Inhibition of acetylcholine-induced activation of extracellular regulated protein kinase prevents the encoding of an inhibitory avoidance response in the rat.

It has been demonstrated that the forebrain cholinergic system and the extracellular regulated kinase signal transduction pathway are involved in the mechanisms of learning, encoding, and storage of information. We investigated the involvement of the cholinergic and glutamatergic systems projecting to the medial prefrontal cortex and ventral hippocampus and of the extracellular regulated kinase signal transduction pathway in the acquisition and recall of the step-down inhibitory avoidance response in the rat, a relatively simple behavioral test acquired in a one-trial session. To this aim we studied by microdialysis the release of acetylcholine and glutamate, and by immunohistochemistry the activation of extracellular regulated kinase during acquisition, encoding and recall of the behavior. Cholinergic, but not glutamatergic, neurons projecting to the medial prefrontal cortex and ventral hippocampus were activated during acquisition of the task, as shown by increase in cortical and hippocampal acetylcholine release. Released acetylcholine in turn activated extracellular regulated kinase in neurons located in the target structures, since the muscarinic receptor antagonist scopolamine blocked extracellular regulated kinase activation. Both increased acetylcholine release and extracellular regulated kinase activation were necessary for memory formation, as administration of scopolamine and of extracellular regulated kinase inhibitors was followed by blockade of extracellular regulated kinase activation and amnesia. Our data indicate that a critical function of the learning-associated increase in acetylcholine release is to promote the activation of the extracellular regulated kinase signal transduction pathway and help understanding the role of these systems in the encoding of an inhibitory avoidance memory.

Proteomic identification of proteins specifically oxidized by intracerebral injection of amyloid beta-peptide (1-42) into rat brain: implications for Alzheimer's disease.

Protein oxidation has been shown to result in loss of protein function. There is increasing evidence that protein oxidation plays a role in the pathogenesis of Alzheimer's disease (AD). Amyloid beta-peptide (1-42) [Abeta(1-42)] has been implicated as a mediator of oxidative stress in AD. Additionally, Abeta(1-42) has been shown to induce cholinergic dysfunction when injected into rat brain, a finding consistent with cholinergic deficits documented in AD. In this study, we used proteomic techniques to examine the regional in vivo protein oxidation induced by Abeta(1-42) injected into the nucleus basalis magnocellularis (NBM) of rat brain compared with saline-injected control at 7 days post-injection. In the cortex, we identified glutamine synthetase and tubulin beta chain 15/alpha, while, in the NBM, we identified 14-3-3 zeta and chaperonin 60 (HSP60) as significantly oxidized. Extensive oxidation was detected in the hippocampus where we identified 14-3-3 zeta, beta-synuclein, pyruvate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase 1. The results of this study suggest that a single injection of Abeta(1-42) into NBM can have profound effects elsewhere in the brain. The results further suggest that Abeta(1-42)-induced oxidative stress in rat brain mirrors some of those proteins oxidized in AD brain and leads to oxidized proteins, which when inserted into their respective biochemical pathways yields insight into brain dysfunction that can lead to neurodegeneration in AD.

Effect of phosphatidylserine on acetylcholine release and content in cortical slices from aging rats.

Cortical slices were prepared from male rats 3 to 28 months old. The slices were superfused with choline-enriched Krebs solution containing physostigmine and electrically stimulated at frequencies of 1, 2 and 5 Hz for 5 min periods preceded and followed by rest periods. The amount of acetylcholine released during the stimulation periods was quantified by bioassay. In some experiments acetylcholine content was measured at the end of the superfusion period in stimulated and unstimulated slices. The evoked acetylcholine release was constant between 3 and 11 months of age at each frequency tested and showed a 50% decrease between 11 and 14 months of age with no further decrease up to 28 months. No difference in the evoked acetylcholine release was detected between 3 and 16 month old rats if the old rats were pretreated with phosphatidylserine 15 mg/kg IP for at least 7 days. The effect of phosphatidylserine lasted for 5 days after interruption of the treatment. There was no difference in acetylcholine content between the stimulated and unstimulated slices in 3 month old rats. In 16 month old rats stimulation brought about a 44% decrease in acetylcholine content. This decrease did not occur in rats pretreated with phosphatidylserine for 7 days. Phosphatidylserine appears to restore acetylcholine release in aging rats by maintaining an adequate acetylcholine supply in the slices.

Differential susceptibility of senile and lesion-induced astrogliosis to phosphatidylserine.

