Differential activation of mitogen-activated protein kinase signalling pathways in the hippocampus of CRND8 transgenic mouse, a model of Alzheimer's disease.

Transgenic Centre for Research in Neurodegenerative Diseases 8 (TgCRND8) mice expressing a double mutant form of human amyloid precursor protein represent a good model of Alzheimer's disease, and can be useful to clarify the involvement of mitogen-activated protein kinases (MAPK) dysregulation in the pathophysiology of this neurodegenerative disorder. Activation of extracellular regulated kinase (ERK) 1/2, jun kinase (JNK) and p38MAPK was studied in the hippocampus of 7-month-old TgCRND8 mice by immunohistochemistry and Western blot analysis using antibodies selective for the phosphorylated, and thus active, forms of the enzymes. We demonstrated that the three main MAPK pathways were differentially activated in cells of the hippocampus of TgCRND8 mice in comparison to wild type (Wt) littermates, p38MAPK and JNK being more activated, while ERK less activated. p38MAPK was significantly activated in microglia, astrocytes and neurons, around and distant from the plaques. JNK was highly activated in cells closely surrounding the plaques. No difference was observed in the activation of the two major bands of JNK, at a molecular weight of 46 kDa and 54 kDa. These data indicate the possible involvement of p38MAPK and JNK pathways dysregulation in the pathogenesis of Alzheimer's disease. The ERK2 isoform of the ERK pathway was less activated in the hippocampal dentate gyrus of Tg mice in basal conditions. Furthermore activation of the ERK pathway by ex vivo cholinergic stimulation with carbachol caused significantly higher activation of ERK in the hippocampus of Wt mice than in Tg mice. These findings may pose a molecular basis for the memory disruption of Alzheimer's disease, since proper functioning of the basal forebrain cholinergic neurons and of ERK2 is critical for memory formation.

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

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

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

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

Comparison between flurbiprofen and its nitric oxide-releasing derivatives HCT-1026 and NCX-2216 on Abeta(1-42)-induced brain inflammation and neuronal damage in the rat.

Brain inflammation is an underlying factor in the pathogenesis of Alzheimers disease (AD). We investigated, in vivo, whether differences exist in the anti-inflammatory and neuroprotective actions of flurbiprofen and its two nitric oxide-donor derivatives, HCT-1026 and NCX-2216, and the ability of these two derivatives to release nitric oxide in the brain. In adult rats injected into the nucleus basalis with preaggregated Abeta(1-42) we investigated glia reaction, the induction of inducible nitric oxide synthase (iNOS), the activation of p38 mitogen-activated protein kinase (p38MAPK) pathway and the number of choline acetyltransferase (ChAT)-positive neurons and, in naive rats we investigated, by microdialysis, cortical extracellular levels of nitrite. Injection of Abeta(1-42) induced iNOS and activation of p38MAPK 7 days after injection and an intense microglia and astrocyte reaction along with a marked reduction in the number ChAT-positive neurons, persisting up to at least 21 days. Flurbiprofen, HCT-1026 and NCX-2216 (15 mg/kg) significantly attenuated the Abeta(1-42)-induced glia reaction, iNOS induction and p38MAPK activation 7 days after treatment and astrocytes reaction 21 days after treatment. On an equimolar basis, HCT-1026 resulted the most active agent in reducing the Abeta(1-42)-induced microglia reaction. The cholinergic cell loss was also significantly reduced by 21 days of HCT-1026 treatment. No differences in body weight were found between the animals treated for 21 days with 15 mg/kg of either HCT-1026 or NCX-2216 and the controls. Oral administration of HCT-1026 (15 mg/kg) or NCX-2216 (100 mg/kg) to naive rats was followed by significant and long lasting increases in cortical nitrite levels. These findings indicate that the addition of a nitric oxide donor potentiates the anti-inflammatory activity of flurbiprofen in a model of brain inflammation.

Experimental brain inflammation and neurodegeneration as model of Alzheimer's disease: protective effects of selective COX-2 inhibitors.

Epidemiological studies indicate that long-term treatment with non-steroidal anti-inflammatory drugs reduces the risk of Alzheimer Disease and may delay its onset or slow its progression. Neuroinflammation occurs in vulnerable regions of the Alzheimer's disease (AD) brain where highly insoluble beta-amyloid (Abeta) peptide deposits and neurofibrillary tangles, as well as damaged neurons and neurites, provide stimuli for inflammation. To elucidate the complex role of inflammation in neurodegenerative processes and the efficacy of selective COX-2 inhibitors in AD, we examined whether the attenuation of brain inflammatory reaction by selective COX-2 inhibitors may protect neurons against neurodegeneration. The data reported in this review show that in in vivo models of brain inflammation and neurodegeneration, the administration of selective COX-2 inhibitors prevent not only the inflammatory reaction, but also the cholinergic hypofunction. Our data may help elucidate the epidemiological findings indicating that anti-inflammatory agents, in particular NSAIDs, reduce the risk of developing AD and may slow its progression.

