound increased significantly VAChT and ChAT protein immunoreactivity. In hippocampus rivastigmine induced a concentration‐dependent increase of VAChT protein expression and no significant changes of ChAT protein expression. A similar pattern of VAChT and ChAT protein expression was observed in control SHR, whereas treatment of SHR with rivastigmine induced a more pronounced increase of VAChT protein immunoreactivity in frontal cortex, hippocampus and striatum compared to normotensive rats. Our data showing an increase of VAChT after treatment with rivastgmine further support the notion of an enhancement of cholinergic neurotransmission by AChE/ChE inhibitors. The observation of a greater expression of this cholinergic marker in SHR suggest that AChE inhibition may provide beneficial effects on cholinergic neurotransmission in an animal model of VaD.     

Melatonin alleviates cognition impairment by antagonizing brain insulin resistance in aged rats fed a high‐fat diet

Brain insulin resistance, induced by neuroinflammation and oxidative stress, contributes to neurodegeneration, that is, processes that are associated with Aβ accumulation and TAU hyperphosphorylation. Here, we tested the effect of chronic administration of melatonin (MLT) on brain insulin resistance and cognition deficits caused by a high‐fat diet (HFD) in aged rats. Results showed that MLT supplementation attenuated peripheral insulin resistance and lowered hippocampal oxidative stress levels. Activated microglia and astrocytes and hippocampal levels of TNF‐α in HFD‐fed rats were reduced by MLT treatment. Melatonin also prevented HFD‐induced increases in beta‐amyloid (Aβ) accumulation and TAU phosphorylation in the hippocampus. In addition, impairments of brain insulin signaling elicited by long‐term HFD were restored by MLT treatment, as confirmed by ex vivo insulin stimulation. Importantly, MLT reversed HFD‐induced cognitive decline as measured by a water maze test, normalized hippocampal LTP and restored CREB activity and BDNF levels as well as cholinergic neuronal activity in the hippocampus. Collectively, these findings indicate that MLT may exhibit substantial protective effects on cognition, via restoration of brain insulin signaling.     

Altered processing of Alzheimer amyloid precursor protein in response to neuronal degeneration.

In the brains of individuals with Alzheimer disease, senile plaques containing aggregates of beta-amyloid peptide, derived from the beta-amyloid precursor protein (APP), are seen in association with degenerating nerve terminals. It is not known whether the degenerating nerve terminals cause the formation of these aggregates or whether beta-amyloid peptide in the aggregates causes nerve-terminal degeneration. In the present study of rat brain, degeneration either of local neurons or of nerve terminals caused decreased levels of a neuron-enriched isoform of APP, increased levels of a glia-enriched isoform of APP, and increased levels of potentially amyloidogenic, as well as nonamyloidogenic, COOH-terminal fragments of APP. Our results demonstrate that neuronal degeneration affects APP processing and suggest that it may contribute to amyloid formation in mammalian brain.     

Improving effects of long-term growth hormone treatment on monoaminergic neurotransmission and related behavioral tests in aged rats

An age-related decline in cognitive functions and physical performance has been associated with reductions in growth hormone (GH) secretion and brain neurotransmitter function. In vivo experiments were performed to study the long-term effects of exogenously administered GH on the central monoaminergic neurotransmitters serotonin, dopamine, and noradrenaline and behavioral tests in old Wistar rats. The accumulation of 5-hydroxytryptophan (5-HTP) and L-3,4-dihydroxyphenylalanine (DOPA) after decarboxylase inhibition was used as a measure of the rate of tryptophan and tyrosine hydroxylation in vivo. Also, the content of the neurotransmitters serotonin, dopamine, and noradrenaline and some metabolites was measured by high-pressure liquid chromatography (HPLC) in the hippocampus and striatum, brain regions involved in adult memory processing and motor coordination. The age-related decline observed in all the neurochemical parameters in control rats was significantly reversed after repeated subcutaneous administration of GH (2 mg/kg per day, 4 weeks). Thus, GH treatment exerted a long-term effect on serotonin, dopamine, and noradrenaline neurotransmission by enhancing neurotransmitter synthesis and metabolism in aged rats. The results obtained after examining working memory tasks in the eight-radial maze and motor ability in the Rotarod treadmill in aged rats were consistent with these neurochemical data; both tests were significantly improved after chronic GH treatment. Overall, these in vivo findings suggest that the positive effects induced by GH on serotonin, dopamine, and noradrenaline neurotransmitters might explain, at least in part, the effects of chronic GH treatment in improving cognitive and motor ability in aged rats, and could aid in preventing or delaying deficits in monoamines associated with learning or motor disabilities.     