The susceptibility of age- and lesion-induced astrogliosis to the treatment with phosphatidylserine was investigated with the use of GFAP immunoblotting. The existence of age-induced upregulation of GFAP content was confirmed in the hippocampus, septum, and corpus callosum of the rat. The Ptd-Ser treatment of the aged rats further increased the GFAP content in the hippocampus and corpus callosum. The GFAP content increase in the corpus callosum was additionally illustrated by the upregulation in GFAP immunostaining. In the septum no further elevation of GFAP was observed after Ptd-Ser treatment, and in the striatum the compound elicited significant GFAP content increase, absent in the untreated aged rat brain striatum. In the intact adult rat brain no effect of Ptd-Ser on GFAP content was observed; neither did the compound elicit any modulation of the astrogliosis related to the mechanical lesion of the brain in the septum, hippocampus, cortex, and striatum. In the corpus callosum, Ptd-Ser potentiated the GFAP content increase related to the mechanical lesion, pointing to the structure-related heterogeneity of astrocytic population. Because it has been previously found that Ptd-Ser partly reverses one of the aspects of rodent brain aging, the aging-induced decrease of the acetylcholine release, the possibility exists that the effects of Ptd-Ser administration on glia and neurons in the aged brain may be related.

Acetylcholine release from rat cortical slices during postnatal development and aging.

Acetylcholine release from cortical slices superfused with choline-enriched Krebs solution containing physostigmine was investigated at birth, at 7, 20 and 30 days, and at 3 and 24 months of age, in order to assess age influence on the functional efficiency of the cortical cholinergic network. The slices were electrically stimulated at frequencies from 1 to 10 Hz for 5 min periods, preceded and followed by rest periods. The superfusate was collected every 5 min and acetylcholine content quantified by bioassay. In the newborn and 7 day-old pups acetylcholine release was approximately 50% lower than that of the 3 month-old rats at all frequencies tested. The highest release was elicited in the 30 day-old rats. Beginning with this age the evoked ACh release underwent a decline which in the 24 month-old rats brought it back to the same level as in the newborn ones. The blockade of the muscarinic autoreceptors by atropine 1.5 X 10(-8) M caused an increase in acetylcholine release at 20 day, 3 and 24 months of age but not in the newborn and 7 day-old pups. Adenosine 3 X 10(-5) M decreased acetylcholine output in newborn and adult but had no effect in the senescent rats.

Decrease of brain acetylcholine release in aging freely-moving rats detected by microdialysis.

In vivo extracellular acetylcholine release from brain hemispheric areas of 2-, 9-, and 18-month-old rats was measured by intracerebral microdialysis coupled with a radioenzymatic assay. Dialysis tubing was inserted transversally through both striata, frontal cortices and dorsal hippocampi 24 hours before the experiments. In the 2-month-old rats, the net average acetylcholine output, corrected for recovery and expressed in fmoles/min/single striatum, cortex and hippocampus, was 902.4 +/- 67, 303.9 +/- 14 and 334 +/- 32, respectively. In 18-month-old rats acetylcholine output was 53, 35 and 37% lower in striatum, cortex and hippocampus, respectively, than in young rats. The release from the striatum in the 9-month-old was intermediate between those of the 2- and 18-month-old rats. The intracerebroventricular injection of hemicholinium-3 caused a marked decrease in acetylcholine release from the striata of 2- and 18-month-old rats. If the decrease with hemicholinium was expressed as percent of the basal release there was no age-related difference between the young and old rats, indicating that the differences observed were due to the lower basal release found in the old rats. The possibility that the deficit in basal acetylcholine release with age may depend on a reduction of acetylcholine synthesis is discussed.

Phosphatidylserine increases acetylcholine release from cortical slices in aged rats.

Acetylcholine release was investigated in cortical slices superfused with choline-enriched Krebs solution containing physostigmine. Slices were prepared from 3 and 24 month old rats treated with either Tris buffer or sonicated suspensions of phosphatidylserine and phosphatidylcholine in Tris buffer. Slices were electrically stimulated at frequencies of 1, 2 and 5 Hz for 5 min periods preceded and followed by rest periods. ACh content of the superfusate was quantified by bioassay. In the 24 month old rats treated with Tris buffer, acetylcholine release, at all frequencies tested, was approximately 50% lower than that in the 3 month old rats. On the contrary, no significant decrease in ACh release was found in the 24 month old rats treated for 30 days with phosphatidylserine (15 mg/kg IP). The same treatment did not increase acetylcholine release in 3 month old rats. Acetylcholine release in 24 month old rats receiving a single administration of phosphatidylserine (15 mg/kg IP) or phosphatidylcholine (15 mg/kg IP) for 30 days was as low as in the 24 month old rats receiving the Tris buffer only. It is proposed that the chronic phosphatidylserine treatment may reduce the age-induced decrease in acetylcholine release by acting on the stimulus-secretion coupling mechanism.