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

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

Rivastigmine antagonizes deficits in prepulse inhibition induced by selective immunolesioning of cholinergic neurons in nucleus basalis magnocellularis.

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.

Effect of subchronic administration of metrifonate, rivastigmine and donepezil on brain acetylcholine in aged F344 rats.

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.

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

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

NO-flurbiprofen attenuates excitotoxin-induced brain inflammation, and releases nitric oxide in the brain.

Brain inflammation underlies the pathogenesis of Alzheimer's disease (AD) and nonsteroidal anti-inflammatory drug therapy may delay the onset of AD. We investigated, in vivo, the effects of NO-flurbiprofen on brain inflammation in rats injected with quisqualic acid into the nucleus basalis and on the release of nitric oxide from the drug in naive rat brains. We showed that the excitotoxin-induced microglia reaction, the expression of inducible nitric oxide synthase-positive cells and the production of interleukin-1beta and prostaglandin-E2 in the injected area were attenuated by the NO-flurbiprofen (15 mg/kg, p.o.) treatment. An oral administration of NO-flurbiprofen (25, 50 and 100 mg/kg) to naive rats was followed by significant increases in cortical nitrite levels. This drug may have important therapeutic implications for the treatment of AD.

Changes in presynaptic proteins, SNAP-25 and synaptophysin, in the hippocampal CA1 area in ischemic gerbils.

A general consensus exists that the presynaptic terminals in the hippocampal CA1 area are resistant to ischemic stress in spite of the loss of their target cells (CA1 pyramidal neurons). We have verified this by immunostaining and Western immunoblotting using the antibodies for presynaptic proteins, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptophysin in gerbils after bilateral carotid artery ligature. In the immunohistochemical analysis, decreases in SNAP-25 and synaptophysin immunoreactivities in the strata radiatum and oriens, especially around the apical dendrite of CA1 neurons, and disappearance of SNAP-25 immunoreactivity in the alveus were observed on day 2 after ischemia. On days 7 and 14, SNAP-25-positive granular materials were expressed in the CA1 area, and intense synaptophysin immunoreactivity around surviving CA1 neurons was observed. Western immunoblot analysis revealed significant decreases of SNAP-25 and synaptophysin (about 60% of control levels) on day 2, and then increase of their proteins (130--140% of control levels) on day 14. These results indicate that presynaptic degeneration occurs in the hippocampal CA1 area after ischemia, and it precedes the delayed neuronal death of CA1 neurons. The presynaptic terminal damage may be responsible for some pathological changes in ischemic brains.

Brain inflammatory reaction in an animal model of neuronal degeneration and its modulation by an anti-inflammatory drug: implication in Alzheimer's disease.

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.

NGF treatment potentiates c-fos expression in the rat nucleus basalis upon excitotoxic lesion with quisqualic acid.

The induction of the c-fos gene in the rat brain by NGF was studied in a model of acute cholinergic hypofunction, i.e., the lesion of the nucleus basalis magnocellularis (NBM) with quisqualic acid. Choline acetyltransferase and Fos immunoreactivity (IR) in the NBM were analyzed at different times after the excitotoxic lesion. NGF treatment induced a potentiation of Fos expression 4 and 24 h after lesion. The possibility is discussed that c-fos induction is one of the early mechanisms of the neuroprotective action of NGF.

Beta(1-40) amyloid peptide injection into the nucleus basalis of rats induces microglia reaction and enhances cortical gamma-aminobutyric acid release in vivo.

The nucleus basalis of adult rats was injected with beta(1-40) amyloid peptide. A marked increase in basal and K(+)-evoked GABA release in the ipsilateral cortex and a significant decrease in GAD activity in the injected NB were found 30 days after injection. An intense activation of microglial cells that surrounded and infiltrated the deposit was observed. These data demonstrate that a local injection of beta(1-40) peptide into the NB induces glia activation and affects GABAergic neurons.

Interleukin-1beta activates forebrain glial cells and increases nitric oxide production and cortical glutamate and GABA release in vivo: implications for Alzheimer's disease.

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.

Effects of novelty and pain on behavior and hippocampal extracellular ACh levels in male and female rats.

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.

