Pepeu GiancarloDepartment of Pharmacology; University of Florence; Pharmacology; Viale Pieraccini 6; Florence ItalyPhone : Fax :
Prof. Giancarlo Pepeu passed away in November 2021 at 91. Born in Milan Pepeu graduated in medicine at the University of Florence in 1954, Full professor of Pharmacology in Cagliari in 1968, he returned to Florence in 1974 where he taught in the Faculty of Medicine until 2005. He has contributed substantially to the understanding of the mechanisms of cholinergic neurotransmission in the central nervous system and of the pathophysiology of Alzheimer's disease. His more than 300 articles have had international resonance and have earned him numerous awards.
Title: Sex and Gender Differences in the Brain Cholinergic System and in the Response to Therapy of Alzheimer Disease with Cholinesterase Inhibitors Giacobini E, Pepeu G Ref: Curr Alzheimer Res, 15:1077, 2018 : PubMed
This review has two aims. First, to examine whether or not sex and gender may influence the brain cholinergic system in animals and in humans. Second, to examine the available evidence of sexually dimorphic response to the therapeutic and toxic effects of cholinesterase inhibitors. Animal research reveals no marked difference in the general morphology of the brain cholinergic system but subtle functional gender differences have been reported. In humans, gender differences in nucleus basalis of Meynert (NBM) exist. In animals, some cholinergic neurons express estrogen alpha receptors in females and androgens in males. It is known that sex hormones exert trophic effects on the cholinergic system. Females show higher frontal cortex cholinergic activity whereas males have higher activity in the hippocampus. Gender differences in the pharmacological effects result in higher sensitivity to the toxic effects of organophosphate cholinesterase inhibitors in males. A stronger and more selective benefit of ChEI treatment in AD has been reported in men by several authors. Sex and estrogen receptor phenotype may both influence the response to donepezil and rivastigmine. Hence, aged male and female individuals might respond differently to ChEI due to either sex-specific differences in structures and function of the cholinergic system, pharmacokinetics, memory function or in the way aging or AD affects these processes.
Title: Do cholinesterase inhibitors act primarily on attention deficit? A naturalistic study in Alzheimer's disease patients Bracco L, Bessi V, Padiglioni S, Marini S, Pepeu G Ref: J Alzheimers Dis, 40:737, 2014 : PubMed
Attention is the first non-memory domain affected in Alzheimer's disease (AD), before deficits in language and visuo-spatial function, and it is claimed that attention deficits are responsible for the difficulties with daily living in early demented patients. The aim of this longitudinal study in a group of 121 Caucasian, community-dwelling, mild-to-moderate AD patients (Mini-Mental State Examination (MMSE) score >17) was to detect which cognitive domains were most affected by the disease and whether one year treatment with cholinesterase inhibitors was more effective in preserving attention than memory. All subjects were evaluated by a neuropsychological battery including global measurements (MMSE, Information-Memory-Concentration Test) and tasks exploring verbal long-term memory, language, attention, and executive functions. The comparison between two evaluations, made 12 months apart, shows statistically significant differences, indicating deterioration compared to baseline, in the following tests: MMSE (with no gender differences), Composite Memory Score, Short Story Delayed Recall, Trail-Making Test A, Semantic Fluency Test, and Token Test. Conversely, there were no differences in the two evaluations of the Digit Span, Corsi Tapping Test, Short Story Immediate Recall, and Phonemic Fluency Tests. It appears that the treatment specifically attenuated the decline in tests assessing attention and executive functions. A stabilization of the ability to pay attention, with the ensuing positive effects on executive functions, recent memory, and information acquisition which depend on attention, appears to be the main neuropsychological mechanism through which the activation of the cholinergic system, resulting from cholinesterase inhibition, exerts its effect on cognition.
Title: Effect of cholinesterase inhibitors on attention Pepeu G, Giovannini MG, Bracco L Ref: Chemico-Biological Interactions, 203:361, 2013 : PubMed
Advantages and limits of the use of cholinesterase inhibitors (ChEI) in Alzheimer's disease (AD) are well established. Their effects result from an increase in extracellular acetylcholine (ACh) whose hydrolysis is prevented by cholinesterase inhibition. In this way, the cholinergic deficit which characterizes AD may be corrected. This overview discusses which components of the cognitive process are improved by ChEI administration. In animal experiments, the increase in ACh release, detected in brain areas during behavioral tasks designed to tax attentional processes, demonstrates that an activation of cholinergic neurons underlies arousal and attention. Since arousal and attention depend on activation of the forebrain cholinergic system, it is to be expected that the loss of cholinergic neurons occurring in AD may lead to impairment of the attentional processes. Indeed, a consensus exists that attention is the first non-memory domain to be affected in AD, before deficits in language and visuo-spatial functions. The difficulties with daily living, which occur even in mild AD, may be related to attentional deficits. ChEIs, by restoring the cholinergic activity, should improve attention. If the cognitive changes resulting from ChEI treatment in AD patients are assessed with appropriate tests or selected items of the scales, a predominant effect on attention and executive functions emerges. In a group of 121 subjects with mild to moderate AD, (MMSE score 21.88+/-3.63) followed in the Alzheimer Unit in Florence, after a year of treatment with standard doses of ChEIs, it was observed a stabilization of the disease, characterized by no changes of the tests evaluating attention and executive functions but a worsening of those involving memory mechanisms. These findings suggest that ChEI treatment preserves attention more than memory. Finally, the electrophysiological and neurochemical mechanisms through which the activation of the cholinergic forebrain neurons enhance attention and create the condition for information acquisition are reviewed.
A consensus exists that cholinesterase inhibitors (ChEIs) are efficacious for mild to moderate Alzheimer's Disease (AD). Unfortunately, the number of non-responders is large and the therapeutic effect is usually short-lasting. In experimental animals, ChEIs exert three main actions: inhibit cholinesterase (ChE), increase extracellular levels of brain acetylcholine (ACh), improve cognitive processes, particularly when disrupted in models of AD. In this overview we shall deal with the cognitive processes that are improved by ChEI treatment because they depend on the integrity of brain cholinergic pathways and their activation. The role of cholinergic system in cognition can be investigated using different approaches. Microdialysis experiments demonstrate the involvement of the cholinergic system in attention, working, spatial and explicit memory, information encoding, sensory-motor gating, skill learning. No involvement in long-term memory has yet been demonstrated. Conversely, memory consolidation is facilitated by low cholinergic activity. Experiments on healthy human subjects, notwithstanding caveats concerning age, dose, and different memory tests, confirm the findings of animal experiments and demonstrate that stimulation of the cholinergic system facilitates attention, stimulus detection, perceptual processing and information encoding. It is not clear whether information retrieval may be improved but memory consolidation is reduced by cholinergic activation. ChEI effects in AD patients have been extensively investigated using rating scales that assess cognitive and behavioural responses. Few attempts have been made to identify which scale items respond better to ChEIs and therefore, presumably, depend on the activity of the cholinergic system. Improvement in attention and executive functions, communication, expressive language and mood stability have been reported. Memory consolidation and retrieval may be impaired by high ACh levels. Therefore, considering that in AD the degeneration of the cholinergic system is associated with alteration of other neurotransmitter systems and a diffuse synaptic loss, a limited efficacy of ChEIs on memory processes should be expected.
Cholinesterase inhibitors (ChEIs) were introduced in the therapy of Alzheimer Disease (AD) in the nineteen nineties with great expectations. The hopes and large interest raised by these drugs are well demonstrated by 12,000 references listed by PubMed under 'ChEI' for 1995-2007. The list is reduced to 2500 if we confine ourselves to 'ChEIs and dementia'. Of them, about 500 were published in the last two years. Whereas an increase in brain acetylcholine and an improvement of cognitive deficits have been consistently demonstrated in animal models of AD, from aging rats to transgenic mice, the clinical effectiveness of ChEIs has been and is still a matter of contrasting opinions. These range from the negative conclusions of the AD2000 trial on donepezil, claiming that it is not cost effective, with benefits below a minimally relevant threshold, to the NICE appraisal of 2007 declaring that donepezil, rivastigmine, galantamine are efficacious for mild to moderate AD, irrespective of their different selectivity for acetyl- (AChE) and butyrylcholinesterase (BCHE). The possibility that ChEIs may exert their effects through mechanisms beyond cholinesterase inhibition has been envisaged. However, according to the information presented in this review, the "classical" ChEIs, donepezil, rivastigmine and galantamine, show no pharmacological actions beyond cholinesterase inhibition which may play an important role in their therapeutic efficacy. The diverging opinions on clinical efficacy do not discourage from developing new ChEIs, and particularly the so called multifunctional ChEIs. They represent the future of the cholinergic therapy for AD but other indications for these drugs may be considered, including vascular dementia, mild cognitive impairment, and the ethically sensitive improvement of memory and learning in healthy subjects.
Title: N1phenethyl-norcymserine, a selective butyrylcholinesterase inhibitor, increases acetylcholine release in rat cerebral cortex: a comparison with donepezil and rivastigmine Cerbai F, Giovannini MG, Melani C, Enz A, Pepeu G Ref: European Journal of Pharmacology, 572:142, 2007 : PubMed
The effects of (-)-N(1)phenethyl-norcymserine (PEC, 5 mk/kg, i.p.) on acetylcholine release and cholinesterase activity in the rat cerebral cortex were compared with those of donepezil (1 mg/kg, i.p.), a selective acetylcholinesterase inhibitor, and rivastigmine (0.6 mg/kg, i.p.), an inhibitor of acetylcholinesterase and butyrylcholinesterase. Acetylcholine extracellular levels were measured by microdialysis coupled with HPLC; acetylcholinesterase and butyrylcholinesterase activity were measured with colorimetric and radiometric methods. It was found that comparable 2-3 fold increases in cortical extracellular acetylcholine level, calculated as areas under the curve, followed the administration of the three drugs at the doses used. At the peak of acetylcholine increase, a 27% acetylcholinesterase inhibition and no butyrylcholinesterase inhibition was found after donepezil (1 mg/kg, i.p) administration. At the same time point, rivastigmine (0.6 mg/kg, i.p.) inhibited acetylcholinesterase by 40% and butyrylcholinesterase by 25%. After PEC (5 mg/kg, i.p.) administration, there was a 39% butyrylcholinesterase inhibition and no effect on acetylcholinesterase. Since in the present study it was also confirmed that in the brain butyrylcholinesterase activity is only about 10% of acetylcholinesterase activity, it is surprising that its partial inhibition is sufficient to increase extracellular acetylcholine levels. The importance of butyrylcholinesterase as a "co-regulator" of synaptic acetylcholine levels should thus be reconsidered.
Title: Acetylcholine release from fetal tissue homotopically grafted to the motoneuron-depleted lumbar spinal cord. An in vivo microdialysis study in the awake rat Gulino R, Cataudella T, Casamenti F, Pepeu G, Stanzani S, Leanza G Ref: Experimental Neurology, 204:326, 2007 : PubMed
Grafts of spinal cord (SC) tissue can survive and develop into the severed SC, but no conclusive data are available concerning the functional activity of transplanted neurons. In the present study, suspensions of prelabeled embryonic ventral SC tissue were grafted to the lumbar SC of rats with motoneuron loss induced by perinatal injection of volkensin. Eight to ten months post-grafting, acetylcholine (ACh) release was measured by microdialysis in awake rats, under either basal or stimulated conditions. In normal animals, baseline ACh output averaged 1.6 pmol/30 microl, it exhibited a 4-fold increase after KCl-induced depolarization or handling, and it was completely inhibited by tetrodotoxin administration. Moreover, ACh levels did not change following acute SC transection performed under anesthesia during ongoing dialysis, suggesting an intrinsic source for spinal ACh. Treatment with volkensin produced a severe (>85%) motoneuronal loss accompanied by a similar reduction in baseline ACh release and almost completely abolished effects of depolarization or handling. In transplanted animals, many motoneuron-like labeled cells were found within and just outside the graft area, but apparently in no case were they able to extend fibers towards the denervated muscle. However, the grafts restored baseline ACh output up to near-normal levels and responded with significantly increased release to depolarization, but not to handling. The present findings indicate that spinal neuroblasts can survive and develop within the motoneuron-depleted SC and release ACh in a near-normal, but apparently non-regulated, manner. This may be of importance for future studies involving intraspinal stem cell grafts.
In 7-month-old TgCRND8 mice, the extracellular cortical acetylcholine levels in vivo, the number and morphology of cholinergic neurons in the nucleus basalis magnocellularis and the ability to acquire an inhibitory avoidance response in the step-down test were studied. The TgCRND8 mouse brain is characterized by many beta-amyloid plaques, reduced neuronal and axonal staining, white matter demyelination, glia reaction and inducible nitric oxide synthase immunoreactivity. Choline acetyltransferase immunoreactivity in the nucleus basalis magnocellularis was significantly decreased. Basal and potassium-stimulated extracellular acetylcholine levels, investigated by microdialysis, and m2 muscarinic receptor immunoreactivity were reduced in the cortex of TgCRND8 mice, and scopolamine administration increased cortical extracellular acetylcholine levels in control but not in TgCRND8 mice. A cognitive impairment was demonstrated in the step-down test. These findings demonstrate that neuronal damage and cholinergic dysfunction in vivo underlie the impairment in learning and memory functions in this mouse model of Alzheimer's disease.
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.
Like acetylcholinesterase, butyrylcholinesterase (BChE) inactivates the neurotransmitter acetylcholine (ACh) and is hence a viable therapeutic target in Alzheimer's disease, which is characterized by a cholinergic deficit. Potent, reversible, and brain-targeted BChE inhibitors (cymserine analogs) were developed based on binding domain structures to help elucidate the role of this enzyme in the central nervous system. In rats, cymserine analogs caused long-term inhibition of brain BChE and elevated extracellular ACh levels, without inhibitory effects on acetylcholinesterase. In rat brain slices, selective BChE inhibition augmented long-term potentiation. These compounds also improved the cognitive performance (maze navigation) of aged rats. In cultured human SK-N-SH neuroblastoma cells, intra- and extracellular beta-amyloid precursor protein, and secreted beta-amyloid peptide levels were reduced without affecting cell viability. Treatment of transgenic mice that overexpressed human mutant amyloid precursor protein also resulted in lower beta-amyloid peptide brain levels than controls. Selective, reversible inhibition of brain BChE may represent a treatment for Alzheimer's disease, improving cognition and modulating neuropathological markers of the disease.