In vivo amino acid release from the striatum of aging rats: adenosine modulation.

The release of glutamate, aspartate, GABA, and taurine from the striatum of young (3 months), mature (12 months), and old (22 months), freely moving male rats was investigated by using a microdialysis fiber inserted transversally in the striatum. In old rats basal extracellular glutamate and aspartate levels were decreased vs. young rats (-38 and -49%, respectively). GABA and taurine levels were unmodified by age. In the presence of the adenosine receptor antagonist 8-phenyltheophilline (8-pT) at the concentration of 50 microM, both K(+)-evoked releases of glutamate and aspartate were more than doubled in young, but not in mature and old rats. 8-pT at the concentration of 500 microM significantly decreased glutamate basal levels and K(+)-evoked aspartate release in old rats only. GABA and taurine releases were not affected by 8-pT at either dose. Our findings indicate a modified adenosine modulation on glutamate and aspartate release in aged rats, that could result from a change in the balance between A1 and A2a adenosine receptor density or an alteration of A1 and A2a receptor-effector coupling.

Effect of cholecystokinin octapeptide and ceruletide on release of acetylcholine from cerebral cortex of the rat in vivo.

The effect of cholecystokinin octapeptide (CCK-8) and its analogue, ceruletide on release of acetylcholine (ACh) from the cerebral cortex was investigated in urethane-anaesthetized and in unanaesthetized rats. Cholecystokinin octapeptide and ceruletide markedly stimulated output of ACh at doses of 1.5 and 5.0 micrograms/kg (i.p.), respectively. This effect was prevented by proglumide (160 mg/kg i.p.), a specific cholecystokinin receptor antagonist. At doses of 10 micrograms/kg (i.p.) and more, both CCK-8 and ceruletide decreased output of ACh from the cerebral cortex. The decrease was prevented by naloxone (1 mg/kg, s.c.), and replaced by a short-lasting increase. Cholecystokinin octapeptide and ceruletide appear therefore to affect the activity of cortical cholinergic fibres by acting upon both specific and opiate receptors. The interaction between CCK-8 and ceruletide, and opiate receptors either direct or through the release of endogenous opiates, was also demonstrated by the antagonism between ceruletide (1, 5 and 10 micrograms/kg, i.p.) and analgesia induced by morphine (5 mg/kg, s.c.), evaluated by the tail-flick test in the rat.

An analysis of cholecystokinin-induced increase in acetylcholine output from cerebral cortex of the rat.

The effect of colecystokinin (CCK-8) on the release of ACh from the cerebral cortex was studied in urethane-anaesthetized rats with the cortical cup technique. The increase in output of ACh brought about by the administration of CCK-8 1.5 micrograms/kg (i.p.) was prevented by pretreatment with haloperidol (1 mg/kg i.p.) and by lesions of the nucleus basalis magnocellularis and substantia nigra but it was reduced only slightly by bilateral vagotomy. Conversely, none of the treatments abolished the decrease in output of ACh brought about by CCK-8 at a dose of 10 micrograms/kg (i.p.). Local injection of CCK-8 into the nucleus basalis had no effect. Therefore, CCK-8 appears to increase cortical cholinergic activity by indirectly stimulating the cholinergic neurones of the nucleus basalis through dopaminergic neurones.

Effect of adenosine, adenosine triphosphate, adenosine deaminase, dipyridamole and aminophylline on acetylcholine release from electrically-stimulated brain slices.

