Change of cholinergic transmission and memory deficiency induced by injection of b-amyloid protein into NBM of rats

The change of cholinergic transmission of b-amyloid protein (b-AP) treated rats was studied by intracerebral microdialysis sampling combined with HPLC analysis. b-AP1-40 was injected into nucleus basalis magnocellularis (NBM). Passive avoidance response test (step-down test) and delayed alternation task were used for memory testing. The impairment of memory after injection of b-AP1-40 into NBM exhibited mainly the deficiency of short-term working memory. One week after injection of b-AP1-40 the release of acetylcholine (ACh) from frontal cortex of freely-moving rats decreased significantly, and the response of cholinergic nerve ending to the action of high [K+] solution was rather weak. In control animals the percentage of increase of ACh- release during behavioral performance was 57%, while in b-AP1-40 - treated rats it was 34%. The temporary in-crease of the ACh-release of the rat put into a new place was also significantly diminished in b-AP1-40 -treated rats. The results show that the injection of b-AP1-40 into NBM impairs the cholinergic transmission in frontal cortex, and the impairment of cholinergic transmission may be the main cause of the deficit of working memory.     

The neurotoxic actions of quinolinic acid in the central nervous system

Excitotoxins such as kainic acid, ibotenic acid, and quinolinic acid are a group of molecules structurally related to glutamate or aspartate. They are capable of exciting neurons and producing axon sparing neuronal degeneration. Quinolinic acid (QUIN), an endogenous metabolite of the amino acid, tryptophan, has been detected in brain and its concentration increases with age. The content of QUIN in the brain and the activity of the enzymes involved in its synthesis and metabolism show a regional distribution. The neuroexcitatory action of QUIN is antagonized by magnesium (Mg2+) and the aminophosphonates, proposed N-methyl-D-aspartate (NMDA) receptor antagonists, suggesting that QUIN acts at the Mg2+ -sensitive NMDA receptor. Like its excitatory effects, QUIN's neurotoxic actions in the striatum are antagonized by the aminophosphonates. This suggests that QUIN neurotoxicity involves the NMDA receptor and (or) another receptor sensitive to the aminophosphonates. The neuroexcitatory and neurotoxic effects of QUIN are antagonized by kynurenic acid (KYN), another metabolite of tryptophan. QUIN toxicity is dependent on excitatory amino acid afferents and shows a regional variation in the brain. Local injection of QUIN into the nucleus basalis magnocellularis (NBM) results in a dose-dependent reduction in cortical cholinergic markers including the evoked release of acetylcholine. A significant reduction in cortical cholinergic function is maintained over a 3-month period. Coinjection of an equimolar ratio of QUIN and KYN into the NBM results in complete protection against QUIN-induced neurodegeneration and decreases in cortical cholinergic markers. In contrast, focal injections of QUIN into the frontoparietal cortex do not alter cortical cholinergic function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trophic factor effects on cholinergic innervation in the cerebral cortex of the adult rat brain

The cholinergic pathway ascending from the nucleus basalis magnocellularis (NBM) to the cortex has been implicated in several important higher brain functions such as learning and memory. Following infarction of the frontoparietal cortical area in the rat, a retrograde atrophy of cholinergic cell bodies and fiber networks occurs in the basalocortical cholinergic system. We have observed that neuronal atrophy in the NBM induced by this lesion can be prevented by intracerebroventricular administration of exogenous nerve growth factor (NGF) or the monosialoganglioside GM₁. In addition, these agents can upregulate levels of cortical choline acetyltransferase (ChAT) activity in the remaining cortex adjacent to the lesion site. Furthermore, an enhancement in cortical high-affinity³H-choline uptake and a sustained in vivo release of cortical acetylcholine (ACh) after K⁺ stimulation are also observed after the application of neurotrophic agents. Moreover, these biochemical changes in the cortex are accompanied by an anatomical remodeling of cortical ChAT-immunoreactive fibers and their synaptic boutons.     