Title: Activation of the cholinergic system during cognitive processes. Giovannini MG, Passani MB, Pepeu G Ref: Cholinergic Mechanisms, CRC Press, :387, 2004 : PubMed
Measuring the changes in neurotransmitter extracellular levels in discrete brain areas is considered a tool for identifying the neuronal systems involved in specific behavioral responses or cognitive processes. Acetylcholine (ACh) is the first neurotransmitter whose diffusion from the central nervous system was investigated and whose extracellular levels variations were correlated to changes in neuronal activity. This was done initially by means of the cup technique and then by the microdialysis technique. The latter, notwithstanding some technical limitations, makes it possible to detect variations in extracellular levels of ACh in unrestrained, behaving animals. This review summarizes and discusses the results obtained investigating the changes in ACh release during performance of operant tasks, exposition to novel stimuli, locomotor activity, and the performance of spatial memory tasks, working memory, and place preference memory tasks. Activation of the forebrain cholinergic system has been demonstrated in many tasks and conditions in which the environment requires the animal to analyze novel stimuli that may represent a threat or offer a reward. The sustained cholinergic activation, demonstrated by high levels of extracellular ACh observed during the behavioral paradigms, indicates that many behaviors occur within or require the facilitation provided by the cholinergic system to the operation of pertinent neuronal pathways.
Title: S 18986, a positive modulator of AMPA receptors with cognition-enhancing properties, increases ACh release in the hippocampus of young and aged rat Rosi S, Giovannini MG, Lestage P, Munoz C, Corte LD, Pepeu G Ref: Neuroscience Letters, 361:120, 2004 : PubMed
The effect of S 18986, positive AMPA receptor modulator, on acetylcholine (ACh), gamma-aminobutyric acid (GABA) and glutamate (Glu) release from the hippocampus of freely moving young and aged rats was investigated by microdialysis coupled to HPLC. The cognition-enhancing properties were evaluated by a passive avoidance test. In 3 month-old rats, S 18986 (10 mg/kg i.p.) increased by 70% ACh release, which returned to basal level within 2 h, while 3 mg/kg had no effect. In 22 month-old rats, both 3 and 10 mg/kg i.p. induced a long lasting increase in ACh release, as large as that induced by 10 mg/kg in young rats. S 18986 did not modify GABA and glutamate release. No effect on general behavior was observed, but S 18986 at both doses prevented the disrupting effect of scopolamine (1 mg/kg i.p.) on passive avoidance acquisition.
The extracellular levels of aspartate, glutamate, gamma-aminobutyric acid (GABA), and acetylcholine (ACh) were investigated by microdialysis, coupled with HPLC, in the ventral hippocampus of rats during two 30-min exploration periods. Motor activity was monitored. During exploration I, an increase in motor activity associated with a 315% increase in aspartate, 181% in glutamate, and 264% in ACh levels, occurred during the first 10 min. The increase in GABA level reached a maximum of 257% during the second 10 min. The neurotransmitter levels returned to basal values within 40 min. During exploration II, 1 h later, a smaller increase in neurotransmitter levels and motor activity was observed. In both explorations, the increase in neurotransmitter levels was completely abolished by 1 and 3 microM TTX. A statistically significant relationship was found between neurotransmitter extracellular levels and motor activity, for aspartate and glutamate in exploration I, and for ACh in exploration I and II. In conclusion, exploratory activity is associated with or depends on the activation of neuronal systems in the ventral hippocampus releasing aspartate, glutamate, GABA, and ACh. The activation is dampened by habituation.
Title: The selective cyclooxygenase-2 inhibitor rofecoxib suppresses brain inflammation and protects cholinergic neurons from excitotoxic degeneration in vivo Scali C, Giovannini MG, Prosperi C, Bellucci A, Pepeu G, Casamenti F Ref: Neuroscience, 117:909, 2003 : PubMed
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.
Title: Rivastigmine antagonizes deficits in prepulse inhibition induced by selective immunolesioning of cholinergic neurons in nucleus basalis magnocellularis Ballmaier M, Casamenti F, Scali C, Mazzoncini R, Zoli M, Pepeu G, Spano PF Ref: Neuroscience, 114:91, 2002 : PubMed
Impairments of cortical cholinergic inputs from the nucleus basalis magnocellularis fundamentally alter information processing and attentional function, thereby advancing the severity of psychopathology in major neuropsychiatric disorders. It was previously shown in adult rats that bilateral 192 IgG saporin-induced selective immunolesioning of the cholinergic neurons in the nucleus basalis produces pronounced and long-lasting deficits in sensorimotor gating measured by prepulse inhibition of the startle reflex. This behavioral paradigm is considered a valid model of sensorimotor gating deficits in the psychotic spectrum and efforts to analyze the significance of the cholinergic basal forebrain in this context are of great interest. In the present study the predictive value of the selective cholinergic immunolesioning model was tested by examining the ability of the cholinesterase inhibitor rivastigmine to restore prepulse inhibition in immunolesioned rats. We report here a pronounced restoring effect of acute (0.75 or 1.5 mg/kg s.c.) as well as repeated (0.75 mg/kg s.c. b.i.d., for 10 days) treatment with rivastigmine in this model of disrupted prepulse inhibition. Intra-nucleus basalis magnocellularis infusions of 192 IgG saporin resulted in extensive loss of basal-cortical cholinergic neurons as shown by the marked decrease in basal telencephalic choline acetyltransferase immunopositive neurons and cortical choline acetyltransferase activity. In this condition, rivastigmine was found to significantly increase cortical acetylcholine extracellular levels in lesioned animals measured by in vivo microdialysis. Taken together, our results strengthen the proposal that the nucleus basalis represents a critical station of the startle gating circuitry. In addition, our findings strongly indicate that even after dramatic decrease of cholinergic neurons, inhibition of acetylcholinesterase restores the cholinergic synaptic function to a point approaching normalization of experimentally induced psychopathology.
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.
The changes in extracellular acetylcholine levels were investigated by microdialysis in the cortex and hippocampus of aging rats after administration of metrifonate (80 mg/kg), rivastigmine (0.75 mg/kg), donepezil (1.5 mg/kg) or vehicle for 21 days (twice daily p.o.). Eighteen h after the last administration, cholinesterase inhibition was 85, 52 and 39% after metrifonate, rivastigmine and donepezil, respectively, and was accompanied by 988, 590 and 75% increase in cortical acetylcholine level. In the hippocampus, metrifonate and rivastigmine brought about a 169 and 108% increase in acetylcholine levels. A challenge dose of metrifonate, rivastigmine and donepezil was followed by a further increase in cortical and hippocampal acetylcholine levels. The retrograde perfusion of the M(2)-M(4) receptor antagonist AFDX-384 (10 microM) induced a 500 and 300% increase in cortical and hippocampal acetylcholine release, in control and rivastigmine-treated rats, respectively, no increase in metrifonate-treated rats, and a 210% increase in donepezil-treated rats. In conclusion, chronic treatment of aging rats with metrifonate, rivastigmine and donepezil induces a long-lasting increase in acetylcholine levels, and reveals marked differences between the three drugs.
Title: Selective immunolesioning of cholinergic neurons in nucleus basalis magnocellularis impairs prepulse inhibition of acoustic startle Ballmaier M, Casamenti F, Zoli M, Pepeu G, Spano P Ref: Neuroscience, 108:299, 2001 : PubMed
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.
Title: Effects of novelty and habituation on acetylcholine, GABA, and glutamate release from the frontal cortex and hippocampus of freely moving rats Giovannini MG, Rakovska A, Benton RS, Pazzagli M, Bianchi L, Pepeu G Ref: Neuroscience, 106:43, 2001 : PubMed
The involvement of the forebrain cholinergic system in arousal, learning and memory has been well established. Other neurotransmitters such as GABA and glutamate may be involved in the mechanisms of memory by modulating the forebrain cholinergic pathways. We studied the activity of cortical and hippocampal cholinergic, GABAergic and glutamatergic systems during novelty and habituation in the rat using microdialysis. After establishing basal release of the neurotransmitters, the animals were transferred to a novel environment and allowed to explore it twice consecutively for 30 min (60 min apart; exploration I and II). The motor activity was monitored. Samples were collected throughout the experiment and the release of acetylcholine (ACh), GABA and glutamate was measured. During the two consecutive explorations of the arena, cortical and hippocampal, ACh release showed a significant tetrodotoxin-dependent increase which was higher during exploration I than II. The effect was more pronounced and longer-lasting in the hippocampus than in the cortex. Cortical GABA release increased significantly only during exploration II, while hippocampal GABA release did not increase during either exploration. Motor activity was higher during the first 10 min of exploration I and II and then gradually decreased during the further 20 min. Both cortical and hippocampal ACh release were positively correlated with motor activity during exploration II, but not during I. During exploration II, cortical GABA release was inversely correlated, while hippocampal GABA release was positively correlated to motor activity. No change in cortical and hippocampal glutamate release was observed. In summary, ACh released by the animal placed in a novel environment seems to have two components, one related to motor activity and one related to attention, anxiety and fear. This second component disappears in the familiar environment, where ACh release is directly related to motor activity. The negative relationship between cortical GABA levels and motor activity may indicate that cortical GABAergic activity is involved in habituation.
Title: NO synthesis inhibition decreases cortical ACh release and impairs retention of a conditioned response Kopf SR, Benton RS, Kalfin R, Giovannini MG, Pepeu G Ref: Brain Research, 894:141, 2001 : PubMed
We investigated in rats the effect N(G)-nitro-L-arginine methyl ester (L-NAME) on retention of a passive avoidance response, and cortical ACh release monitored using the microdialysis technique. Post-training administration of L-NAME impaired 24 h retention of a passive avoidance and decreased cortical ACh release. Both effects of L-NAME were reversed by L-Arg. These results suggest that nitric oxide is involved in retention of the passive avoidance response through the modulation of the forebrain cholinergic system.
Title: Overview and perspective on the therapy of Alzheimer's disease from a preclinical viewpoint Pepeu G Ref: Prog Neuropsychopharmacol Biological Psychiatry, 25:193, 2001 : PubMed
1. Drugs effective in Alzheimer's disease (AD) should have several aims: to improve the cognitive impairment, control the behavioural and neurological symptoms, delay the progression of the disease, and prevent the onset. In order to attain these targets, cell and animal models are needed on which to test pathogenetic hypothesis and demonstrate the potential effectiveness of new drugs. This overview examines the results obtained in animal models. They are the link between the molecular and biochemical studies on the disease and the reality of human pathology. 2. The development of animal models reproducing the complexity of AD pathogenetic mechanisms and clinical symptoms still represents a challenge for the preclinical investigators. Moreover, the succession of different animal models well documents the progressive widening of our knowledge of the disease with the identification of new therapeutic targets. 3. The main animal models are listed, and their contribution to the understanding of the pathogenic mechanisms and development of the drugs presently used in AD therapy is described. Moreover, their role in the study of future drugs is analysed 4. Preclinical studies on cholinesterases and animal models mimicking the cholinergic hypofunction occurring in AD have been instrumental in developing cholinesterase inhibitors, which are the only recognised drugs for the symptomatic treatment of AD. 5. Artificially created beta-amyloid (A beta) deposits in normal rats, and transgenic mice overexpressing amyloid precursor protein (APP) are the models on which the future treatment are tested. They are aimed to prevent formation of A beta deposits or its transformation in neuritic plaques. 6. Models of brain inflammation, aging animals, and models of brain glucose and energy metabolism impairment make it possible to identify and assess the activity of anti-inflammatory agents, antioxidants, ampakines and other potentially active agents. 7. It is concluded that the present level of information on AD could never have been reached without preclinical studies, and the development of new drugs will always require extensive preclinical investigations.
Title: Brain inflammatory reaction in an animal model of neuronal degeneration and its modulation by an anti-inflammatory drug: implication in Alzheimer's disease Scali C, Prosperi C, Vannucchi MG, Pepeu G, Casamenti F Ref: European Journal of Neuroscience, 12:1900, 2000 : PubMed
Brain inflammatory processes underlie the pathogenesis of Alzheimer's disease, and nonsteroidal anti-inflammatory drugs have a protective effect in the disease. The aim of this study was to characterize in vivo in the rat brain the inflammatory reaction in response to excitotoxic insult and to investigate the efficacy of nimesulide treatment. Quisqualic acid was injected into the right nucleus basalis of rats. The excitotoxin induced cholinergic degeneration, an intense glial reaction and the production of inflammatory mediators. Three hours after injection, a five-fold elevation in the concentration of interleukin-1beta in the injected area was observed. This elevation was reduced by 50% by nimesulide (10 mg/kg, i.m.) pretreatment. Electron microscope examination and immunocytochemical staining revealed an intense activation of microglia and astrocytes at both 24 h and 7 days after injection. Cyclooxygenase-2-immunoreactivity was induced in the blood vessels of the injected hemisphere in perivascular microglial and endothelial cells 24 h after injection. Seven days postinjection, a cyclooxygenase-2-positive signal was induced in the parenchymal microglia and large amounts of prostaglandin-E2 were measured in the injected area. Twenty-four hours and 7 days after injection, many inducible nitric oxide synthase-positive cells and a high level of nitrite were detected at the injection site. Seven days of nimesulide (10 mg/kg/day, i.m.) treatment strongly attenuated the microglial reaction, reduced the number of inducible nitric oxide synthase-positive cells and completely abolished the increase in prostaglandin-E2 formation. These data provide valuable support in vivo for the potential efficacy of cyclooxygenase-2 inhibitors in Alzheimer's disease therapy.