Chronic Ethanol Exposure Increases 3H-GABA Release in Rat Hippocampus by Presynaptic Muscarinic Receptor Modulation

BACKGROUND: Chronic ethanol treatment (CET) for 28 weeks significantly increases electrically-stimulated 3H-GABA release from hippocampal slices. This increase in GABA release may be one of the mechanisms by which CET decreases the magnitude of long-term potentiation (LTP) in the hippocampus. The present study examined whether CET increases GABA release via an alteration in heterologous presynaptic cholinergic regulation. METHODS: Animals were treated with ethanol or sucrose diet for 28 weeks followed by either no withdrawal or a 48-hr withdrawal period. The electrically-stimulated 3H-GABA release from preloaded superfused hippocampal slices of naive and CET rats was measured. RESULTS: Carbachol increased 3H-GABA release in a concentration-dependent manner, and atropine modulated 3H-GABA release in a biphasic concentration-dependent manner. Atropine (10 microM) significantly blocked the effects of carbachol. Oxotremorine, a selective muscarinic receptor agonist, also increased 3H-GABA release. Mecamylamine, a selective nicotinic antagonist, did not modulate 3H-GABA release and did not block the effects of carbachol. The effects of these agents were also tested in rats 0 or 48 hrs after withdrawal from CET. The biphasic effects of atropine were decreased, whereas the facilitating effects of carbachol were significantly increased. There were no changes in the effects of these agents on 3H-acetylcholine release from hippocampal slices of CET rats compared to sucrose-treated rats. CONCLUSION: These results suggest that presynaptic muscarinic receptors facilitate GABA release, whereas nicotinic receptors do not play a significant role in modulating GABA release in hippocampus. CET selectively alters presynaptic muscarinic regulation of GABA release in hippocampus and may help us to further understand the mechanism underlying the disruption of LTP by CET.     

Effects of repeated administration of rolipram, a cAMP-specific phosphodiesterase inhibitor, on acetylcholinergic indices in the aged rat brain

The effects of repeated treatment of rolipram, a cAMP specific phosphodiesterase inhibitor (0.1 mg/kg/day i.p., 14 days), on several neuronal cholinergic indices, especially on those in aged rats were examined. In young-adult rats, rolipram treatment increased choline acetyltransferase (ChAT) activity (V(max)value) in the striatum as well as in thalamus + midbrain, whereas it decreased choline esterase activity in the hippocampus. The ChAT activity (V(max)value) and the M1-R binding (B(max)value) in the aged control rats were significantly reduced in all the brain regions examined, compared with the young-adult rats, but consecutive rolipram treatment ameliorated the reductions of both indices in the frontal cortex and the hippocampus to approximately the young-adult control levels. Since high membrane binding site concentrations for rolipram itself were revealed in the frontal cortex and the hippocampus, where the rolipram treatment showed ameliorating effects on the ChAT activity and the M1-R binding, the present findings indicate that repeated rolipram administration easily affects these two brain regions. Thus, repeated rolipram administration could restore both the presynaptic ChAT activity and the postsynaptic muscarinic cholinergic M1-R binding which are decreased with aging.     

Hippocampus-dependent spatial learning and memory are impaired in growth hormone-deficient spontaneous dwarf rats

Growth hormone (GH)/insulin-like growth factor-I deficiencies are known to cause alterations in brain development resulting in impairment of cognitive function. In order to investigate the behavioral phenotype of GH-deficient spontaneous dwarf rats (SDRs), we examined the behavior of the SDRs in the Morris water maze and Y-maze tasks. The SDRs showed severe deficits in spatial learning and memory compared to normal rats. The possibility that the cognitive impairment is associated with alteration of neurotransmitter systems was examined histologically following completion of the behavioral tests, using choline acetyltransferase (ChAT), vesicular glutamate transporter 1 (VGlut1) and glutamic acid decarboxylase (GAD6) immunohistochemistry as markers. In the SDRs the number of ChAT-stained basal forebrain cholinergic neurons was decreased. ChAT staining was also decreased in the hippocampus, one of the target areas of basal forebrain cholinergic neurons. Next, we examined the number of glutamatergic and GABAergic boutons in the hippocampal molecular layer and found a significant reduction in the density of VGlut1+ boutons and an increase in GAD6+ profiles, leading to a significantly reduced ratio in glutamatergic/GABAergic synapses. Finally, the number of newly generated cells in the subgranular zone of the hippocampus was significantly lower than in normal rats. Taken together, our data suggest that GH is an important regulator of hippocampus-dependent spatial learning and memory. The behavioral deficits in the SDRs may be explained by altered basal forebrain cholinergic innervation, imbalance in hippocampal glutamatergic/GABAergic synapses, and decreased neurogenesis in the hippocampus.     