Change of cholinergic transmission and memory deficiency induced by injection of β-amyloid protein into NBM of rats

The change of cholinergic transmission of β-amyloid protein (β-AP) treated rats was studied by intracerebral microdialysis sampling combined with HPLC analysis. β-AP_1-40 was injected into nucleus basalis magnocellularis (NBM). Passive avoidance response test (step-down test) and delayed alternation task were used for memory testing. The impairment of memory after injection of β-AP_1-40 into NBM exhibited mainly the deficiency of short-term working memory. One week after injection of β-AP^1-40 the release of acetylcholine (ACh) from frontal cortex of freely-moving rats decreased significantly, and the response of cholinergic nerve ending to the action of high [K⁺] solution was rather weak. In control animals the percentage of increase of AChrelease during behavioral performance was 57%, while in β-AP^1-40-treated rats it was 34%. The temporary increase of the ACh-release of the rat put into a new place was also significantly diminished in β-AP_1-40 -treated rats. The results show that the injection of β-AP_1-40 into NBM impairs the cholinergic transmission in frontal cortex, and the impairment of cholinergic transmission may be the main cause of the deficit of working memory.     

Change of cholinergic transmission and memory deficiency induced by injection of β-amyloid protein into NBM of rats

The change of cholinergic transmission of ?-amyloid protein (β-AP) treated rats was studied by intracerebral microdialysis sampling combined with HPLC analysis. β-AP_1—40 was injected into nucleus basalis magnocellularis (NBM). Passive avoidance response test (step-down test) and delayed alternation task were used for memory testing. The impairment of memory after injection of β-AP_1—40 into NBM exhibited mainly the deficiency of short-term working memory. One week after injection of β-AP_1—40 the release of acetylcholine (ACh) from frontal cortex of freely-moving rats decreased significantly, and the response of cholinergic nerve ending to the action of high [K⁺] solution was rather weak. In control animals the percentage of increase of ACh-release during behavioral performance was 57%, while in β-AP_1—40-treated rats it was 34%. The temporary increase of the ACh-release of the rat put into a new place was also significantly diminished in β-AP_1—40-treated rats. The results show that the injection of β-AP_1—40 into NBM impairs the cholinergic transmission in frontal cortex, and the impairment of cholinergic transmission may be the main cause of the deficit of working memory.     

Acute Brain Inflammation and Oxidative Damage Are Related to Long-Term Cognitive Deficits and Markers of Neurodegeneration in Sepsis-Survivor Rats

Survivors from sepsis present long-term cognitive deficits and some of these alterations resemble the pathophysiological mechanisms of neurodegenerative diseases. For this reason, we analyzed beta-amyloid peptide (Aβ) and synaptophysin levels in the brain of rats that survived from sepsis and their relation to cognitive dysfunction and to acute brain inflammation. Sepsis was induced in rats by cecal ligation and puncture, and 30 days after surgery, the hippocampus and prefrontal cortex were isolated just after cognitive evaluation by the inhibitory avoidance test. The immunocontent of Aβ and synaptophysin were analyzed by Western blot analysis. Aβ increased and synaptophysin decreased in septic animals both in the hippocampus and prefrontal cortex concurrent with the presence of cognitive deficits. Prefrontal levels of synaptophysin correlated to the performance in the inhibitory avoidance. Two different treatments known to decrease brain inflammation and oxidative stress when administered at the acute phase of sepsis decreased Aβ levels both in the prefrontal cortex and hippocampus, increased synaptophysin levels only in the prefrontal cortex, and improved cognitive deficit in sepsis-survivor animals. In conclusion, we demonstrated that brain from sepsis-survivor animals presented an increase in Aβ content and a decrease in synaptophysin levels and cognitive impairment. These alterations can be prevented by treatments aimed to decrease acute brain inflammation and oxidative stress.     