Interleukin-1beta (10 U) was injected into the nucleus basalis of adult male Wistar rats. The inflammation-induced changes in glial cell morphology and expression of inducible nitric oxide synthase in the injected area, the release of acetylcholine, GABA and glutamate from the ipsilateral cortex, the production of nitrite levels in the injected area and ipsilateral cortex, and changes in motor activity were investigated. Saline-injected rats were used as control. Interleukin-1beta induced an activation of both microglia and astrocytes which was already evident 24 h after injection. Seven days after injection, many reactive microglial cells and astrocytes were seen in the injected area and in other brain regions of the same hemisphere. Microglia reaction, but not astrocyte activation, disappeared 30 days post-injection. Seven days after interleukin-1beta injection, many cells immunopositive for inducible nitric oxide synthase were found surrounding the injection site. Inducible nitric oxide synthase-positive cells were identified, by double staining immunohistochemistry, in the reactive microglial cells and, by electron microscope examination, in the perineuronal subpopulation of resident activated microglia. Microdialysis investigations revealed a transient increase in reactive nitrogen intermediates (at seven days post-injection), a delayed (at 30 days post-injection) increase in GABA and glutamate release, and no changes in acetylcholine release in the ipsilateral cortex in interleukin-1beta, but not saline, injected rats. Inhibition of inducible nitric oxide synthase expression by N(G)-nitro-L-arginine methyl ester administration prevented the increase in nitrogen intermediates and GABA release, but not in glutamate release. Our findings suggest that an inflammatory reaction of the basal forebrain facilitates GABA release through the production of nitric oxide.
In vivo microdialysis was used to assess the effects of novelty and pain on hippocampal ACh release in male and female rats. Experiments were carried out during the dark phase and consisted of 2 days of tests: on Day 1, after Baseline 1, animals were exposed to a new cage (Novelty) to which, 30 min later, a plastic cylinder (Object) was introduced. On Day 2, after Baseline 2, the Formalin test (50 microl of formalin 10%, s.c. injected in the dorsal hindpaw) was carried out in the animal's home cage. All behaviors were recorded. The extracellular levels of ACh in the dorsal hippocampus were estimated, in 10-min samples, by assay of ACh in the dialysates by HPLC. On Day 1 the raw values of ACh were higher in females than in males, but no sex difference was present when the percentage of change was considered. In both sexes the Novelty and Object tests induced an increase in ACh levels with respect to Baseline. Higher levels of exploration were present in females than males during the first 10 min of Novelty. On Day 2, ACh release increased in both sexes during the Formalin test. No sex difference in either ACh raw values or the percentages of change were found. Females showed higher levels of licking and lower levels of activity than males. The present study shows that novelty and pain induce similar hippocampal cholinergic activation in male and female rats but different behaviors. The results are discussed in light of the several anatomical and functional sex differences present in the hippocampus.
Title: Systemic chlorophenylpiperazine increases acetylcholine release from rat hippocampus-implication of 5-HT2C receptors Zhelyazkova-Savova M, Giovannini MG, Pepeu G Ref: Pharmacol Res, 40:165, 1999 : PubMed
The release of acetylcholine (ACh) from the hippocampus of freely moving rats was studied after the systemic and local administration of the 5-HT agonist chlorophenylpiperazine (mCPP), utilising the in vivo microdialysis coupled to HPLC. Intraperitoneally (i.p.) given mCPP at a dose of 8 mg kg(-1)increased the release of ACh from the hippocampus by approximately 96%. This effect was not observed when the agonist was delivered locally through the dialysis tube (reverse dialysis). The mCPP-induced increase of ACh release was prevented by i.p. mesulergine, a 5-HT2A/2C receptor antagonist, at a dose of 2 mg kg(-1). A similar effect was found with the i.p. administration of isoteoline-a putative serotonergic antagonist. Both mesulergine and isoteoline have been shown to prevent also the mCPP-induced increase of ACh release from rat cortex. In the cortex experiments both antagonists were inactive by themselves. In the hippocampus, however, isoteoline, unlike mesulergine, increased significantly the output of ACh when used alone. This effect was haloperidol-sensitive, which implies a possible dopaminergic mechanism. The results of the present work suggest that (i) the effect of mCPP on ACh release could be attributed to stimulation of 5-HT2C receptors located outside the hippocampus and (ii) isoteoline antagonizes this mCPP-induced effect irrespective of its own enhancing action on ACh release.
The long-term effects of beta-amyloid peptide 1-40 injection into the rat forebrain were studied. Ten micrograms of pre-aggregated peptide were injected into the right nucleus basalis of male Wistar rats which were then killed four or six months later. Congo Red staining of histological sections showed that the peptide deposit was aggregated in a fibrillary form four months post-surgery, whereas at six months almost no trace of birefringency was detected at the deposit site, indicating a loss of fibril organization. This result was confirmed by electron microscopic analysis of the peptide deposits. The presence of the peptide at the injection site six months post-surgery was demonstrated by both Haematoxylin staining and beta-amyloid immunoreactivity. The number of choline acetyltransferase-immunoreactive neurons was reduced by 66% in the injected nucleus basalis four months after injection. A decrease in cortical acetylcholine release was also found at this time. Concomitantly with the loss of fibril conformation, a complete recovery of choline acetyltransferase immunoreactivity in the nucleus basalis and of acetylcholine release in the cortex was observed at six months. These data provide in vivo evidence that beta-amyloid neurotoxicity is related to the fibrillary conformation of the peptide aggregates, thus confirming previous in vitro studies.
Title: Effect of subchronic treatment with metrifonate and tacrine on brain cholinergic function in aged F344 rats Giovannini MG, Scali C, Bartolini L, Schmidt B, Pepeu G Ref: European Journal of Pharmacology, 354:17, 1998 : PubMed
The effects of 21-day treatment with the acetylcholinesterase inhibitors metrifonate (80 mg kg(-1) per os (p.o.)) and tacrine (3 mg kg(-1) p.o.), twice daily, on cortical and hippocampal cholinergic systems were investigated in aged rats (24-26 months). Extracellular acetylcholine levels were measured by transversal microdialysis in vivo; choline acetyltransferase and acetylcholinesterase activities were measured ex vivo by means of radiometric methods. Basal cortical and hippocampal extracellular acetylcholine levels, measured 18 h after the last metrifonate treatment, were about 15 and two folds higher, respectively, than in control and tacrine-treated rats. A challenge with metrifonate further increased cortical and hippocampal acetylcholine levels by about three and four times, respectively. Basal extracellular acetylcholine levels, measured 18 h after the last treatment with tacrine were not statistically different from those of the control rats. A challenge with tacrine increased cortical and hippocampal extracellular acetylcholine levels by about four and two times. A 75% inhibition of cholinesterase activity was found 18 h after the last metrifonate administration, while only a 15% inhibition was detectable 18 h after the last tacrine administration. The challenge with metrifonate or tacrine resulted in 90 and 80% cholinesterase inhibition, respectively. These results demonstrate that in aging rats a subchronic treatment with metrifonate results in a long-lasting, cholinesterase inhibition, and a persistent increase in acetylcholine extracellular levels which compensate for the age-associated cholinergic hypofunction. Metrifonate is therefore a potentially useful agent for the cholinergic deficit accompanying Alzheimer's disease.
The activation of the cortical cholinergic system was investigated in 3- and 25-month-old male Wistar rats, by measuring by transversal microdialysis the changes in cortical extracellular acetylcholine (ACh) levels during the performance of simple spontaneous tasks involving exploratory activity and working memory. Two days after implantation of the microdialysis probe in the frontal cortex, object recognition was investigated by either moving the rats from the home cage to the arena containing the objects or keeping the rats in the arena and introducing the objects. Spontaneous alternation was investigated in a Y runway. Young rats discriminated between familiar and novel objects and alternated in the Y runway, while aged rats were unable to discriminate. Whenever rats were moved from the home cage to the arena, ACh release increased (+70-80%) during the exploratory activity. Handling per se had no effect on extracellular ACh levels. When young rats were left in the arena, introduction of the objects caused some exploratory activity and object recognition but no increase in ACh release. ACh release increased by about 300% during spontaneous alternation. In aging rats basal extracellular ACh levels and their increase after placement in the arena were less than half that in young rats. Our work demonstrates that a novel environment activates the cortical cholinergic system, which presumably is associated with arousal mechanisms and selective attentional functions. It also demonstrates that in aging rats the cortical cholinergic hypofunction is associated with a loss of non-spatial working memory.
Title: Activation of non-NMDA receptors stimulates acetylcholine and GABA release from dorsal hippocampus: a microdialysis study in the rat Giovannini MG, Rakovska A, Della Corte L, Bianchi L, Pepeu G Ref: Neuroscience Letters, 243:152, 1998 : PubMed
The effect of the non-N-methyl-D-aspartate (NMDA) agonists (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and quisqualate (QUIS) on the release of acetylcholine (ACh), gamma-amino butyric acid (GABA), aspartate (Asp) and glutamate (Glu) from the hippocampus of freely moving rats was studied by transversal microdialysis. Intracerebroventricular (i.c.v.) administration of the non-NMDA receptor agonist AMPA (0.5 nmol) enhanced (by about 200%) ACh release from the hippocampus. The effect of AMPA was completely antagonized by 6-nitro-7-sulphamoyl-benz(f)quinoxaline-2,3-dione (NBQX; 2 nmol, i.c.v). No effect was seen when AMPA was perfused through the septum. However, AMPA (200 microM) locally applied to the hippocampus, increased (by about 200%) ACh release. QUIS (200 microM) applied locally to the hippocampus produced a long-lasting increase in the release of ACh (by about 215%) and GABA (by about 460%). Local infusion of tetrodotoxin (1 microM) decreased ACh and GABA basal extracellular levels, and abolished the QUIS-induced increase in ACh and GABA. Our results demonstrate that non-NMDA glutamatergic receptors in the hippocampus regulate hippocampal release of GABA and ACh.
Title: The acetylcholine, GABA, glutamate triangle in the rat forebrain Pepeu G, Blandina P Ref: Journal de Physiologie (Paris), 92:351, 1998 : PubMed
The present overview demonstrates that stress, fear, novelty, and learning processes are associated with arousal and increases of extracellular levels of cortical and hippocampal ACh, independently of increases of motor activity. Forebrain cholinergic systems appears to be regulated by GABAergic and glutamatergic inputs. However, several other neurotransmitter systems play a role.
The effects of neurotensin (NT) on the release of acetylcholine (ACh), aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) from the hippocampus of freely moving rats were studied by transversal microdialysis. ACh was detected by High Performance Liquid Chromatography (HPLC) with electrochemical detection while GABA, glutamate and aspartate were measured using HPLC with fluorometric detection. Neurotensin (0.2 and 0.5 microM) administered locally through the microdialysis probe to the hippocampus produced a long-lasting and concentration-dependent increase in the basal extracellular levels of GABA and ACh but not of glutamate and aspartate. The increase in the extracellular levels of GABA and ACh produced by 0.5 microM neurotensin in the hippocampus reached a maximum of about 310% for GABA and 250% for ACh. This stimulant effect of NT was antagonized by the NT receptor antagonist SR 48692 (100 microg/kg, i.p.). Local infusion of tetrodotoxin (1 microM) decreased the basal release of ACh, GABA, Asp, Glu and prevented the 0.2 microM NT-induced increase in GABA and ACh release. The effect of NT on the release of ACh was blocked by the GABA(A) receptor antagonist bicuculline (2-10 microM). Our findings indicate for the first time that neurotensin plays a neuromodulatory role in the regulation of GABAergic and cholinergic neuronal activity in the hippocampus of awake and freely moving rats. The potentiating effects of neurotensin on GABA and ACh release in the hippocampus are probably mediated by (i) NT receptors located on GABAergic cell bodies and (ii) through GABA(A) receptors located on cholinergic nerve terminals.
Title: Effects of novelty, pain and stress on hippocampal extracellular acetylcholine levels in male rats Aloisi AM, Casamenti F, Scali C, Pepeu G, Carli G Ref: Brain Research, 748:219, 1997 : PubMed
In vivo microdialysis was used to assess the effects of Novelty, persistent pain (Formalin test) and stress (Restraint) on hippocampal acetylcholine (ACh) release. Experiments were carried out during the dark phase, i.e. during the active period of the animal, and consisted of four experimental phases: Baseline (30 min), Novelty (30 min), Formalin test (90 min) and Restraint (30 min); each animal was consecutively exposed to all phases. The extracellular levels of ACh in the dorsal hippocampus were estimated by measurement of its concentration in the perfusion fluid by high-performance liquid chromatography with electrochemical detection. The introduction to a new environment (Novelty) induced in all rats higher ACh levels than Baseline. Formalin treatment decreased ACh release only in animals considered 'Inactive' during the Novelty phase while no modification in ACh release was observed in the 'Active' ones. Restraint did not produce any modification of ACh release but increased Corticosterone plasma levels both in sham- and formalin-treated animals. Results indicate that Novelty, but not Formalin or Restraint, increases ACh release in the hippocampus and that the type of behavioral state displayed by the animal at the time of formalin injection determines the response of the septo-hippocampal cholinergic pathway.
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.
Title: Glutamatergic modulation of cortical acetylcholine release in the rat: a combined in vivo microdialysis, retrograde tracing and immunohistochemical study Giovannini MG, Giovannelli L, Bianchi L, Kalfin R, Pepeu G Ref: European Journal of Neuroscience, 9:1678, 1997 : PubMed
The microdialysis technique with one or two probes was used to investigate the modulation of cortically projecting cholinergic neurons by glutamatergic input in the rat in vivo. Male albino Wistar rats (250-300 g) were used. Under chloral hydrate anaesthesia microdialysis membranes were positioned in the parietal cortex, nucleus basalis magnocellularis (NBM) or medial septum. Acetylcholine was assayed using high-performance liquid chromatography (HPLC) with electrochemical detection while GABA was detected using HPLC with fluorimetric detection after derivatization of the amino acid with o-phthalaldehyde. Septo-cortical neurons were retrogradely labelled with fluoro-gold. Double labelling with choline acetyltransferase (ChAT) immunoreactivity was performed to identify these neurons. Our main findings were that: (i) i.c.v. administration of the NMDA antagonist 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 1-5 nmol) increased cortical acetylcholine outflow; (ii) local administration of CPP (100 microM) to the cortex had no effect on cortical acetylcholine outflow; (iii) local administration of CPP (100 microM) to the NBM decreased cortical acetylcholine outflow; (iv) local administration of CPP (100-200 microM) to the septum increased cortical GABA and acetylcholine outflow; (v) administration of muscimol to the septum prevented the effect of CPP on cortical acetylcholine outflow; (vi) retrograde tracing with fluoro-gold labelled cell bodies in the medial septum; (vii) septal fluoro-gold-positive neurons were not ChAT-immunoreactive. Our in vivo neurochemical results, in combination with retrograde tracing and immunohistochemistry, indicate that the cortically projecting cholinergic system is indirectly regulated by a glutamatergic input via a polysynaptic GABAergic circuitry located in the septum.