The effect of adenosine on release of acetylcholine (ACh) was investigated in slices of rat cortex perfused with Krebs solution, at rest and during electrical stimulation at frequencies between 0.2 and 20 Hz. Electrical stimulation brought about a linear increase in release of ACh. Adenosine, in concentrations ranging from 1 to 100 microM, reduced in a dose-dependent manner the release of ACh and was more active on the stimulated than on the resting release. However, the fractional reduction by adenosine of stimulated release of ACh did not vary with increasing stimulation rate. Adenosine triphosphate was less active than adenosine in reducing release of ACh. The inhibitory effect of adenosine was antagonized by aminophylline (0.5 mM) and did not occur when the stimulated release of ACh was enhanced by blocking muscarinic autoreceptors with atropine (15 nM). Aminophylline (0.1 and 0.5 mM) itself exerted a biphasic effect on release of ACh, increasing it at rest and during stimulation at low frequencies, and decreasing it at higher stimulation rates. The manipulation of endogenous adenosine concentrations by adding adenosine deaminase or diphyridamole, an inhibitor of adenosine uptake, had little effect on release of ACh. Dipyridamole, (4 microM), only significantly decreased release of ACh at the 20 Hz stimulation rate.

Stereoselective increase in cholinergic transmission by R-(+)-hyoscyamine.

R-(+)-hyoscyamine, the dextro enantiomer of atropine, has been shown to amplify cholinergic transmission. R-(+)-hyoscyamine, unlike S-(-)-hyoscyamine, was able to increase acetylcholine release both in vitro and in vivo at a range of concentrations (10(-14) to 10(-12) M) and doses (5 microg/kg i.p.) which were inadequate for blocking muscarinic receptors. The increase over control values in ACh release was 15.9 +/- 2.1% in in vitro experiments performed in rat phrenic nerve-hemidiaphragm preparations (n = 6), and 63.3 + 16.3% in cortical microdialysis performed in free-moving rats (n = 5). The maximum ACh release was reached 60 min after R-(+)-hyoscyamine administration in in vivo experiments. At the same doses and concentrations, R-(+)-hyoscyamine was also able to elicit: antinociception of a cholinergic type (55.6-112.7% depending on the test used); complete prevention of scopolamine- and dicyclomine-induced amnesia; potentiation of muscular contractions electrically evoked in isolated guinea-pig ileum (16.7 +/- 3.6%) and in rat phrenic nerve-hemidiaphragm (19.9 +/- 3.2%) preparations. Antinociception was performed using the hot-plate and acetic acid abdominal constriction tests in mice, and the paw pressure test in rats, while prevention of induced amnesia was evaluated in mice using the passive-avoidance test. The respective affinities (pA2) for R-(+)- and S-(-)-hyoscyamine vs M1 (rabbit vas deferens), M2 (rat atrium) and M3 (rat ileum) receptor subtypes were as follows: 7.05 +/- 0.05/9.33 +/- 0.03 for M1; 7.25 +/- 0.04/8.95 +/- 0.01 for M2; 6.88 +/- 0.05/9.04 +/- 0.03 for M3. The respective pKi values for R-(+)- and S-(-)-hyoscyamine vs the five human muscarinic receptor subtypes expressed in Chinese hamster oocytes (CHO-K1) were as follows: 8.21 +/- 0.07/9.48 +/- 0.18 for m1; 7.89 +/- 0.06/9.45 +/- 0.31 for m2; 8.06 +/- 0.18/9.30 +/- 0.19 for m3; 8.35 +/- 0.11/9.55 +/- 0.13 for m4; 8.17 +/- 0.08/9.24 +/- 0.30 for m5.

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.

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

The nucleus basalis of male Charles River Wistar rats was injected with 10 micrograms of the beta-amyloid peptides beta-(1-40) and beta-(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 beta-peptides injection site, which lasted for about 21 days in the case of the beta-(25-35) peptide and at least two months for beta-(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 beta-(1-40) but not in the beta-(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 beta-(25-35) peptide injection, whereas the beta-(1-40) peptide impaired the performance only two months after lesion. Rats with lesions induced by beta-peptides may be a useful animal model of amyloid deposition for investigation of the pathogenetic mechanisms leading to Alzheimer's disease.

Unilateral and bilateral nucleus basalis lesions: differences in neurochemical and behavioural recovery.

