Carla Scali

β-Amyloid-Induced Inflammation and Cholinergic Hypofunction in the Rat Brain in Vivo: Involvement of the p38MAPK Pathway

Injection into the nucleus basalis of the rat of preaggregated Abeta(1-42) produced a congophylic deposit and microglial and astrocyte activation and infiltration and caused a strong inflammatory reaction characterized by IL-1beta production, increased inducible cyclooxygenase (COX-2), and inducible nitric oxide synthase (iNOS) expression. Many phospho-p38MAPK-positive cells were observed around the deposit at 7 days after Abeta injection. Phospho-p38MAPK colocalized with activated microglial cells, but not astrocytes. The inflammatory reaction was accompanied by cholinergic hypofunction. We investigated the protective effect of the selective COX-2 inhibitor rofecoxib in attenuating the inflammatory response and neurodegeneration evoked by Abeta(1-42). Rofecoxib (3 mg/kg/day, 7 days) reduced microglia and astrocyte activation, iNOS induction, and p38MAPK activation to control levels. Cholinergic hypofunction was also significantly attenuated by treatment with rofecoxib. We show here for the first time in vivo the pivotal role played by the p38MAPK microglial signal transduction pathway in the inflammatory response to the Abeta(1-42) deposit.

Differential effects of amyloid peptides β-(1-40) and β-(25-35) injections into the rat nucleus basalis

Abstract The nucleus basalis of male Charles River Wistar rats was injected with 10 μg of the β-amyloid peptides β-(1–40) and β-(25–35) and changes in the morphology of the lesioned area, the release of acetylcholine from the cortex, and in behavior were investigated. Injections of saline and a scrambled (25–35) peptide were used as controls. One week after lesioning, a Congo Red-positive deposit of aggregated material was found at the β-peptides injection site, which lasted for about 21 days in the case of the β-(25–35) peptide and at least two months for β-(1–40). No deposit was detected after scrambled peptide injection. At one week post injection, an extensive glial reaction surrounded the injection site of all peptides and saline as well. Such a reaction was still present but rather attenuated after two months. A decrease in the number of cholinergic neurons was detected in the nucleus basalis after one week with all treatments except saline. After two months, a reduction in the number of choline acetyltransferase-immunopositive neurons was still detectable in the rats injected with β-(1–40) but not in the β-(25–35)- or scrambled-injected. The reduction in choline acetyltransferase immunoreactivity was closely paralleled by a decrease in basal acetylcholine release from the parietal cortex ipsilateral to the lesion. Disruption of object recognition was observed in the first weeks after β-(25–35) peptide injection, whereas the β-(1–40) peptide impaired the performance only two months after lesion. Rats with lesions induced by β-peptides may be a useful animal model of amyloid deposition for investigation of the pathogenetic mechanisms leading to Alzheimers disease.

Selective muscarinic antagonists differentially affect in vivo acetylcholine release and memory performances of young and aged rats.

Brain acetylcholine release and memory performance were investigated in young (three- to six-months) and old (20- to 24-months) rats. Acetylcholine release was measured in vivo in the cortex and hippocampus of freely-moving animals, under basal conditions and in the presence of the following muscarinic antagonists: scopolamine, (+/-)-5,11-dihydro-11-[[(2-[2-[(dipropylamino) methyl]-1-piperidinyl]ethyl) amino] carbonyl]-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one (AFDX 384) and pirenzepine. The amount of acetylcholine released from the cortex and hippocampus of old rats was significantly reduced. In the presence of scopolamine and AFDX 384 but not of pirenzepine, the acetylcholine release was significantly higher in the old than the young rats, suggesting that changes in presynaptic M2/M4 muscarinic receptor function occur with ageing in the two brain regions. Cognitive capacities were evaluated using two different behavioural tasks: object recognition and passive avoidance response. Old rats were unable to discriminate between familiar and novel objects and had impaired performance in the passive avoidance test. AFDX 384 restored the performance in both tests. Furthermore, in young rats AFDX 384 reversed the impairment of both object recognition and passive avoidance response induced by scopolamine. The effect of AFDX 384 on acetylcholine release and behaviour in the old rats offers further support to a relationship between the age-related cholinergic hypofunction and cognitive impairment and indicates the blockade of presynaptic muscarinic receptors as a possible selective target for therapeutic strategies aimed at improving age-associated memory deficits.