Title: Differential susceptibility of senile and lesion-induced astrogliosis to phosphatidylserine Jeglinski W, Pepeu G, Oderfeld-Nowak B Ref: Neurobiology of Aging, 18:81, 1997 : PubMed
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.
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.
Title: Tacrine administration enhances extracellular acetylcholine in vivo and restores the cognitive impairment in aged rats Scali C, Giovannini MG, Prosperi C, Bartolini L, Pepeu G Ref: Pharmacol Res, 36:463, 1997 : PubMed
The effect of oral tacrine administration on cortical and hippocampal extracellular acetylcholine (ACh) levels has been investigated by a microdialysis technique, coupled to a HPLC method, in 6- and 22-24-month-old rats. In order to assess whether the increase in extracellular ACh levels was associated with an improvement in the age-related cognitive impairment, the object recognition and step-trough passive avoidance tests were carried out in the treated rats. The extracellular ACh levels measured in the cortex and hippocampus of aged rats without cholinesterase inhibitor in the perfusion Ringer solution were 39 and 54% lower, respectively, than in the young rats. At the dose of 3 mg kg-1, tacrine brought about a three- to four-fold increase in extracellular ACh levels, both in young and aged rats, which peaked 60-80 min after administration and disappeared within the next 60 min. At the same dose, tacrine caused a twofold increase in extracellular ACh levels in the hippocampus of young rats and a sixfold increase in aged rats. The absolute ACh levels at the peak in aged rats were not significantly different from those of young rats. In the object recognition test, aging rats were unable to discriminate between the familiar and novel object. Discrimination was restored by the administration of tacrine at the dose of 1 and 3 mg kg-1, but not 0. 3 mg kg-1 given 30 min before the first trial. Tacrine (3 mg kg-1 p. o.) administered to aging rats before the training trial significantly improved the acquisition of the passive avoidance conditioned response. Our findings demonstrate that tacrine increased both cortical and hippocampal extracellular ACh levels and improved behavioural functions in aged rats.
Title: Selective muscarinic antagonists differentially affect in vivo acetylcholine release and memory performances of young and aged rats Vannucchi MG, Scali C, Kopf SR, Pepeu G, Casamenti F Ref: Neuroscience, 79:837, 1997 : PubMed
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.
Title: Increase of cortical acetylcholine release after systemic administration of chlorophenylpiperazine in the rat: an in vivo microdialysis study Zhelyazkova-Savova M, Giovannini MG, Pepeu G Ref: Neuroscience Letters, 236:151, 1997 : PubMed
The changes in acetylcholine (ACh) release from the cortex of freely moving rats after systemic administration of chlorophenylpiperazine (mCPP), a 5-HT2C agonist, were measured utilising microdialysis coupled to high performance liquid chromatography. mCPP administered intraperitoneally (i.p.) increased cortical ACh release, but failed to do so when applied locally in the cortex. The effect of i.p. administered mCPP on cortical ACh release was prevented by i.p. injection of mesulergine, a 5-HT2A/2C receptor antagonist, and isoteoline, a compound previously shown to antagonize behavioural effects of mCPP. An increase of cortical ACh release was also found after the local administration of mCPP in nucleus basalis magnocellularis (NBM). The results of the present work suggest that 5-HT2C receptors located in NBM are involved in the modulation of cortical ACh release in the rat.
1. The effects of histamine and agents at histamine receptors on spontaneous and 100 mM K(+)-evoked release of acetylcholine, measured by microdialysis from the cortex of freely moving, rats, and on cognitive tests are described. 2. Local administration of histamine (0.1-100 microM) failed to affect spontaneous but inhibited 100 mM K(+)-stimulated release of acetylcholine up to about 50%. The H3 receptor agonists (R)-alpha-methylhistamine (RAMH) (0.1-10 microM), imetit (0.01-10 microM) and immepip (0.01-10 microM) mimicked the effect of histamine. 3. Neither 2-thiazolylethylamine (TEA), an agonist showing some selectivity for H1 receptors, nor the H2 receptor agonist, dimaprit, modified 100 mM K(+)-evoked release of acetylcholine. 4. The inhibitory effect of 100 microM histamine was completely prevented by the highly selective histamine H3 receptor antagonist, clobenpropit but was resistant to antagonism by triprolidine and cimetidine, antagonists at histamine H1 and H2 but not H3 receptors. 5. The H3 receptor-induced inhibition of K(+)-evoked release of acetylcholine was fully sensitive to tetrodotoxin (TTX). 6. The effects of intraperitoneal (i.p.) injection of imetit (5 mg kg-1) and RAMH (5 mg kg-1) were tested on acetylcholine release and short term memory paradigms. Both drugs reduced 100 mM K(+)-evoked release of cortical acetylcholine, and impaired object recognition and a passive avoidance response. 7. These observations provide the first evidence of a regulatory role of histamine H3 receptors on cortical acetylcholine release in vivo. Moreover, they suggest a role for histamine in learning and memory and may have implications for the treatment of degenerative disorders associated with impaired cholinergic function.
Title: A2 adenosine receptors: their presence and neuromodulatory role in the central nervous system Latini S, Pazzagli M, Pepeu G, Pedata F Ref: General Pharmacology, 27:925, 1996 : PubMed
1. Adenosine is an endogenous neuromodulator that exerts its depressant effect on neurons by acting on the A1 adenosine receptor subtype. Excitatory actions of adenosine, mediated by the activation of the A2 adenosine receptor subtype, have also been shown in the central nervous system. 2. Adenosine A2a receptors are highly localized in the striatum, as demonstrated by the binding assay of the A2a selective agonist, CGS2680, and by analysis of the A2 receptor mRNA localization with in situ hybridization histochemistry. However, adenosine A2a, receptors, albeit at lower levels, are also localized in other brain regions, such as the cortex and the hippocampus. 3. In the striatum, adenosine A2a, receptors are implicated in the control of motor activity. Evidences exists of an antagonistic interaction between adenosine A2a and dopamine D2 receptors. 4. Utilizing selective agonists and antagonists for adenosine A2a receptors, their role in the modulation of the release of several neurotransmitters (acetylcholine, dopamine, glutamate, GABA) has been extensively studied in the brain (striatum, cortex, hippocampus). Controversial results have been obtained and, because the overall effect of endogenous adenosine in the brain is that of an inhibitory tonus, the physiological meaning of the excitatory A2 receptor remains to be clarified.
Title: Enhanced acetylcholine release in the hippocampus and cortex during acquisition of an operant behavior Orsetti M, Casamenti F, Pepeu G Ref: Brain Research, 724:89, 1996 : PubMed
The activity of the septo-hippocampal and nucleus basalis-cortical cholinergic pathways was investigated by measuring changes in the extracellular acetylcholine levels in the hippocampus and parietal cortex, by means of transversal microdialysis, during the acquisition and recall of a positively reinforced operant behavior. Adult male Wistar rats were trained in a sound-isolated operant chamber equipped with a single lever. The positive reinforcement was represented by food pellets and the number of cumulative reinforced responses was recorded every 30 min. Five groups of rats were used. Unoperated animals were used as controls. In two groups of untrained animals, the microdialysis tubes were transversally implanted in the parietal cortex, and hippocampus and the training in the operant behavior chamber began 24 h after surgery. In two further groups the microdialysis tubes were implanted in the parietal cortex, and hippocampus after training for 15 days in the operant chamber. Food was removed 12 h before training. The time needed by the control rats to reach a stable baseline of reinforced responses was 83 +/- 12 min, while in the untrained rats implanted with dialysis probes in the cerebral cortex and in the hippocampus was 621 +/- 129 and 521 +/- 126 min, respectively, and in those pretrained and implanted in cerebral cortex and in the hippocampus was 116 +/- 38 and 217 +/- 59 min, respectively. In the untrained operated rats, both cortical and hippocampal extracellular acetylcholine levels remained constant until the number of reinforced responses was low but increased significantly (+156% in the cortex and +183% in the hippocampus) in the first 30 min period in which there was a sharp rise in the reinforced responses. In the pretrained operated rats, neither in the cortex nor in the hippocampus was the increase in response rate accompanied by a statistically significant increase in extracellular acetylcholine levels. Our findings demonstrate that activation of the forebrain cholinergic pathways occurs during the acquisition of a rewarded operant responses, while recall of the same behavior is not associated with the activation of the cholinergic system.
Title: Amyloid beta-peptides injection into the cholinergic nuclei: morphological, neurochemical and behavioral effects Pepeu G, Giovannelli L, Casamenti F, Scali C, Bartolini L Ref: Prog Brain Res, 109:273, 1996 : PubMed
The enantiomers of 3-alpha-tropyl 2-(phenylthio)butyrate (SM32, 1) were prepared by chiral synthesis and tested for analgesic, cognition-enhancing, and ACh-releasing properties. They show enantioselectivity in some of the tests, the eutomer being related in configuration to R-(+)-hyoscyamine.
The enantiomers of two alpha-tropanyl esters, SM21 (1) and PG9 (2), derived from (+)-R-hyoscyamine, that act by increasing the central cholinergic tone, were obtained by esterification after resolution of the corresponding racemic acids [(-)-S-1, (-)-R-2 and (+)-S-2] and by stereospecific synthesis [(+)-R-1]. Their analgesic and cognition-enhancing activities were tested in mice and their ACh-releasing properties determined on rat parietal cortex. These compounds show enantioselectivity in analgesic and cognition-enhancing tests on mice, the eutomers being the isomers which possess the same spatial arrangement of the groups on the chiral atom as (+)-R hyoscyamine [(+)-R-SM21, (+)-S-PG9]. The ACh-releasing effect of the enantiomers of SM21 in rats is in agreement with the results in mice, while PG9 enantiomers do not show any appreciable enantioselectivity in this test. On the basis of the different effects of the 5-HT4 antagonist SDZ 205557 on analgesia induced by the enantiomers of 1 and 2 and by (+)-R-hyoscyamine and the alpha-tropanyl ester of 2-phenylpropionic acid 3, a mechanism of action is proposed for this class of compounds.
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.
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).
Title: Peripherally injected scopolamine differentially modulates acetylcholine release in vivo in the young and aged rats Scali C, Vannucchi MG, Pepeu G, Casamenti F Ref: Neuroscience Letters, 197:171, 1995 : PubMed
The effect of intraperitoneal administration of scopolamine (1 mg/kg) on acetylcholine (ACh) release in vivo in 3- and 24-month-old freely behaving rats was investigated in the cerebral cortex, hippocampus and striatum by means of transverse microdialysis. In the parietal cortex, the increase in ACh release after scopolamine administration was significantly greater in the old than in the young rats, reaching a maximum increase of about 600 and 300% in the old and young animals, respectively. In the hippocampus, scopolamine caused a larger increase in ACh release in the young (+900%) than in the old rats (+600%). In the striatum of aged rats, a 40% increase occurred only at 40 min after scopolamine administration while in the striatum of young animals the increase lasted for at least 2 h, reaching a maximum of about 100%. These findings demonstrate that the modulation of ACh release in vivo is affected in a different manner in the cerebral cortex than in the hippocampus and striatum by aging.
Title: Muscarinic receptor modulation of acetylcholine release from rat cerebral cortex and hippocampus Vannucchi MG, Pepeu G Ref: Neuroscience Letters, 190:53, 1995 : PubMed
An attempt to identify the muscarinic receptor subtypes involved in presynaptic modulation of acetylcholine (ACh) release from cortical and hippocampal slices was made by means of several muscarinic antagonists. Cortical and hippocampal slices prepared from adult rats were superfused with Krebs solution containing physostigmine; ACh content of the superfusate at rest and after electrical stimulation (1 Hz) was quantified by high performance liquid chromatography. The antagonists were added to the Krebs at the concentration of 1 microM. ACh release at rest was enhanced only in the cortex by (+/-)-5,11-dihydro-11-([(2-[2-[(dipropylamino)methyl]-1- piperidinyl)ethyl)amino]carbonyl)-6H-pyrido[2,3-b](1,4)- benzodiazepine-6-one (AFDX384), an M2/M4 selective antagonist. The evoked ACh release from the cerebral cortex was significantly increased by AFDX384, methoctramine, pirenzepine, M2/M4, M2 and M1 selective antagonists, respectively, and scopolamine. This finding suggests that M1, M2 and M4 presynaptic receptor subtypes could regulate evoked ACh release in the cortex. In hippocampal slices, the evoked ACh release was enhanced by AFDX384, pirenzepine and scopolamine but not by methoctramine. In this region ACh release seems therefore regulated only by M1 and M4 receptor subtypes. The M3 antagonist (+/-)-p-fluorohexahydro-sila-difenidol hydrochloride did not affect ACh release.
Title: Administration of amyloid beta-peptides into the medial septum of rats decreases acetylcholine release from hippocampus in vivo Abe E, Casamenti F, Giovannelli L, Scali C, Pepeu G Ref: Brain Research, 636:162, 1994 : PubMed
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.
Title: Glutamatergic regulation of acetylcholine output in different brain regions: a microdialysis study in the rat Giovannini MG, Camilli F, Mundula A, Pepeu G Ref: Neurochem Int, 25:23, 1994 : PubMed
The glutamatergic regulation of cortical and striatal cholinergic neurons was investigated by measuring ACh output from the parietal cortex and striata of freely moving rats after administration of the competitive NMDA-receptor antagonist 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). It has been shown that intracerebroventricular administration of 5 nmol of CPP brings about a long lasting 100% increase in ACh output from the parietal cortex but does not affect ACh output from the striatum. Conversely, local perfusion of the striata with 50 microM CPP results in a 45% decrease in ACh output from the striatum but has no effect on parietal ACh output. The decrease in striatal ACh output induced by CPP is antagonized by concurrent perfusion with NMDA. In conclusion, glutamate may exert both inhibitory and excitatory modulatory effects on ACh output, through NMDA receptors, according to the neuronal circuitry existing in different brain regions.