Structural analogs of hemicholinium: effects on cholinergic and GABAergic markers in the CNS

The synthesis and neurotoxic effects of several structural analogs of hemicholinium were studied. All compounds were injected unilaterally into the lateral ventricle (4 nmol) and the effects of the hemicholinium derivatives on choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activity were compared with those of AF64A in an equimolar concentration. Structures of the newly synthesized compounds were confirmed by i.r., NMR and u.v. spectrometry and elemental analysis. The most specific cholinotoxic effects were observed with a,a-bis[di(2-chloroethyl)amino]4,4-biacetophenone (toxin 7). This compound causes specific decrease of ChAT activity in the brain structures (hippocampus and cortex) containing cholinergic terminals deriving from septum and the nucleus basalis magnocellularis (NBM), respectively. Large ChAT-positive magnocellular neurons in the NBM became paler and lost their processes following treatment with toxin 7 after 1 week.     

Environmental enrichment reduces the response to stress of the cholinergic system in the prefrontal cortex during aging

The present study was designed to evaluate the release of acetylcholine in the prefrontal cortex (PFC) induced by handling stress during aging and also to investigate whether this response changed as a result of the animals living in an enriched environment. Male Wistar rats of 3 months of age were housed in control and enriched conditions during the entire period of their adult life and experiments were performed at 6, 15 and 24 months of age. Spontaneous motor activity was first monitored in an open field arena. Then, rats were stereotaxically implanted with guide cannula to perform microdialysis experiments in the PFC and to evaluate the effects of stress on extracellular concentrations of acetylcholine. Handling stress increased the extracellular concentrations of acetylcholine in the PFC of control and enriched rats. These increases were not modified by aging in control rats. However, environmental enrichment (EE) reduced the effects of stress on acetylcholine concentrations in all groups of age. Spontaneous motor activity in the open field was reduced by aging. EE also decreased motor activity in all groups of age. These results suggest that EE reduces the reactivity to stress of the cholinergic system in the prefrontal cortex during aging.     

Basal forebrain lesions with or without reserpine injection inhibit cortical reorganization in rat hindpaw primary somatosensory cortex following sciatic nerve section.

To test the hypothesis that cortical reorganization depends on acetylcholine and one or more of the monoamines, the hindpaw cortex was mapped in eight different groups of mature rats: (1) untreated; (2) after sciatic nerve transection; (3) after intraperitoneal injections of reserpine, to reduce the level of cortical monoamines; (4) after ibotenic acid lesion of the nucleus basalis of Meynert (NBM), to destroy cholinergic cells projecting to the cortex; (5) after reserpine treatment and transection; (6) after ibotenic acid lesion and transection; (7) after reserpine treatment and ibotenic acid lesion; and (8) after reserpine treatment, ibotenic acid lesion, and transection. Four days after transection, the cortex had reorganized in the transected group. However, this process of reorganization was prevented in transected animals with NBM lesions. Treatment with reserpine alone did not inhibit the process of reorganization, nor did it enhance the effect of NBM lesion. Nonetheless, the animals treated with reserpine and transected had higher response thresholds in the reorganized cortex than did the animals that were treated but not transected. These data suggest that acetylcholine plays an important role in the early reorganization that follows deafferentation, and that one or more of the monoamines may have other influences on reorganization of the primary somatosensory cortex of adult rats.     

Some Long-Lasting Neurochemical Efafects of Prenatal or Early Postnatal Exposure to Diazepam

Changes in the uptake of various neurotransmitters were measured in the frontal cortex and hippocampus of male and female rats that were exposed to diazepam through the placenta or through the mother's milk during the prenatal or early postnatal period of rapid brain development. Earlier studies from our laboratory showed that early diazepam exposure has long-lasting behavioral consequences. The present results show that prenatally diazepam-exposed rat pups show significant reduction in choline uptake in the frontal cortex at 10 days of age. At 60 days of age, both pre- and postnatally exposed males, but not females, show significant differences from controls in terms of choline uptake, whereas postnatally exposed females whose behavior was shown previously to be profoundly affected by the diazepam exposure showed significant increase in gamma-aminobutyric acid (GABA) uptake in the hippocampus and reduction of 5-hydroxytryptamine (5HT) uptake in the cortex at 60 days of age.     