Muscarinic receptors in the preoptic area are sensitive to 17 beta-estradiol during the critical period

The relationship between the steroid hormone 17 beta-estradiol and the muscarinic cholinergic receptors present in the preoptic area (POA), median hypothalamus and posterior hypothalamus of female rats was examined in vitro at various stages of the estrous cycle. Muscarinic receptors varied in a cyclic manner, specifically in the POA, as shown by an increase in the proportion of high-affinity agonist binding sites (RH) to 60% during the proestrus, as compared to RH proportion observed during diestrus-2 and during the afternoon of proestrus (35%). Exposure of POA homogenates to 17 beta-estradiol resulted in conversion of RH to low-affinity agonist binding sites (RL). This effect of the hormone was also restricted to the POA taken from rats during the morning of proestrus. It was blocked by the antiestrogenic drug, clomiphene, and could be prevented by preoccupation of the muscarinic receptors by their own ligands prior to the addition of hormone. It follows that significant changes in POA muscarinic receptors in situ exactly coincided with the known critical time period characterized by high estrogen levels and high levels of estrogen receptors in the POA. These changes in muscarinic receptors might thus conceivably reflect variations in cholinergic activity in the POA during the estrous cycle.     

Altered levels of amyloid protein precursor transcripts in the basal forebrain of behaviorally impaired aged rats.

The beta/A4 protein is a constituent of plaque and vascular amyloid deposits in Alzheimer disease. Previous studies have shown increased levels of amyloid protein precursor (APP) mRNA in basal forebrain neurons in the disease. Morphological and neurochemical changes occur within the forebrain in Alzheimer disease and are also correlated with behavioral impairments in aged rats. Recent studies suggest that decreased nerve growth factor responsiveness of basal forebrain neurons is a feature of normal aging and of Alzheimer disease. We have used in situ hybridization to show that the abundance of specific forms of APP mRNA, which contain an inserted Kunitz-type serine protease inhibitor motif (APP-751, APP-770, and APP-related 563), are increased relative to the noninserted form (APP-695) of APP mRNA in the basal forebrain of aged rats. This increase appears to be specific to animals who exhibit spatial memory deficits but not aged rats without behavioral impairments.     

Investigations of the cholinergic deficit hypothesis in the hippocampus of the aged rat brain with physostigmine and scopolamine

Using histochemically demonstrated acetylcholinesterase activity and (14)C-2-deoxyglucose uptake as the respective indices, a study was set up to determine whether cerebral (hippocampal) metabolism was stimulated by a cholinergic agonist and/or inhibited by a cholinergic antagonist. For this 36 12-month-old (adult) and 48 27-month-old (aged) Fischer 344 rats were given intraperitoneal injections of physostigmine 0.05, 0.1 or 0.2 mg/kg or scopolamine 0.01, 0.03 or 0.1 mg/kg for 5 days. In the aged rats there was a slight increase in acetylcholinesterase activity after physostigmine but no convincing evidence of enhanced (14)C-2-deoxyglucose uptake. In neither age group was glucose uptake significantly reduced by scopolamine; it was in fact increased, as was - slightly but significantly - acetylcholinesterase activity. Findings for acetylcholinesterase activity and (14)C-2-deoxyglucose uptake in aged Fischer 344 rats thus do not provide firm corroboration of physostigmine-induced stimulation of mental performance found in behavioural studies, while scopolamine did not adversely affect the hippocampal variables studied. It is concluded that cholinergic agents such as physostigmine and scopolamine have only a marginal effect on the functional and metabolic deficits associated with cerebral aging.     