Title: NMDA receptor antagonists decrease GABA outflow from the septum and increase acetylcholine outflow from the hippocampus: a microdialysis study Giovannini MG, Mutolo D, Bianchi L, Michelassi A, Pepeu G Ref: Journal of Neuroscience, 14:1358, 1994 : PubMed
The modulation of the septohippocampal cholinergic pathway by glutamatergic or GABAergic inputs was studied by monitoring the outflow of ACh collected via a transversal microdialysis probe implanted into the hippocampus and other brain areas of freely moving rats. In one set of experiments a transversal microdialysis membrane was inserted in the dorsal hippocampus, drugs were administered intracerebroventricularly through a cannula implanted in the lateral ventricle, and ACh outflow in the dialysate was measured by an HPLC method with an electrochemical detector. The dialysis membrane was usually perfused with Ringer's solution containing 7 microM physostigmine sulfate. Intracerebroventricular injections of the NMDA antagonists 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 1-50 nmol), MK801 (0.5-20 nmol), and D(-)-2-amino-7-phosphonoheptanoic acid (100 nmol) brought about an increase in hippocampal ACh outflow while the non-NMDA antagonist 6,7-dinitroquinoxaline-2,3-dione (0.25-20 nmol) was without effect. The increase in ACh outflow following CPP administration was dose dependent and reached a maximum of about 500%. It was abolished by TTX (0.5 microM) delivered locally to the hippocampus via the dialysis membrane and prevented by intracerebroventricular injection of the GABA agonist muscimol (5 nmol). In a second set of experiments, one microdialysis membrane was inserted in the dorsal hippocampus to detect ACh outflow and another in the septum to administer drugs locally and at the same time detect septal GABA outflow. The septal dialysis membrane was perfused with Ringer's solution without physostigmine, and GABA levels in the dialysate were measured by an HPLC method with a fluorescence detector. CPP (100 microM) perfused through the septum resulted in a decrease in septal GABA outflow and a concomitant increase in hippocampal ACh outflow. Muscimol (100 microM) administration into the septum abolished the effect of CPP on hippocampal ACh outflow but did not affect septal GABA outflow. These results demonstrate that in the septum NMDA receptors tonically activate GABAergic neurons which in turn inhibit the cholinergic septohippocampal neurons.
Title: In vivo regulation of extracellular adenosine levels in the cerebral cortex by NMDA and muscarinic receptors Pazzagli M, Corsi C, Latini S, Pedata F, Pepeu G Ref: European Journal of Pharmacology, 254:277, 1994 : PubMed
The adenosine concentration in samples of perfusate was determined 24 h after implantation of microdialysis fibre in the cortex. High performance liquid chromatography coupled with a fluorometric detector was used. K+ (100 mM) depolarization was followed by a 2- to 4-fold increase in adenosine efflux. The addition of tetrodotoxin (1 microM) to the perfusate was followed by a decrease in spontaneous and K(+)-evoked adenosine efflux. The increase induced by high K+ was markedly inhibited by the NMDA receptor antagonist, D(-)-2-amino-7-phosphonoheptanoic acid (1 mM, D-AP7), but not by the muscarinic receptor antagonist, atropine (1.5 microM). The acetylcholine esterase inhibitor, physostigmine (7 microM), and the muscarinic receptor agonist, oxotremorine (100 microM), significantly enhanced the K(+)-evoked increase in adenosine. The spontaneous efflux of adenosine was not modified by any of the drugs tested. A neurotoxic lesion of the cholinergic pathway innervating the cortex, although inducing a marked decrease in cortical choline acetyltransferase activity, did not significantly modify the cortical adenosine efflux. It is concluded that, under K(+)-depolarizing conditions, adenosine efflux is triggered by excitatory amino acids and enhanced by muscarinic activation.
Title: Memory disorders: novel treatments, clinical perspective Pepeu G Ref: Life Sciences, 55:2189, 1994 : PubMed
The aging of the society is accompanied by a strong pressure to develop drugs to treat age-related memory disorders. This paper examines the discrepancy between the results of preclinical tests, which have identified a large number of putative cognition enhancers, and the limited clinical efficacy of most of them. The predictivity of the preclinical tests is discussed, and the criteria for evaluating the therapeutic efficacy of the cognition enhancers are examined. An analysis is made of the novel treatments presently available or under investigation. In light of the results of the trials with tacrine a warning is given not to expect striking clinical improvements by only attempting to restore signal transmission, disrupted by aging and dementia, without modifying the underlying morphological alterations.
Title: Dysfunction of the brain cholinergic system during aging and after lesions of the nucleus basalis of Meynert Pepeu G, Marconcini Pepeu I Ref: J Neural Transm Suppl, 44:189, 1994 : PubMed
In order to establish how closely the models mimic aging and Alzheimer's disease, a comparison was made, using the extensive literature available, between brain cholinergic dysfunction in aging animals and man, and between that in animals with lesions of the nucleus basalis of Meynert and in subjects affected by Alzheimer's disease. It is concluded that cholinergic dysfunction in the aging rat closely resembles that in aging man. A similarity can also be found between the cortical dysfunction induced by lesions of the nucleus basalis and that occurring in patients with Alzheimer's disease. However, cholinergic dysfunction only represents a limited aspect of the neorotransmitter deficits and neuropathological alterations of the disease.
Title: Nerve growth factor increases extracellular acetylcholine levels in the parietal cortex and hippocampus of aged rats and restores object recognition Scali C, Casamenti F, Pazzagli M, Bartolini L, Pepeu G Ref: Neuroscience Letters, 170:117, 1994 : PubMed
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.
Title: Long-term ethanol consumption by rats: effect on acetylcholine release in vivo, choline acetyltransferase activity, and behavior Casamenti F, Scali C, Vannucchi MG, Bartolini L, Pepeu G Ref: Neuroscience, 56:465, 1993 : PubMed
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.
Title: Overview and future directions of CNS cholinergic mechanisms Pepeu G Ref: Prog Brain Res, 98:455, 1993 : PubMed
The complex picture of age-associated brain cholinergic deficiency in humans and animals, and the possibilities of correcting it, are presented in this article. The changes that occur during ageing and senile dementias in cholinergic neurones and receptors and in the release and synthesis of acetylcholine are described and discussed. The drugs that have so far been administered to humans to correct cholinergic deficiency are listed and the effects of cholinesterase inhibitors, nerve growth factor and phosphatidylserine are discussed in some detail.
Title: Phosphatidylserine reverses the age-dependent decrease in cortical acetylcholine release: a microdialysis study Casamenti F, Scali C, Pepeu G Ref: European Journal of Pharmacology, 194:11, 1991 : PubMed
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.
Title: Effect of thyrotropin releasing hormone (TRH) on acetylcholine release from different brain areas investigated by microdialysis Giovannini MG, Casamenti F, Nistri A, Paoli F, Pepeu G Ref: British Journal of Pharmacology, 102:363, 1991 : PubMed
1. The effect of thyrotropin releasing hormone (TRH) administration upon acetylcholine (ACh) release in freely moving rats was investigated by means of transversal microdialysis coupled to h.p.l.c. TRH administered either s.c. or via local perfusion increased the ACh release from cortex and hippocampus but not from the striatum. The increase in ACh release was maintained after 7 days of s.c. administration of TRH. 2. After s.c. injection of the neuropeptide, the increase in ACh release was dose-dependent and reached a maximum at 40 min after administration. The maximal percentage increases were 18, 52, 66 and 89% at doses of 1, 2.5, 5 and 10 mg kg-1 and 35, 48 and 54% at doses of 2.5, 5 and 10 mg kg-1 in the cortex and hippocampus, respectively. The effect of TRH was dependent on neuronal activity since it was completely inhibited by perfusion with tetrodotoxin (TTX), 5 X 10(-7) M. 3. Perfusion with TRH, 2.5 micrograms microliters-1, caused 198% and 150% increase in ACh release 60 and 80 min after the beginning of the perfusion in the cortex and hippocampus, respectively. After this initial peak, a 100% increase in ACh release persisted throughout the perfusion. 4. Systemic TRH administration was followed by marked hyperactivity and stereotyped behaviour that showed a time course shorter than that of the increase in ACh release. 5. These findings demonstrate that TRH exerts a strong stimulant action on cortical and hippocampal cholinergic pathways.
Title: A decrease in brain catecholamines prevents oxiracetam antagonism of the effects of scopolamine on memory and brain acetylcholine Giovannini MG, Spignoli G, Carla V, Pepeu G Ref: Pharmacol Res, 24:395, 1991 : PubMed
The effect of oxiracetam on passive avoidance conditioned response and acetylcholine (ACh) levels in rats with selective lesions of the central monoaminergic pathways was investigated. The lesions were followed by a marked decrease in cortical serotonin (-88%), noradrenaline (-54%) and striatal dopamine (-57%) levels, while neither the performance of a passive avoidance conditioned response nor brain ACh levels were affected. Scopolamine (hyoscine) administration (0.63 mg/kg, s.c.) to lesioned rats exerted the expected amnesic effect, associated with a decrease in hippocampal, cortical and striatal ACh levels. In the rats with degeneration of dopaminergic and noradrenergic but not serotoninergic pathways, oxiracetam (50 and 100 mg/kg, s.c.) was unable to prevent both amnesia and the decrease in brain ACh levels caused by scopolamine. The effect of oxiracetam was prevented by haloperidol (0.2 mg/kg, s.c.). Our findings support the hypothesis that an interaction between monoaminergic and cholinergic neurotransmitter systems may be involved in the actions of nootropic drugs on cognitive functions.
Title: Decrease of acetylcholine release from cortical slices in aged rats: investigations into its reversal by phosphatidylserine Vannucchi MG, Casamenti F, Pepeu G Ref: Journal of Neurochemistry, 55:819, 1990 : PubMed
The release of total acetylcholine (ACh) and [3H]ACh was investigated in electrically stimulated cortical slices prepared from 4- and 18-month-old male Wistar rats. The slices were prelabeled with [3H]choline ([3H]Ch) and perfused with Krebs solution containing physostigmine. Total ACh was measured and the nature of the tritium efflux identified by HPLC. The total tritium content in the slices at the end of the incubation period was half as great in the old as in young rats. A linear relationship was found between stimulation frequencies (2, 5, and 10 Hz) and fractional [3H]ACh release in both young and old rats. In the latter the release was significantly smaller. At 10 Hz stimulation frequency the ratio between the two 2-min stimulation periods, S2/S1, was higher in the 18-month-old rats than in the young rats. Specific activity of the evoked ACh release was significantly smaller in S2 than in S1 in 4-month-old rats only. These findings indicate that the young synthetize ACh from endogenous unlabeled Ch more than older rats. In 18-month-old rats both the evoked total ACh and [3H]ACh release, expressed as picograms per minute, showed an approximately 50% decrease in both S1 and S2 stimulation periods, with no significant difference in specific activity. Phosphatidylserine (PtdSer) administration (15 mg/kg, i.p. daily) for 1 week to 18-month-old rats prevented the reduction in total evoked ACh release but not the reduction in evoked [3H]ACh release. The specific activity of ACh release was therefore significantly smaller than that of the young and untreated old rats.
Title: Cholinergic and noradrenergic denervations decrease labelled purine release from electrically stimulated rat cortical slices Pedata F, Di Patre PL, Giovannini MG, Pazzagli M, Pepeu G Ref: Neuroscience, 32:629, 1989 : PubMed
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.
1. This review has two aims: first, to marshal and discuss evidences demonstrating an interaction between nootropic drugs and brain cholinergic mechanisms; second, to define the relationship between the effects on cholinergic mechanisms and the cognitive process. 2. Direct or indirect evidences indicating an activation of cholinergic mechanisms exist for pyrrolidinone derivatives including piracetam, oxiracetam, aniracetam, pyroglutamic acid, tenilsetam and pramiracetam and for miscellaneous chemical structures such as vinpocetine, naloxone, ebiratide and phosphatidylserine. All these drugs prevent or revert scopolamine-induced disruption of several learning and memory paradigms in animal and man. 3. Some of the pyrrolidinone derivatives also prevent amnesia associated with inhibition of acetylcholine synthesis brought about by hemicholinium. Oxiracetam prevents the decrease in brain acetylcholine and amnesia caused by electroconvulsive shock. Oxiracetam, aniracetam and pyroglutamic acid prevent brain acetylcholine decrease and amnesia induced by scopolamine. Comparable bell-shaped dose-effect relationships result for both actions. Phosphatidylserine restores acetylcholine synthesis and conditioned responses in aging rats. 4. The mechanisms through which the action on cholinergic systems might take place, including stimulation of the high affinity choline uptake, are discussed. The information available are not yet sufficient to define at which steps of the cognitive process the action on cholinergic system plays a role and which are the influences of the changes in cholinergic function on other neurochemical mechanisms of learning and memory.
Title: The relationship between the behavioral effects of cognition-enhancing drugs and brain acetylcholine. Nootropic drugs and brain acetylcholine Pepeu G, Spignoli G, Giovannini MG, Magnani M Ref: Pharmacopsychiatry, 22 Suppl 2:116, 1989 : PubMed
The effect of different doses of the cognition-enhancing agent oxiracetam on the disruption of acquisition of a passive avoidance conditioned response (step-through) by scopolamine 0.6 mg/kg s.c., and on the impairment of performance by scopolamine 0.2 mg/kg s.c. in an eight-arm maze was investigated in adult male Wistar rats. At the end of the experiments, ACh levels were measured in the cerebral cortex and hippocampus by an HPLC method, in order to ascertain whether a relationship exists between the effects of the two drugs on behavior and cholinergic function. In the step-through test, oxiracetam at doses of 50 and 100 mg/kg s. c., given 30 min. before scopolamine, antagonized both scopolamine-induced behavioral disruption and decrease in ACh levels but was inactive at the dose of 300 mg/kg s. c. In the eight-arm maze test, oxiracetam prevented scopolamine-induced disruption of the performance and decrease in brain ACh at the dose of 30 mg/kg s. c. but was inactive at the dose of 100 mg/kg s. c. In conclusion, when scopolamininduced disruption of behavior is prevented or reduced, the scopolamininduced decrease in ACh level, particularly in the hippocampus, is also reduced. The present results therefore offer further confirmation of a relationship between the cognition-enhancing effects of oxiracetam and its effects on hippocampal and cortical cholinergic mechanisms.