The neurochemical and behavioural recovery following unilateral and bilateral lesions of the nucleus basalis was investigated in adult male Wistar rats 20 days and 6 months after surgery. The lesions were made by stereotaxic injections of ibotenic acid. Twenty days after surgery there was a statistically significant choline acetyltransferase decrease in the frontal and parietal ipsilateral cortex of the unilaterally lesioned rats and in the cortex of both hemispheres after bilateral lesions. Cortical high affinity choline uptake rate was significantly decreased 4 days after lesions but showed a rapid recovery within 20 days post-lesion in unilaterally and bilaterally lesioned rats. However, at this time both groups of lesioned rats showed a marked impairment in the acquisition of passive and active (shuttle-box) avoidance conditioned responses. Six months after surgery the decrease in choline acetyltransferase activity was smaller and statistically significant in the ipsilateral frontal cortex only in the unilaterally lesioned rats and in the frontal and parietal cortex of both hemispheres in the bilaterally lesioned rats. High affinity choline uptake was increased in the contralateral hemispheres of the unilaterally lesioned rats and was significantly larger than in the bilaterally lesioned rats. There was no difference in the acquisition of both passive and active avoidance conditioned responses between the sham operated and unilaterally lesioned rats, while the bilaterally lesioned rats could only negotiate the active avoidance conditioned response. In conclusion, our experiments demonstrate a remarkable neurochemical and behavioural recovery within 6 months in rats with a unilateral lesion of the nucleus basalis and only a limited recovery in the bilateral lesioned rats.

Serotonin blocks the long-term potentiation induced by primed burst stimulation in the CA1 region of rat hippocampal slices.

The effect of 5-hydroxytryptamine on the induction of long-term potentiation by a train of high frequency pulses (100 Hz; 1 s) or by a stimulation consisting of one burst of five pulses at 100 Hz delivered 170 ms after a single pulse (primed burst) was investigated in the CA1 region of the rat hippocampal slice in vitro with extracellular recordings. Superfusion with 5-hydroxytryptamine (3-30 microM) produced a concentration-dependent decrease in amplitude of the population spikes evoked by test stimuli. The presence of 5-hydroxytryptamine (30 microM) did not affect the magnitude of long-term potentiation produced by the high-frequency stimulation but it prevented the long-term potentiation induced by a primed burst. The action of 5-hydroxytryptamine was mimicked by the 5-hydroxytryptamine1A agonist 5-carboxamidotryptamine (0.3 microM) and blocked by the 5-hydroxytryptamine2/5-hydroxytryptamine1A antagonist spiperone (3 microM) or by the 5-hydroxytryptamine1/5-hydroxytryptamine2 antagonist methiothepin (1-10 microM). The selective 5-hydroxytryptamine2 antagonist ritanserin (1 microM) did not antagonize the block of long-term potentiation produced by 5-hydroxytryptamine. The selective 5-hydroxytryptamine3 antagonists (3-tropanyl)-1H-indole-3-carboxylic acid ester (ICS 205-930; 1 nM) and ondansetron (GR-38032; 30 nM) did not affect the reduction in the population spike produced by application of 5-hydroxytryptamine. In contrast, a primed burst delivered at the fifth minute of 5-hydroxytryptamine application in the presence of a 5-hydroxytryptamine3 antagonist induced a long-term potentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective immunolesioning of cholinergic neurons in nucleus basalis magnocellularis impairs prepulse inhibition of acoustic startle.

Information processing and attentional abnormalities are prominent in neuropsychiatric disorders. Since the cholinergic neurons located in the nucleus basalis magnocellularis have been shown to be involved in attentional performance and information processing, recent efforts to analyze the significance of the basal forebrain in the context of schizophrenia have focused on this nucleus and its projections to the cerebral cortex. We report here that bilateral selective immunolesioning of the cholinergic neurons in the nucleus basalis magnocellularis is followed by significant deficits in sensorimotor gating measured by prepulse inhibition of the startle reflex in adult rats. This behavioral approach is used in both humans and rodents and has been proposed as a valuable model contributing to the understanding of the neurobiological substrates of schizophrenia. The disruption of prepulse inhibition persisted over repeated testing. The selective lesions were induced by bilateral intraparenchymal infusions of 192 IgG saporin at a concentration having minimal diffusion into adjacent nuclei of the basal forebrain. The infusions were followed by extensive loss of choline acetyltransferase-immunopositive neurons. Our results show that the cholinergic neurons of the nucleus basalis magnocellularis represent a critical station of the startle gating circuitry and suggest that dysfunction of these neurons may result in impaired sensorimotor gating characteristic of schizophrenia.

The selective cyclooxygenase-2 inhibitor rofecoxib suppresses brain inflammation and protects cholinergic neurons from excitotoxic degeneration in vivo.