C-fos expression in the rat nucleus basalis upon excitotoxic lesion with quisqualic acid: a study in adult and aged animals.

A unilateral quisqualic acid lesion was placed in the nucleus basalis magnocellularis of 3- and 24-month-old rats, and the animals were sacrificed at different times post-surgery. The morphology and the number of the cholinergic neurons of the nucleus basalis were analyzed by means of immunohistochemistry for cholineacetyltransferase, in order to evaluate the size and severity of the lesion. Immunohistochemistry for the immediate early gene c-fos was also performed in order to clarify its role in the process of neurodegeneration following the excitotoxin injection. The DNA laddering and TUNEL techniques were used to define the type of cell death involved. At short times (4 hr) the lesion induced alterations in the morphology of cholinergic neurons of the nucleus basalis. Subsequently, a significant decrease in the number of neurons was found in comparison to the contralateral unlesioned side. In the older animals the loss of cholineacetyltransferase immunoreactivity had an earlier onset (4 hr) than in the young (24 hr). C-fos expression was induced by the lesion and not by saline injection in the nucleus basalis and in neighbouring areas of the brain as early as 4 hr after surgery. The c-fos protein was no longer present by 24 hr. Furthermore, the c-fos gene product was consistently absent from the nuclei of cholinergic cells. The aged animals exhibited a slower and smaller increase in c-fos as measured by counting the labelled nuclei in the injected area. Analysis of DNA fragmentation did not provide any evidence for apoptosis as the type of cell death involved in the cholinergic degeneration. These results indicate that the c-fos protein might have a protective role in the response to excitotoxic lesions. Furthermore, we have shown that the aged brain displays a reduced ability to produce a c-fos-mediated plastic response to the lesion.

Cholinergic function in the hippocampus of juvenile rats chronically deprived of NGF.

Intracellular and extracellular recordings were used to assess the cholinergic function in hippocampal slices from juvenile rats chronically deprived of NGF. NGF was neutralised by implanting into the lateral ventricle of postnatal (P) day 2 rats, alphaD11 hybridoma cells (secreting monoclonal antibodies specific for NGF). Parental myeloma cells (P3U) were used as controls. At P15-P18, slow cholinergic EPSPs could be elicited in cells from both alphaD11- and P3U-treated rats. However, slices from alphaD11-implanted rats exhibited a 50% reduction in acetylcholine release following stimulation of cholinergic fibres. This effect was associated to a significant increase in the sensitivity of pyramidal cells to carbachol, as suggested by the shift to the left of the dose/response curve. This may reflect a compensatory mechanism for the reduced efficacy of cholinergic innervation in NGF-deprived rats. In both alphaD11- and P3U-treated rats, carbachol was able to induce a similar concentration-dependent depression of the field EPSPs, evoked by Schaffer collateral stimulation, suggesting that presynaptic muscarinic receptors were not altered. In rats implanted with alphaD11 cells at P15 and sacrificed at P21-P24, no changes in the sensitivity to carbachol were found. At this developmental stage, no differences in acetylcholine release were observed between P3U- and alphaD11-treated animals. These results provide physiological evidence for a regulatory role of NGF in the cholinergic function of the hippocampus during postnatal development.

Long-term changes in the aggregation state and toxic effects of beta-amyloid injected into the rat brain.

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.

Morphological, biochemical and behavioural changes induced by neurotoxic and inflammatory insults to the nucleus basalis.

Interest in the basal forebrain cholinergic system has greatly increased since neuropathological studies in humans provided evidence that this system is severely affected in Alzheimer's disease and other dementing disorders. In laboratory animals, disruption of the nucleus basalis cholinergic neurones has been produced by several neurotoxic insults in order to obtain a model reproducing the behavioural impairment related to the cholinergic deficits. The experiments reported in this review demonstrate that excitotoxic amino acids, beta-amyloid and lipopolysaccharide, injected directly in the nucleus basalis are toxic to the cholinergic neurones in the rat. The excitotoxin lesions of the nucleus basalis, although not selective, are a useful tool for producing experimental animals with cholinergic hypofunction and for investigating drugs able to ameliorate the cholinergic functions. Local injections of amyloid peptides in the rat nucleus basalis produced cholinergic hypofunction and some behavioural impairment. Finally, an intense glia reaction with a limited cholinergic hypofunction and no behavioural impairment was induced by a 4-week infusion of lipopolysaccharide in the nucleus basalis. In conclusion, all three models, in spite of their limitations, offer useful tools for the study of the pathogenetic mechanisms of Alzheimer's disease and for investigating potentially useful drugs.