Title: Purinergic modulation of cortical acetylcholine release is decreased in aging rats Giovannelli L, Giovannini MG, Pedata F, Pepeu G Ref: Experimental Gerontology, 23:175, 1988 : PubMed
The effect of adenosine, N-ethylcarboxamide adenosine (NECA), and caffeine on acetylcholine (ACh) release was investigated in cortical slices prepared from 3 and 22-24-month-old rats. The slices were perfused with Krebs solution and electrically stimulated at 0.2, 1, and 5 Hz stimulation frequency. In old rats, ACh released by stimulation at 1 and 5 Hz was about half as large as in adult rats. In 22-24-month-old rats, the potency of adenosine was strongly reduced, and a similar significant inhibition of ACh release was obtained with concentrations of 1 microM adenosine in adult and 300 microM in old rats. Conversely, NECA, which has no effect on ACh release in adult rats, brought about a 40% decrease in old rats. Caffeine at 50 microM concentration enhanced, and at 500 microM inhibited, the evoked ACh release in adult rats, but was inactive in old rats. The possibility is envisaged that aging may modify purinergic modulation of ACh release by inducing conformational changes in purinergic receptors or changing adenosine metabolism.
Title: Acetylcholine and brain aging Pepeu G Ref: Pharmacol Res Commun, 20:91, 1988 : PubMed
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.
Title: Recovery of cortical acetylcholine output after ganglioside treatment in rats with lesion of the nucleus basalis Florian A, Casamenti F, Pepeu G Ref: Neuroscience Letters, 75:313, 1987 : PubMed
The changes in acetylcholine release from the cerebral cortex induced by a unilateral electrolytic lesion of the nucleus basalis and by a treatment with GM1 monosialoganglioside (30 mg/kg i.p. for 20 days) were investigated. Acetylcholine release was measured using the cortical cup technique in rats transected at midpontine level. In the lesioned rats treated with saline an ipsilateral 38% decrease in acetylcholine release was observed. GM1 treatment prevented the decrease and brought about a 30% contralateral increase. These results indicate that GM1 induces a functional recovery of the cholinergic neurons ascending to the cortex.
Title: An analysis of cholecystokinin-induced increase in acetylcholine output from cerebral cortex of the rat Magnani M, Florian A, Casamenti F, Pepeu G Ref: Neuropharmacology, 26:1207, 1987 : PubMed
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.
Title: Effect of pyroglutamic acid stereoisomers on ECS and scopolamine-induced memory disruption and brain acetylcholine levels in the rat Spignoli G, Magnani M, Giovannini MG, Pepeu G Ref: Pharmacol Res Commun, 19:901, 1987 : PubMed
The acquisition of a passive avoidance conditioned response was disrupted in the rat by electroconvulsive shock (ECS) and scopolamine administration. D,L-pyroglutamic acid (D,L-PCA) 500 and 1000 mg/kg, administered as arginine salt 120 min before the retest, prevented both the ECS and scopolamine-induced amnesia. Arginine alone was ineffective. Scopolamine brought about a 52 and 39% decrease, respectively, in cortical and hippocampal acetylcholine (ACh) levels, measured by means of a gas-chromatographic method. D,L-PCA 500 and 1000 mg/kg also prevented the decrease in brain ACh level. When the two isomers were studied separately, D-PCA was more effective than L-PCA and antagonized scopolamine-induced amnesia at the doses of 250 and 500 mg/kg. In conclusion, D,L-PCA is active on cortical and hippocampal cholinergic mechanisms and, like other 2-oxopyrrolidone derivatives, shows cognition-enhancing properties.
Title: Interactions between oxiracetam, aniracetam and scopolamine on behavior and brain acetylcholine Spignoli G, Pepeu G Ref: Pharmacol Biochem Behav, 27:491, 1987 : PubMed
The effect of cognition-enhancing agents oxiracetam and aniracetam on scopolamine-induced amnesia and brain acetylcholine decrease was investigated in the rat. Acetylcholine levels were measured by means of a gas-chromatographic method. Scopolamine (0.63 mg/kg IP 60 min before training) prevented the acquisition of a passive avoidance conditioned response ("step through": retest 30 min after training) and brought about a 64, 56 and 42% decrease in acetylcholine level in the cortex, hippocampus and striatum respectively. Oxiracetam (50 and 100 mg/kg IP) administered 30 min before scopolamine reduced the scopolamine-induced amnesic effect and decrease in acetylcholine level in the cortex and hippocampus, but not in the striatum. Lower and higher doses of oxiracetam were ineffective. Aniracetam (100 mg/kg PO) also prevented scopolamine-induced amnesia but attenuated acetylcholine decrease in the hippocampus only. Aniracetam (300 mg PO) reduced acetylcholine decrease in the hippocampus but did not prevent scopolamine-amnesia. In conclusion, oxiracetam and aniracetam exert a stimulatory effect on specific central cholinergic pathways. However, a direct relationship between cognition-enhancing properties and cholinergic activation needs further confirmation.
Title: Effect of phosphatidylserine on acetylcholine release and content in cortical slices from aging rats Vannucchi MG, Pepeu G Ref: Neurobiology of Aging, 8:403, 1987 : PubMed
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.
Title: Effect of BM-5, a presynaptic antagonist-postsynaptic agonist, on cortical acetylcholine release Casamenti F, Cosi C, Pepeu G Ref: European Journal of Pharmacology, 122:288, 1986 : PubMed
The effect of N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl) acetamide (BM-5) on acetylcholine release from the cerebral cortex was investigated in unanaesthetized and urethane-anaesthetized rats. BM-5 at doses ranging from 0.3 to 5 mg/kg i.p. enhanced acetylcholine output in both groups of rats. The maximum increase occurred with 0.5 mg/kg in the unanaesthetized and 2 mg/kg in the anaesthetized rats. The effect lasted approximately 60 min. At the largest doses peripheral muscarinic effects including salivation, chromodachryorrhea and rhinorrhea were also seen. These results demonstrate that BM-5 exerts presynaptic antagonistic and postsynaptic agonistic effects on muscarine receptors in vivo also.
Title: Changes in cortical acetylcholine output induced by modulation of the nucleus basalis Casamenti F, Deffenu G, Abbamondi AL, Pepeu G Ref: Brain Research Bulletin, 16:689, 1986 : PubMed
The modulatory inputs of the cholinergic neurons of the nucleus basalis have been investigated in midpontine transected and freely moving rats by measuring acetylcholine release from the cerebral cortex using the cortical cup technique. Acetylcholine release was found to be the same in both groups of rats indicating similar levels of activity of the cholinergic neurons ascending to the cortex. The electrical stimulation of the nucleus basalis was always followed by an increase in acetylcholine release. Conversely, in some experiments only the stimulation of the midbrain reticular formation enhanced acetylcholine output. The stimulation of the nucleus accumbens prevented the increase in acetylcholine release elicited by amphetamine. The dose-dependent increase in acetylcholine output following IP administration of amphetamine was also prevented by the 6-hydroxydopamine induced degeneration of the dopaminergic fibres. However injection of apomorphine in the nucleus basalis did not modify acetylcholine output. Direct injection of the GABAergic agonist muscimol resulted in a decrease in acetylcholine output which was prevented by picrotoxin. In conclusion, the cholinergic neurons ascending to the cortex can be inhibited by GABA receptors located in the nucleus basalis and stimulated indirectly by dopaminergic fibres.
Title: Biochemical and Behavioral Effects of Ethylcholine Mustard Aziridinium Ion Casamenti F, Bracco L, Pedata F, Pepeu G Ref: Advances in Behavioral Biology, 30:1137, 1986 : PubMed
The effects of both adenosine and caffeine on the release of acetylcholine (ACh) were investigated in slices of cerebral cortex taken from rats pretreated for 30 days with caffeine (100 mg kg-1 daily, dissolved in their drinking water) at rest and during electrical stimulation at frequencies of 0.2, 1 and 5 Hz. The effect of this treatment on adenosine binding sites was also investigated in cortical membranes using N-cyclohexyl-[3H]-adenosine ([3H]-CHA) as a ligand. The chronic caffeine treatment did not change animal growth patterns. Spontaneous exploratory activity appeared to be increased at the 3rd day but was unchanged at the 30th day when compared with controls. Caffeine-treatment increased the number of high affinity binding sites for [3H]-CHA by 64% over the control values. Low affinity binding site density and affinity constants were unaffected. Adenosine 30 microM added to the superfusion fluid decreased electrically stimulated ACh release both in rats drinking tap water and rats drinking caffeine. In rats drinking tap water, caffeine added to the superfusion fluid at a concentration of 50 microM enhanced ACh release, while at 0.5 mM it decreased ACh output from the slices. Both effects were abolished by pretreatment with caffeine in vivo. The results indicate that prolonged consumption of high doses of caffeine causes changes in the responsiveness of cholinergic neurones to caffeine. The change is not shared by adenosine, through whose recognition sites caffeine is believed to act. It is therefore possible that the adaptive changes following repeated caffeine administration involve either only the coupler-transducer mechanism activated by the antagonist, or effects unrelated to receptors.
Title: Choline Fluxes to and from the Rat Cerebral Cortex Studied with the Cup Technique in Vivo Corradetti R, Brehm R, Loffelholz K, Pepeu G Ref: Advances in Behavioral Biology, 30:817, 1986 : PubMed
Title: Effect of adenosine, adenosine derivatives, and caffeine on acetylcholine release from brain synaptosomes: interaction with muscarinic autoregulatory mechanisms Pedata F, Giovannelli L, De Sarno P, Pepeu G Ref: Journal of Neurochemistry, 46:1593, 1986 : PubMed
Synaptosomes, prepared from rat cerebral cortex and hippocampus, were preincubated with [methyl-3H]choline. The effect of adenosine, cyclohexyladenosine, N-ethylcarboxamide adenosine, 2'-deoxyadenosine, and oxotremorine on K+-evoked 3H efflux was investigated. High-voltage electrophoretic separation showed that in the presence of physostigmine, the K+-evoked 3H efflux from hippocampal synaptosomes was 90% [3H]acetylcholine and 10% [3H]choline. Adenosine (30 microM) and oxotremorine (100 microM) both decreased [3H]acetylcholine release from hippocampal synaptosomes. The effect was inversely proportional to the KCl concentration and disappeared at a KCl concentration of 50 mM. Cyclohexyladenosine was approximately 3,000 times more active than adenosine, whereas N-ethylcarboxamide adenosine and 2'-deoxyadenosine were inactive. This indicates that A1 adenosine receptors were involved in the inhibitory effect. Caffeine antagonized the adenosine effect, and at a concentration of 100 microM, it stimulated [3H]acetylcholine efflux. The inhibitory effect of oxotremorine was as great in cortical as in hippocampal synaptosomes. In contrast, adenosine was much less active in cortical than in hippocampal synaptosomes. When inhibitory concentrations of adenosine and oxotremorine were added together into the incubation medium, the effect of adenosine on [3H]acetylcholine release was consistently reduced. An interaction between muscarinic and A1 adenosine presynaptic receptors at a common site modulating acetylcholine release can be assumed.
Title: Neuroregulators and electrical activity: transmitter and modulator influence on the cortical cholinergic system Pepeu G, Pepeu IM Ref: Electroencephalography & Clinical Neurophysiology Suppl, 38:406, 1986 : PubMed
Oxiracetam at 100 and 300 mg/kg i.p. dose levels increased acetylcholine (ACh) utilization in the rat cerebral cortex and hippocampus. ACh utilization was assessed by measuring, with a gas chromatographic method, the decrease in ACh level after inhibiting its synthesis by 15 micrograms intracerebroventricularly (i.c.v.) injection of hemicholinium (HC-3). ACh steady state levels were not affected. Piracetam (300 mg/kg i.p.) also increased ACh utilization in the hippocampus. Repeated daily administration of oxiracetam 100 mg/kg i.p. caused a 31% increase in high-affinity choline uptake (HACU) in the hippocampus. A single administration of 300 mg/kg i.p. of oxiracetam and piracetam also increased HACU rate in the hippocampus. However, the effect of piracetam was over within 3 h, while 3 h after its administration oxiracetam still caused a 40% increase in HACU rate. Oxiracetam (100 mg/kg i.p.) significantly antagonized the impairment in the acquisition of an active-avoidance conditioned response (pole climbing) associated with the inhibition of ACh synthesis by HC-3. These results indicate that oxiracetam enhances the activity of the septohippocampal cholinergic pathways, and to a lesser extent, of the cortical cholinergic network.
Title: Oxiracetam prevents electroshock-induced decrease in brain acetylcholine and amnesia Spignoli G, Pepeu G Ref: European Journal of Pharmacology, 126:253, 1986 : PubMed
In the rat, 1 min following electroshock (ECS) a 46 and 39% decrease in acetylcholine levels was found in the hippocampus and cerebral cortex, respectively. The decrease in the hippocampus was still statistically significant 30 min after ECS. The ECS applied 1 min after training also disrupted the performance of a passive avoidance conditioned response ('step down') tested 30 min later. Oxiracetam (100 and 300 mg/kg i.p.) administered 90 min before training prevented, in a dose-dependent manner, the decrease of acetylcholine in the cerebral cortex and hippocampus. Oxiracetam prevented the ECS disruption of the acquisition of a passive avoidance response. At the dose of 300 mg/kg the acetylcholine level 1 min after ECS was significantly higher than in the sham-treated rats. Piracetam at the same doses was inactive. These results support the hypothesis that oxiracetam may prevent the disruption of the conditioned response by acting on cortical and hippocampal cholinergic mechanisms.