Long-term ethanol consumption by rats: Effect on acetylcholine release in vivo, choline acetyltransferase activity, and behavior

The extent and duration of cholinergic hypofunction induced by long-term ethanol consumption was investigated in the rat. Ethanol (20% v/v) was administered to male adult Wistar rats as a sole source of fluid for three or six months. Control rats received tap water. The body weight, food and fluid intake in ethanol-treated rats were lower than in control rats throughout the treatment. After three months of ethanol consumption, and one week withdrawal, acetylcholine release in freely moving rats, investigated by microdialysis technique coupled to high-performance liquid chromatography quantification, was significantly decreased by 57 and 32% in the hippocampus and cortex, respectively, while choline acetyltransferase activity was significantly decreased (-30%) only in the hippocampus. A complete recovery of choline acetyltransferase activity and acetylcholine release was found after four ethanol-free weeks. Conversely, after four weeks of withdrawal following six months of ethanol treatment, the recovery in acetylcholine release was not accompanied by that in choline acetyltransferase activity, which remained significantly lower than in control rats in both cortex and hippocampus. The ability of rats to negotiate active and passive avoidance conditioned response tasks, tested after four ethanol-free weeks, was strongly impaired in both three- and six-month ethanol-treated rats. In conclusion, our experiments demonstrate that the development of a long-lasting cholinergic hypofunction requires at least six months of ethanol administration. The hypofunction affects choline acetyltransferase activity and acetylcholine release differently, and undergoes a remarkable recovery.

Effect of metrifonate on extracellular brain acetylcholine and object recognition in aged rats.

The effects of metrifonate were investigated in 4-6- and 22-24-month-old rats. Extracellular acetylcholine levels were measured by transversal microdialysis in vivo. Baseline extracellular acetylcholine levels in the cerebral cortex and hippocampus were 42% and 60% lower, respectively, in old than in young rats. Old rats did not discriminate between familiar and novel objects. In old rats, metrifonate (80 mg/kg p.o.) brought about 85% inhibition of cholinesterase activity in the cortex and hippocampus, a 4-fold increase in extracellular acetylcholine levels in the cortex only, and restored object recognition. In young rats, metrifonate caused 75% cholinesterase inhibition in the cerebral cortex and hippocampus, a 2-fold increase in cortical and hippocampal extracellular acetylcholine levels, and no effect on object recognition. The slight cholinesterase inhibition following metrifonate (30 mg/kg) in aged rats had no effect on cortical acetylcholine levels and object recognition. In conclusion, metrifonate may improve the age-associated cholinergic hypofunction and cognitive impairment.

Phospatidylserine reverses the age-dependent decrease in cortical acetylcholine release: a microdialysis study

In vivo basal acetylcholine (ACh) and choline (Ch) output from the parietal cortex of 3- and 19-month-old freely moving rats was measured by microdialysis. A dialysis tubing was inserted transversally through the parietal cortex 24 h before the experiment. ACh and Ch concentrations were determined in the same perfusate samples by HPLC with electrochemical detection. In 19-month-old rats treated with Tris buffer, ACh and Ch outputs were 39 and 16% lower, respectively, than in 3-month-old rats. Phosphatidylserine (PtdSer) administration (15 mg/kg i.p. daily) for 8 days to 19-month-old rats markedly attenuated the decrease in ACh release. The same treatment did not affect ACh and Ch outputs in 3-month-old rats. ACh and Ch outputs in 19-month-old rats administered either phosphatidylcholine (PtdCho) or o-phospho-dl-serine (P-Ser) (15 mg/kg i.p. daily) for 8 days were as low as in 19-month-old rats receiving Tris buffer only. It is possible that chronic PtdSer treatment improve ACh release in aging rats by increasing the availability of Ch for ACh synthesis.