Polarized secretion of beta-amyloid precursor protein and amyloid beta-peptide in MDCK cells.

The beta-amyloid precursor protein (beta APP) is awidely expressed integral membrane protein that is proteolytically processed toyield several secreted derivatives, including soluble APP (APPs), the 4-kDaamyloid beta-peptide (A beta), and a related 3-kDa peptide (p3). To understandbeta APP trafficking and processing, we analyzed the sorting of beta APP inMadin-Darby canine kidney (MDCK) cells, an epithelial cell known to possessphysiologically distinct apical and basolateral plasma membranes. Processing ofbeta APP resulted in highly polarized secretion of APPs. More than 90% of APPswas detected in the basolateral compartment, and less than 10% was found in theapical compartment. This was associated with a preferential localization of betaAPP on the basolateral cell surface. Activation of protein kinase C, which isknown to enhance the secretion of APPs, did not change the polarity of APPsrelease but significantly increased the amount secreted. A beta and p3 peptideswere also secreted predominantly basolaterally. In addition, MDCK cells secreteda truncated form of A beta beginning at Arg-5. These data show that theproteolytic processing products of beta APP undergo polarized secretion.Moreover, the results suggest that the amyloidogenic A beta peptide is generatedfollowing the polarized sorting of beta APP. The polarized basolateral secretionof A beta in these epithelial cells provides a potential mechanism for theaccumulation of A beta in the abluminal basement membrane of brain microvesselsduring Alzheimer disease.     

Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus

The levels of molecules crucial for signal transduction processing change in the brain with aging. Lipid rafts are membrane microdomains involved in cell signaling. We describe here substantial biophysical and biochemical changes occurring within the rafts in hippocampus neurons from aging wild-type rats and mice. Using continuous sucrose density gradients, we observed light-, medium-, and heavy raft subpopulations in young adult rodent hippocampus neurons containing very low levels of amyloid precursor protein (APP) and almost no caveolin-1 (CAV-1). By contrast, old rodents had a homogeneous age-specific high-density caveolar raft subpopulation containing significantly more cholesterol (CHOL), CAV-1, and APP. C99-APP-Cter fragment detection demonstrates that the first step of amyloidogenic APP processing takes place in this caveolar structure during physiological aging of the rat brain. In this age-specific caveolar raft subpopulation, levels of the C99-APP-Cter fragment are exponentially correlated with those of APP, suggesting that high APP concentrations may be associated with a risk of large increases in beta-amyloid peptide levels. Citrulline (an intermediate amino acid of the urea cycle) supplementation in the diet of aged rats for 3 months reduced these age-related hippocampus raft changes, resulting in raft patterns tightly close to those in young animals: CHOL, CAV-1, and APP concentrations were significantly lower and the C99-APP-Cter fragment was less abundant in the heavy raft subpopulation than in controls. Thus, we report substantial changes in raft structures during the aging of rodent hippocampus and describe new and promising areas of investigation concerning the possible protective effect of citrulline on brain function during aging.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-012-9462-2) contains supplementary material, which is available to authorized users.     

Exposure of Rats to Ethanol from Postnatal Days 4 to 8: Alterations of Cholinergic Neurochemistry in the Hippocampus and Cerebellum at Day 20

Brief neonatal ethanol exposure (3.0 g/kg/dose, twice daily; postnatal day (PN) 4 to PN8) resulted in cholinergic neurochemical alterations in the cerebellum, but not the hippocampus of rats assayed on PN20. Analysis revealed that the binding affinity of cerebellar muscarinic receptors for [3H]quinuclidinyl benzilate was decreased by ethanol, but only in female pups. Other gender-specific but treatment-independent cerebellar differences were identified as well, including lower levels of choline acetyltransferase activity and S1-level (1,000 x g) crude protein in males and females, respectively. No evidence of ethanol-induced cholinergic change was noted in the hippocampus of the same pups on PN20. However, collapsed across treatment, male hippocampi were found to contain less S1-level protein than their female counterparts. Neither muscarinic receptor density nor acetyl cholinesterase activity were found to differ between treatments or genders, in either brain region. Consistent with the developmental timetables for regional cholinergic synaptogenesis in the rat, observations on PN20 confirm a hypothesis of cerebellar cholinergic vulnerability and hippocampal cholinergic resilience to neonatal ethanol insult.