Title: Noradrenergic modulation of 4-aminopyridine-induced acetylcholine release from rat cerebral cortex Magnani M, Mantovani P, Pepeu G Ref: Pharmacol Res Commun, 17:653, 1985 : PubMed
The noradrenergic influence on cortical acetylcholine (ACh) release was investigated by the cortical cup technique in urethane anaestetized rats treated with 4-aminopyridine (4-AP). The following results were obtained: 1) The increase in ACh release induced by 4-AP (3 mg/kg i.p.) was strongly potentiated by pretreatment with -methyl-p-tyrosine (alpha-MPT) which inhibits catecholamine biosynthesis or by N-(2-chloroethyl)-N-ethyl-bromobenzylamine (DSP4) bringing about a selective degeneration of noradrenergic fibres. Neither pretreatment enhanced the spontaneous ACh output. 2) Pretreatment with p-chlorophenylalanine (PCPA), an inhibitor of serotonin synthesis, did not modify 4-AP effect on ACh output. 3) The alpha blockers, yohimbine (1 mg/kg i.p.) and prazosin (4 mg/kg i.p.), did not enhance the 4-AP effect on ACh release but only delayed its onset. 4) Yohimbine (7 mg/kg i.p.) completely reversed 4-AP effect on ACh release which was significantly decreased. It is concluded therefore that pretreatments with alpha-MPT and DSP4 remove an inhibitory noradrenergic control on cortical ACh release. On the other hand, the alpha blockers might interfere with the ionic mechanisms underlaying the 4-AP effect thus, masking the removal of the noradrenergic control, due to an alpha blockade.
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.
Title: Effect of ranitidine on ileal myenteric plexus preparation and on acetyl- and butyrylcholinesterase Galli A, Mantovani P, Pepeu G Ref: Biochemical Pharmacology, 33:1845, 1984 : PubMed
Ranitidine at concentrations from 1 microM to 0.1 mM brought about a dose-dependent potentiation of the twitch responses elicited by electrical stimulation of the ileal myenteric preparation. At higher concentrations (0.3-3 mM) ranitidine also caused irregular slow contractions of the unstimulated ileal preparation which were potentiated by eserine and blocked by atropine and tetrodotoxin. In order to identify the mechanism of these apparently cholinomimetic actions, the effects of ranitidine on AChE and BuChE were studied. Ranitidine showed an instantaneous and promptly reversible inhibitory action at concentrations between 0.5 and 30 microM. Double reciprocal plots were prepared and equilibrium dissociation constants calculated. It appears that ranitidine exerts an inhibition of the "mixed" type on both AChE and BuChE, but the dissociation constants for BuChE were markedly higher than those for AChE. Since AChE inhibition occurs in the same concentration range potentiating the twitch responses on the ileal myenteric preparation, it may explain the cholinomimetic effect of ranitidine.
Title: Effect of cholecystokinin octapeptide and ceruletide on release of acetylcholine from cerebral cortex of the rat in vivo Magnani M, Mantovani P, Pepeu G Ref: Neuropharmacology, 23:1305, 1984 : PubMed
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.
Title: Poster 64 Ranitidine is a rapidly reversible inhibitor of acetylcholineterase Mantovani P, Galli A, Pepeu G Ref: In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases, (Brzin M, Barnard EA, Sket D, Eds) De Gruyter:, 1984 : PubMed
Title: Biphasic effect of methylxanthines on acetylcholine release from electrically-stimulated brain slices Pedata F, Pepeu G, Spignoli G Ref: British Journal of Pharmacology, 83:69, 1984 : PubMed
The effect of caffeine and aminophylline on the release of acetylcholine (ACh) was investigated in slices of rat cortex perfused with Krebs solution at rest and during electrical stimulation at frequencies of 0.2, 1 and 5 Hz. Both methylxanthines added to the superfusing Krebs solution at a concentration of 50 microM enhanced ACh release. Conversely, at a concentration of 0.5 mM both caffeine and aminophylline decreased ACh release. Neither caffeine nor aminophylline affected the unstimulated ACh release. Dipyridamole 10 microM potentiated the inhibitory effect of adenosine 30 microM on ACh release and antagonized both the stimulatory and inhibitory effects of caffeine on ACh release. The inhibitory effect of caffeine was antagonized by cyclohexyladenosine (CHA) 0.5 microM and N-ethylcarboxamideadenosine (NECA) 5 microM. The results indicate that methylxanthines exert both stimulatory and inhibitory effects on ACh release by acting on adenosine receptors. Methylxanthines may enhance the electrically-evoked ACh release by antagonizing the effect of endogenous adenosine on inhibitory adenosine receptors. On the other hand the mechanism through which methylxanthines decrease ACh release remains obscure.
Title: Effect of adenosine and caffeine on acetylcholine release from electrically stimulated brain slices Pedata F, Spignoli G, Pepeu G Ref: Annali dell Istituto Superiore di Sanita, 20:33, 1984 : PubMed
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.
Title: Acetylcholine release from rat cortical slices during postnatal development and aging Pedata F, Slavikova J, Kotas AM, Pepeu G Ref: Neurobiology of Aging, 4:31, 1983 : PubMed
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.
1 The effects of 4-aminopyridine (4AP) on the output of acetylcholine (ACh) from the cerebral cortex were investigated in unanaesthetized freely moving rats and in anaesthetized rats by means of the ;cup technique'. ACh was determined by bioassay on the dorsal muscle of the leech.2 In unanaesthetized rats intraperitoneal injection of 4AP (3 mg/kg) had no effect on the cortical output of ACh.3 After injection of morphine (10 mg/kg s.c.), which depressed the spontaneous output of ACh, 4AP increased the cortical output to a level significantly higher than that determined before morphine injection.4 In rats anaesthetized with either urethane or pentobarbitone, drugs known to decrease cortical output of ACh, 4AP (i.v. or i.p.) elicited a significant increase in the output of ACh. The time-courses of the 4AP-induced effects were different depending on the anaesthetic drug used: an immediate increase slowly fading in urethane anaesthesia and a gradual increase after delayed onset in pentobarbitone-anaesthetized rats.5 In some urethane-anaesthetized rats, respiratory frequency was kept constant (tracheotomy, connection to respirator, bilateral vagotomy) and prazosin (1 mg/kg i.v.) was administered to reduce the 4AP-induced increase of blood pressure. Cortical output of ACh was not related to changes in blood pressure. Moreover, the 4AP-induced increase in cortical ACh output was not related to changes in respiratory frequency.6 In summary systemic administration of 4AP in subconvulsive doses (1 and 3 mg/kg) increased cortical output of ACh in rats anaesthetized with urethane or pentobarbitone or after injection of morphine, but not in untreated freely moving rats. It is suggested that the anaesthetic agents and morphine may cause an imbalance between excitatory and inhibitory central pathways, and that this imbalance may play a role in their depressant effect on cortical output of ACh and/or in the 4AP-induced facilitation described in this paper.
The acquisition of active (shuttle-box) and passive avoidance conditioned responses and the effects of scopolamine on acetylcholine (ACh) output in freely moving rats and on conditioned responses were investigated 20 days after placing a unilateral lesion in the magnocellular forebrain nuclei (MFN). In the lesioned rats spontaneous ACh output from the cerebral cortex ipsilateral to the lesion was slightly decreased, while on the other hand the increase in ACh output elicited by scopolamine was strongly reduced. Sham operated rats always performed more active avoidance responses than MFN lesioned rats in the daily training shuttle-box sessions, and the facilitating effect of scopolamine (1 mg/kg IP) on the shuttle-box performance was suppressed. However the lesion did not disrupt the shuttle-box performance whenever training had taken place before the lesion. In the lesioned rats retested 30 min after the training trial, an impairment of the passive avoidance response was found. The effect of the lesion was potentiated by scopolamine. The results show therefore that MFN lesions impair the cortical cholinergic mechanisms, whose activity seems to play an important role in cognitive functions.
Title: Effect of magnocellular forebrain nuclei lesions on acetylcholine output from the cerebral cortex, electrocorticogram and behaviour Lo Conte G, Casamenti F, Bigl V, Milaneschi E, Pepeu G Ref: Archives Italiennes de Biologie, 120:176, 1982 : PubMed
ACh output from the cerebral cortex, electrocortical activity, spontaneous alternation and the acquisition of a conditioned avoidance response have been investigated in rats 20 days after the placement of a unilateral lesion of the magnocellular forebrain nuclei (MFN). ACh output from the hemisphere ipsilateral to the lesion was 40% lower than in sham operated rats. Electrocortical activity quantified with a frequency analyzer supplemented by a period and power spectrum analysis showed a marked asymmetry between the two hemispheres of the lesioned rats. The total electrical activity was strongly reduced over the lesioned hemisphere. The reduction involved all frequencies but was more evident in the high frequencies. In the lesioned rats spontaneous alternation was not impaired and spontaneous motility was enhanced while the acquisition of a conditioned active avoidance in a two-way shuttle box was significantly hampered. The possibility is envisaged that the destruction of cholinergic fibres impinging upon the cerebral cortex may decrease cortical activation and impair the selective awareness necessary for information acquisition and exclusion of irrelevant output.
Title: Effect of GABA-mimetic drugs on acetylcholine release in vitro Mantovani P, Santicioli P, Pepeu G Ref: Acta Physiol Pharmacol Bulg, 8:24, 1982 : PubMed
Title: Changes in synaptosomal high affinity choline uptake following electrical stimulation of guinea-pig cortical slices: effect of atropine and physostigmine Antonelli T, Beani L, Bianchi C, Pedata F, Pepeu G Ref: British Journal of Pharmacology, 74:525, 1981 : PubMed
1 Superfused guinea-pig cortical slices were electrically stimulated at different frequencies and the changes in acetylcholine (ACh) content measured. Synaptosomes were prepared at the end of the stimulation period and high affinity choline uptake (HACU) rate was measured. 2 The effect of increasing KC1 concentrations was compared on ACh content of the slices and on synaptosomal HACU. 3 Electrical stimulation (2, 5, 10, 20 Hz) elicited a frequency-dependent linear increase in synaptosomal HACU rate and a decrease in ACh content of the slices. 4 The addition of atropine (1.5 x 10(-8) M) to the slices enhanced and that of physostigmine (3 x 10(-5) M) reduced the frequency-dependent increase in HACU rate. Atropine (1.5 x 10(-6) M) not only antagonized the effect of physostigmine, but the HACU rate measured after treatment with both drugs was larger than that found after atropine alone. 5 These results indicate that in the cortical cholinergic nerve endings, depolarization caused by electrical stimulation is coupled with an increase in choline transport which can be modulated by the addition of atropine or physostigmine. Furthermore, within given experimental conditions a linear relationship exists between the reciprocal of ACh content in the slices and synaptosomal HACU.
Title: Interactions between ionophores, Mg2+ and Ca2+ on acetylcholine formation and release in brain slices Mantovani P, Pepeu G Ref: Pharmacol Res Commun, 13:175, 1981 : PubMed
Title: Effect of haloperidol and pimozide on acetylcholine output from the cerebral cortex in rats and guinea pigs Casamenti F, Bianchi C, Beani L, Pepeu G Ref: European Journal of Pharmacology, 65:279, 1980 : PubMed
The effect of haloperidol, pimozide and amphetamine on acetylcholine (ACh) output from the cerebral cortex was investigated in unanaesthetized, freely moving and urethane-anaesthetized rats and guinea pigs. Haloperidol (1 mg/kg i.p.) decreased ACh output only in the anaesthetized rats and increased it only in unanaesthetized guinea pigs. Pimozide (1 mg/kg i.p.) stimulated ACh output in unanaesthetized rats and guinea pigs and anaesthetized guinea pigs, but not in anaesthetized rats. Amphetamine (1 mg/kg i.p.) in all cases, stimulated ACh output. In rats with a septal lesion, the effect of amphetamine on ACh output was suppressed but that of pimozide was still present.
Title: Acetylcholine input from the cerebral cortex, choline uptake and muscarinic receptors in morphine-dependent, freely-moving rats Casamenti F, Pedata F, Corradetti R, Pepeu G Ref: Neuropharmacology, 19:597, 1980 : PubMed
Title: Stimulation of acetilcholine (ACh) output from isolated brain slices by anticholinergic drugs: influence of septal lesion, changes in ionic environment and GABA Mantovani P, Santicioli P, Lo Conte G, Pepeu G Ref: Pharmacol Res Commun, 12:605, 1980 : PubMed
Title: Choline high-affinity uptake and metabolism and choline acetyltransferase activity in the striatum of rats chronically treated with neuroleptics Pedata F, Sorbi S, Pepeu G Ref: Journal of Neurochemistry, 35:606, 1980 : PubMed
High-affinity uptake of choline and choline acetyltransferase activity (ChAT) were measured in the striatum of rats treated for 45-60 days with haloperidol (1 mg/kg per os) and pimozide (1 mg/kg per os) daily and with fluspirilene (1 mg/kg i.m.) twice a week. Haloperidol and fluspirilene caused a 20%, and pimozide a 38%, increase in high-affinity uptake of choline. They also caused a significant decrease in ChAT activity: haloperidol, 20%; pimozide, 27%; and fluspirilene, 42%. In rats treated with fluspirilene for 65-80 days the metabolism of [3H] choline taken up by striatal synaptosomes was investigated. A 33% increase in total radioactivity, a significant increase in labelled acetylcholine (ACh), a relative decrease in labelled choline, and no change in labelled phosphorylcholine and betaine were found. It is concluded that the increase in high-affinity choline uptake caused by chronic administration of neuroleptic drugs is associated with a parallel increase in choline utilization for ACh formation. On the other hand, the decrease in ChAT activity does not appear to influence ACh formation.
1. Dementias have a complex etiology and pathogenesis which should be carefully evaluated in order to attempt a medical treatment. 2. Drugs are among the most frequent causes of dementias, particularly drugs with anticholinergic properties. 3. A similarity between the mental symptoms of anticholinergic poisoning and senile dementia has been observed. 4. Recent investigations have also presented a decrease of cholinergic activity in postmortem material of subjects with Alzheimer's disease. Also a number of data suggest that recent memory would be related to changes in the metabolism of brain acetylcholine (Ach). 5. The possibility that drugs stimulating brain cholinergic mechanisms might be used in the treatment of dementias is envisaged.