Brain inflammatory processes underlie the pathogenesis of Alzheimer's disease, and non-steroidal anti-inflammatory drugs have a protective effect in the disease. The aim of this work was to study in vivo whether attenuation of brain inflammatory response to excitotoxic insult by the selective cyclooxygenase-2 inhibitor, rofecoxib, may prevent neurodegeneration, as a contribution to a better understanding of the role inflammation plays in the pathology of Alzheimer's disease. We investigated, by immunohistochemical methods, glia reaction, the activation of p38 mitogen-activated protein kinase (p38MAPK) pathway with an antibody selective for the phosphorylated form of the enzyme and the number of choline acetyltransferase-positive neurons and, by in vivo microdialysis, cortical extracellular levels of acetylcholine following the injection of quisqualic acid into the right nucleus basalis of adult rats. Seven days after injection, a marked reduction in the number of choline acetyltransferase-positive neurons was found, along with an intense glia reaction, selective activation of p38MAPK at the injection site and a significant decrease in the extracellular levels of acetylcholine in the cortex ipsilateral to the injection site. The loss of cholinergic neurons persisted for at least up to 28 days. Rofecoxib (3 mg/kg/day, starting 1 h prior to injection of quisqualic acid) treatment for 7 days significantly attenuated glia activation and prevented the loss of choline acetyltransferase-positive cells and a decrease in cortical acetylcholine release. The prevention of cholinergic cell loss by rofecoxib occurred concomitantly with the inhibition of p38MAPK phosphorylation. Our findings suggest an important role of brain inflammatory reaction in cholinergic degeneration and demonstrate a neuroprotective effect of rofecoxib, presumably mediated through the inhibition of p38MAPK phosphorylation.

Differential regulation by N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors of acetylcholine release from the rat striatum in vivo.

The modulation of striatal cholinergic neurons by glutamatergic inputs was studied by monitoring the output of acetylcholine collected via a transversal microdialysis probe implanted into the striatum of freely moving rats. A transversal microdialysis membrane was inserted in the striatum and acetylcholine or GABA levels in the dialysate were measured. Acetylcholine levels in the dialysate were quantified by a high-performance liquid chromatography method with an electrochemical detector, while GABA levels were measured by a high-performance liquid chromatography method with a fluorescence detector. The dialysis membrane was perfused with Ringer solution containing 7 microM physostigmine sulphate and drugs, dissolved in the perfusion solution, were administered locally via the dialysis membrane. Local administration of the N-methyl-D-aspartate antagonist 3-[(RS)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (25-100 microM) brought about a decrease in striatal acetylcholine output which was dose-dependent, reversible and partially antagonized by 100 microM N-methyl-D-aspartate. On the other hand, local administration of the non-N-methyl-D-aspartate antagonist 2,3-dihydroxy-6-nitro-7-sulfamoil-benzo(F)quinoxaline was followed by an increase in acetylcholine output which reached a maximum of about +55% at 12.8 microM 2,3-dihydroxy-6-nitro-7-sulfamoil-benzo(F)quinoxaline and was readily reversed when the drug was withdrawn from the perfusion solution. Local administration of the non-N-methyl-D-aspartate receptor agonist (S)-alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (50 and 200 microM) decreased acetylcholine output and this effect was reversed by simultaneous perfusion with the GABA antagonist bicuculline (50 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic and noradrenergic denervations decrease labelled purine release from electrically stimulated rat cortical slices.

The origin of cortical purine release was investigated by measuring [3H]purine release from electrically stimulated cortical slices of rats after neurotoxic lesions of cholinergic, noradrenergic and serotoninergic pathways innervating the cortex. Purines were labelled by incubating the cortical slices with [3H]adenine. The 3H efflux at rest and during stimulation, analysed by high performance liquid chromatography, consisted of adenosine, inosine, hypoxanthine and a small amount of nucleotides. Twenty days after unilateral or bilateral lesion of the nucleus basalis a marked decrease in choline acetyltransferase activity was associated with a decrease in [3H]purine release. A linear relationship was found between the decrease in choline acetyltransferase activity and [3H]purine release. A partial recovery in both choline acetyltransferase activity and [3H]purine release was observed eight months after the lesion. Twenty days after intra-cerebroventricular injection of 6-hydroxydopamine a 59% decrease in cortical noradrenaline content was associated with a 44% decrease in [3H]purine release. Conversely, no change in [3H]purine release was found in rats in which a 89% decrease in cortical serotonin content was induced by intra-cerebroventricular injection of 5,7-dihydroxytryptamine. The decrease in [3H]purine release after the lesion of the cholinergic and noradrenergic pathways may depend on metabolic changes, a loss of a stimulating influence of acetylcholine and noradrenaline or may indicate a release of [3H]purine from cholinergic and noradrenergic fibres.