Procognitive and Neuroprotective Activity of a Novel α7 Nicotinic Acetylcholine Receptor Agonist for Treatment of Neurodegenerative and Cognitive Disorders

The α7 nicotinic acetylcholine receptor (nAChR) is a promising target for treatment of cognitive dysfunction associated with Alzheimers disease and schizophrenia. Here, we report the pharmacological properties of 5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-amide [SEN12333 (WAY-317538)], a novel selective agonist of α7 nAChR. SEN12333 shows high affinity for the rat α7 receptor expressed in GH4C1 cells (Ki = 260 nM) and acts as full agonist in functional Ca2+ flux studies (EC50 = 1.6 μM). In whole-cell patch-clamp recordings, SEN12333 activated peak currents and maximal total charges similar to acetylcholine (EC50 = 12 μM). The compound did not show agonist activity at other nicotinic receptors tested and acted as a weak antagonist at α3-containing receptors. SEN12333 treatment (3 mg/kg i.p.) improved episodic memory in a novel object recognition task in rats in conditions of spontaneous forgetting as well as cognitive disruptions induced via glutamatergic [5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); MK-801] or cholinergic (scopolamine) mechanisms. This improvement was blocked by the α7-selective antagonist methyllycaconitine, indicating that it is mediated by α7 activation. SEN12333 also prevented a scopolamine-induced deficit in a passive avoidance task. In models targeting other cognitive domains, including attention and perceptual processing, SEN12333 normalized the apomorphine-induced deficit of prepulse inhibition. Neuroprotection of SEN12333 was demonstrated in quisqualate-lesioned animals in which treatment with SEN12333 (3 mg/kg/day i.p.) resulted in a significant protection of choline acetyltransferase-positive neurons in the lesioned hemisphere. Cumulatively, our results demonstrate that the novel α7 nAChR agonist SEN12333 has procognitive and neuroprotective properties, further demonstrating utility of α7 agonists for treatment of neurodegenerative and cognitive disorders.

Nerve growth factor increases extracellular acetylcholine levels in the parietal cortex and hippocampus of aged rats and restores object recognition

Male Wistar rats (3- and 20-month-old) were perfused i.c.v. with 1.5 micrograms of either nerve growth factor (NGF) or cytochrome C daily for 14 days. At the end of the infusion, the object-recognition test was carried out and extracellular acetylcholine levels (ACh) were measured in the cortex and hippocampus by transversal microdialysis technique. In 20-month-old control rats, the cortical and hippocampal ACh levels were 35 and 45% lower, respectively, than in 3-month-old rats and the ability to discriminate between a familiar and new object was impared. In the old rats treated with NGF, the ACh release as well as the behavioral performance showed no difference from those of young rats. These findings indicate that both ACh levels and memory impairment are improved in aged rats by NGF treatment and suggest that there is a relationship between object recognition and the activity of the forebrain cholinergic system.

Administration of amyloid β-peptides into the medial septum of rats decreases acetylcholine release from hippocampus in vivo

The septum of male Wistar rats was injected with synthetic beta-amyloid fragments, beta 12-28, beta 25-35 and beta 1-40, and hippocampal acetylcholine (ACh) release was evaluated by transversal microdialysis. A marked decrease in basal and K(+)-evoked ACh release was found 7 or 21 days after injection of 5 nmol of beta 12-28 and beta 25-35, or 3 nmol of beta 1-40, respectively. These data indicate that septal injection of beta-amyloid peptides causes hypofunction of the septo-hippocampal cholinergic system.

Increased Dickkopf-1 expression in transgenic mouse models of neurodegenerative disease

J. Neurochem. (2010) 112, 1539–1551.