Title: Effect of phosphatidylserine on acetylcholine output from the cerebral cortex of the rat Casamenti F, Mantovani P, Amaducci L, Pepeu G Ref: Journal of Neurochemistry, 32:529, 1979 : PubMed
Title: Stimulation of acetylcholine output from brain slices caused by the ionophores BrX-537A and A23187 Casamenti F, Mantovani P, Pepeu G Ref: British Journal of Pharmacology, 63:259, 1978 : PubMed
1 The effect of two ionophores, BrX-537A (Bromolasolacid) and A 23187, on acetylcholine (ACh) output from brain slices was studied. 2 The slices were prepared from rat cerebral cortex, incubated in Krebs solution containing physostigmine and ACh output determined by bioassay. 3 Both ionophores enhanced ACh output. BrX-537A exerted its maximal effect, a six fold increase, at a concentration of 1.8 micron, while A 23187 caused a three fold increase at a concentration of 58 micron. 4 When the slices were incubated in a Ca-free medium, the effect of A 23187 on ACh output was only reduced, BrX-537A was abolished while that of BrX-537A was also active when disodium edetate (EDTA) was added to the the Ca-free medium. 5 The activity of BrX-537A was not affected by the presence of tetrodotoxin in the incubation medium. 6 The stimulation of ACh output elicited by KCl (25 mM) was increased further by hyoscine, but not by BrX-537A. Hyoscine however had no effect when ACh output was stimulated by BrX-537A. 7 The effect of BrX-537A on ACh output was potentiated by the addition of Mg2+ (9.3 mM) to the incubation medium and was reduced in a Mg-free medium. 8 It is concluded that A 23187 stimulates ACh output by transporting extracellular Ca2+ into cholinergic nerve endings. The effect of BrX-537A does not depend only on Ca2+ but also on other mechanisms.
Title: Drug effect on cholinergic mechanisms in the cerebral cortex Pepeu G Ref: Annali dell Istituto Superiore di Sanita, 14:157, 1978 : PubMed
Investigations aimed to define the components of the cholinergic network in the cerebral cortex and their functional modifications under the effect of a number of psychotropic drugs are described. By measuring acetylcholine levels and acetylcholine output from the cerebral cortex in cats and rats with various subcortical lesions, at least two systems of cholinergic neurons ascending to the cortex were demonstrated. One of the systems originates from the septum and its activity is affected by drugs acting on dopaminergic receptors.
Title: Effect of bromocriptine on acetylcholine output from the cerebral cortex Pepeu G, Mantovani P Ref: Pharmacology, 16 Suppl 1:204, 1978 : PubMed
The effect of bromocriptine on acetylcholine (ACh) output from the cerebral cortex was investigated in anaesthetized rats. Bromocriptine caused a brief decrease in ACh output at the dose of 0.1 mg/kg i.p. and a dose-related long-lasting increase at doses from 1.25 to 10 mg/kg i.p. Apomorphine elicited an increase in ACh output when injected intraperitoneally at doses from 0.1 to 10 mg/kg. When bromocriptine was administered to rats in which a septal lesion had been made 12 days prior to the test, no increase in ACh output was observed. Bromocriptine seems therefore to stimulate a cortical cholinergic pathway originating from or passing through the septum.
Title: Drug Stimulation of Acetylcholine Output from the Cerebral Cortex Pepeu G, Mantovani P, Pedata F Ref: Advances in Behavioral Biology, 24:605, 1978 : PubMed
Title: Interelationships between dopaminergic and cholinergic systems in the cerebral cortex Mantovani P, Bartolini A, Pepeu G Ref: Adv Biochem Psychopharmacol, 16:423, 1977 : PubMed
Title: Effect of choline, phosphorylcholine and dimethylaminoethanol on brain acetylcholine level in the rat Pedata F, Wieraszko A, Pepeu G Ref: Pharmacol Res Commun, 9:755, 1977 : PubMed
Title: Proceedings: Effect of some phospholipids on acetyl-choline output from the cerebral cortex in the rat Amaducci L, Mantovani P, Pepeu G Ref: British Journal of Pharmacology, 56:379P, 1976 : PubMed
Title: Effect of single and repeated electroshock applications on brain acetylcholine levels and choline acetyltransferase activity in the rat Longoni R, Mulas A, Novak BO, Pepeu IM, Pepeu G Ref: Neuropharmacology, 15:283, 1976 : PubMed
Title: Investigations into the relationship between phospholipids and brain acetylcholine Mantovani P, Pepeu G, Amaducci L Ref: Advances in Experimental Medicine & Biology, 72:285, 1976 : PubMed
The effect of a phospholipid mixture extracted from the bovine cerebral cortex and of pure phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) on ACh output from the cerebral cortex was investigated in rats anaesthetized with urethane. All the phospholipids were administered after sonication. ACh was determined by bioassay. The following results were obtained: after i.p. administration of 100 mg/kg of the phospholipid mixture a marked increase in ACh output I asting approximately 30 min was observed. The i.v. administration of PS caused a dose-dependent increase in ACh. At the dose of 150 mg/kg a 3-fold increase over the spontaneous ACh output was found. PE was less active than PS and PC showed no effect on ACh output. The possibility that PS and PE might increase ACh output through a mechanism facilitating Ca penetration is discussed.
Title: Changes in regional brain acetylcholine levels during drug-induced convulsions Pedata F, Mulas A, Pepeu IM, Pepeu G Ref: European Journal of Pharmacology, 40:329, 1976 : PubMed
Acetylcholine (ACh) levels were determined in the brain of rats killed by decapitation or focussed microwave radiation during drug-induced convulsions. During metrazol or strychnine-induced convulsions a diffuse decrease in ACh levels was found in rats killed by decapitation. When the rats were killed by radiation and the brain was only divided into three large regions, strychnine caused no changes in ACh levels; metrazol caused a decrease in the cerebral cortex and lower brainstem. When discrete brain regions were investigated in rats killed by radiation, metrazol-induced convulsions were associated with a decrease in ACh level in all regions dissected and strychnine-induced convulsions with a decrease in the hippocampus and caudate nucleus only. Picrotoxin-induced convulsions were associated with a decrease in ACh level in the cerebral cortex, hippocampus, midbrain and medulla-pons, those induced by bicuculline with an increase in ACh level in the frontal cortex, hippocampus, midbrain and medulla-pons, by dimefline with an increase in the frontal cortex, midbrain and medulla-pons and a decrease in the caudate nucleus. The experiments show that each type of convulsant affects ACh levels in discrete brain regions in a different way.
Title: The influence of raphe lesions on the effect of morphine on nociception and cortical ACh output Garau L, Mulas ML, Pepeu G Ref: Neuropharmacology, 14:259, 1975 : PubMed
Title: Stimulation by morphine of acetylcholine output from the cerebral cortex of septal rats Pepeu G, Garau L, Mulas ML, Marconcini-Pepeu I Ref: Brain Research, 100:677, 1975 : PubMed
Title: Proceedings: Drug effect on acetylcholine level in discrete brain regions of rats killed by microwave irradiation Longoni R, Mulas A, Pepeu G Ref: British Journal of Pharmacology, 52:429P, 1974 : PubMed
Title: Effect of limbic system lesions on acetylcholine release from the cerebral cortex of the rat Mulas A, Mulas ML, Pepeu G Ref: Psychopharmacologia, 39:223, 1974 : PubMed
Title: Acetylcholine levels in the frog spinal cord following the administration of different convulsants Nistri A, Pepeu G Ref: European Journal of Pharmacology, 27:281, 1974 : PubMed
Title: Proceedings: Brain acetylcholine levels and nociceptive threshold in frogs after the administration of morphine, nalorphine and naloxone Nistri A, Pepeu G Ref: The Journal of Physiology, 241:51P, 1974 : PubMed
Title: Effects of morphine on brain and spinal acetylcholine levels and nociceptive threshold in the frog Nistri A, Pepeu G, Cammelli E, Spina L, de Bellis AM Ref: Brain Research, 80:199, 1974 : PubMed
Title: Does 5-hydroxytryptamine influence cholinergic mechanisms in the central nervous system? Pepeu G, Garau L, Mulas ML Ref: Adv Biochem Psychopharmacol, 10:247, 1974 : PubMed
Title: Increase in brain and spinal acetylcholine levels without antinociceptive actions following morphine administration in the frog Nistri A, Pepeu G Ref: British Journal of Pharmacology, 47:650P, 1973 : PubMed
Title: The release of acetylcholine from the brain: an approach to the study of the central cholinergic mechanisms Pepeu G Ref: Prog Neurobiol, 2:259, 1973 : PubMed
Title: Changes in the acetylcholine content in the rat brain after lesions of the septum, fimbria and hippocampus Pepeu G, Mulas A, Mulas ML Ref: Brain Research, 57:153, 1973 : PubMed
Title: Investigations into the release of acetylcholine from the cerebral cortex of the cat: effects of amphetamine, of scopolamine and of septal lesions Nistri A, Bartolini A, Deffenu G, Pepeu G Ref: Neuropharmacology, 11:665, 1972 : PubMed
Title: Effect of bicuculline and strychnine on the acetylcholine content of the amphibian spinal cord Nistri A, Pepeu G Ref: British Journal of Pharmacology, 45:173P, 1972 : PubMed
Title: Cholinergic neurotransmission in the central nervous system Pepeu G Ref: Archives Internationales de Pharmacodynamie et de Therapie, 196:Suppl 196:229, 1972 : PubMed
Title: Effect of septal lesions on acetylcholine output from the cerebral cortex in the cat Bartolini A, Defenu G, Nistri A, Pepeu G Ref: British Journal of Pharmacology, 41:399P, 1971 : PubMed
Title: The effect of oxotremorine on the acetylcholine content of different parts of cat brain Bartolini A, Bartolini R, Pepeu G Ref: J Pharm Pharmacol, 22:59, 1970 : PubMed
Title: Investigations into the increase of acetylcholine output from the cerebral cortex of the cat caused by amphetamine Pepeu G, Bartolini A, Deffenu G Ref: Drugs and Cholinergic Mechanisms in the CNS, :387, 1970 : PubMed
Title: Effect of psychoactive drugs on the output of acetylcholine from the cerebral cortex of the cat Pepeu G, Bartolini A Ref: European Journal of Pharmacology, 4:254, 1968 : PubMed
Title: Investigations into the acetylcholine output from the cerebral cortex of the cat in the presence of hyoscine Bartolini A, Pepeu G Ref: British Journal of Pharmacology and Chemotherapy, 31:66, 1967 : PubMed
Title: Acetylcholine and 5-hydroxytryptamine levels of the lateral geniculate bodies and superior colliculus of cats after visual deafferentation Deffenu G, Bertaccini G, Pepeu G Ref: Experimental Neurology, 17:203, 1967 : PubMed
Title: [Effect of some psychopharmacological agents on acetylcholine release from cerebral cortex in cats] Pepeu G, Bartolini A Ref: Bollettino Societa Italiana Biologia Sperimentale, 43:1409, 1967 : PubMed
Title: [Amnesic action of some anti-depressive drugs and their effect on cerebral acetylcholine in the rat] Pacini S, Gliozzi E, Pepeu G Ref: Bollettino Societa Italiana Biologia Sperimentale, 42:1078, 1966 : PubMed
Title: Amnesic properties of scopolamine and brain acetylcholine in the rat Pazzagli A, Pepeu G Ref: International Journal of Neuropharmacology, 4:291, 1965 : PubMed
Title: [Incorporation of P32 into the cat brain with pretrigeminal section and mesencephalic hemi-section] Amaducci L, Deffenu G, Pazzagli A, Pepeu G Ref: Bollettino Societa Italiana Biologia Sperimentale, 40:Suppl:2065, 1964 : PubMed
Title: THE INFLUENCE OF CENTRALLY ACTING CHOLINOLYTIC DRUGS ON BRAIN ACETYLCHOLINE LEVELS Giarman NJ, Pepeu G Ref: British Journal of Pharmacology and Chemotherapy, 23:123, 1964 : PubMed
A number of centrally acting cholinolytic drugs reduced levels of cerebral acetylcholine in the rat. Among its naturally occurring analogues, hyoscine had the greatest potency, producing a decrease of 31% at a dose of 0.63 mg/kg. Atropine methyl nitrate, which acts as a cholinolytic drug in the periphery, had no effect on brain acetylcholine levels. The fall in acetylcholine produced by hyoscine was greatest after 60 min and disappeared at about 120 min. The animals tended to show a partial tolerance to this effect of hyoscine when the drug was administered repeatedly. The reduction in acetylcholine after hyoscine was restricted to the cerebral hemispheres, and did not appear in subcortical regions of the brain. Hyoscine had no influence on the net synthesis of acetylcholine by acetone-extracted powder of rat brain. In a series of four synthetic cholinolytic drugs, only the two with conspicuous psychotomimetic actions in man produced a decrease in brain acetylcholine comparable to that seen with hyoscine and related alkaloids.
Title: [Correlations between the modifications of behavior induced by scopolamine and cerbral acetylcholine contents in the rat] Pazzagli A, Pepeu G Ref: Rass Studi Psichiatr, 53:261, 1964 : PubMed
A midbrain hemisection in a midpontine pretrigeminal preparation of the cat causes an asymmetry in the electroencephalogram with a marked increase in the acetylcholine content of the cortex from the synchronized hemisphere. It is suggested that the increase in acetylcholine is related to the decrease of nervous activity caused by the hemisection.
Title: Effect of tremorine and some anti-parkinson's disease drugs on acetylcholine in the rat's brain Pepeu G Ref: Nature, 200:895, 1963 : PubMed
In rats, drug-induced depression of the central nervous system has been shown generally to be associated with an elevation in level of total acetylcholine in the brain. This generalization held true for a wide variety of depressant drugs with one notable exception: the subacute administration of reserpine, with which there was an increase in cerebral acetylcholine after the first dose, but a return to normal levels after subsequent doses, despite continued depression of the animals. Reduction in the level of total acetylcholine in the brain followed the administration of certain convulsants (pentylenetetrazole and 3,5-dimethylbutylethylbarbiturate); but no change was seen after the administration of several mildly exciting agents. The notable exceptions to this generalization were atropine and scopolamine, which significantly lowered brain acetylcholine in doses producing mild excitation in only some of the animals and no gross manifestations in the rest.
Title: [Variations of the intracellular distribution of cerebral acetylcholine caused by drugs] Giarman NJ, Pepeu G Ref: Bollettino Societa Italiana Biologia Sperimentale, 37:128, 1961 : PubMed
Title: [Variations in the total acetylcholine content of the guinea pig small intestine induced by various drugs] Pepeu G Ref: Arch Ital Sci Farmacol, 11:382, 1961 : PubMed