Laboratory handling and its influence on hormonal and metabolic parameters during acute inflammation in rats. A critique of long-term treatment by repeated injections

Twice a day, rats were exposed to handling stress by sham i.p. injections during a period of five days. This procedure progressively drove up the basic blood levels of free adrenaline, noradrenaline and dopamine, while insulin decreased to below normal. Blood glucose returned successively from hyperglycemia during the beginning of the experiment to normal later on. If rats subjected to acute inflammation were handled, the parameters blood catecholamines, glucose and the lymphocyte ingress into the site of inflammation all showed the same patterns of changes, suggesting a certain synchronization of the metabolic events. This synchronization failed to occur in animals with inflammation, but without handling. In any case, the disturbed metabolism of handled animals did not normalize during the test period of five days. Therefore, application of test substances by repeated injections is a useless method for the correct investigation of chronic exogenous influences.     

Ethylcholine mustard aziridinium ion lesions of the rat cortex result in retrograde degeneration of basal forebrain cholinergic neurons: Implications for animal models of neurodegenerative disease

Multiple injections of 2 nmols of cyclised ethylcholine mustard aziridinium ion (ECMA), a putative cholinergic neurotoxin, were made (unilaterally) into the cortical terminal field of cholinergic neurons projecting from the nucleus basalis of Meynert (NBM) in the rat basal forebrain. After 30 days, choline acetyltransferase enzymatic activity, a marker for cholinergic function, was significantly lowered in both ipsilateral cortex and NBM, and cholinergic cell bodies in the latter reduced in cross-sectional area, a spectrum of effects characteristic of retrograde degeneration of this pathway. These results are discussed in the context of neurodegenerative diseases affecting cholinergic function.     

Maturation and maintenance of cholinergic medial septum neurons require glucocorticoid receptor signaling

Glucocorticoids have been shown to influence trophic processes in the nervous system. In particular, they seem to be important for the development of cholinergic neurons in various brain regions. Here, we applied a genetic approach to investigate the role of the glucocorticoid receptor (GR) on the maturation and maintenance of cholinergic medial septal neurons between P15 and one year of age by using a mouse model carrying a CNS-specific conditional inactivation of the GR gene (GRNesCre). The number of choline acetyltransferase and p75NTR immuno-positive neurons in the medial septum (MS) was analyzed by stereology in controls versus mutants. In addition, cholinergic fiber density, acetylcholine release and cholinergic key enzyme activity of these neurons were determined in the hippocampus. We found that in GRNesCre animals the number of medial septal cholinergic neurons was significantly reduced during development. In addition, cholinergic cell number further decreased with aging in these mutants. The functional GR gene is therefore required for the proper maturation and maintenance of medial septal cholinergic neurons. However, the loss of cholinergic neurons in the medial septum is not accompanied by a loss of functional cholinergic parameters of these neurons in their target region, the hippocampus. This pinpoints to plasticity of the septo-hippocampal system, that seems to compensate for the septal cell loss by sprouting of the remaining neurons.     

Effects of Melatonin on Memory and Learning Deficits Induced by Exposure to Thinner

The neurotoxic effects of thinner, a mixture including aromatic compounds (in particular, toluene) and widely used as an industrial solvent, were examined. Exposure of rats to high inhalation concentrations (3000 p.p.m.) of thinner for 45 days (1 h per day) significantly influenced the cognitive functions and levels of neural cell adhesion molecules (NCAM) in the hippocampus, cortex, and cerebellum of experimental animals. These exposures also caused dramatic increases in levels of LPO (malondialdehyde and 4-hydroxyalkenals) in these cerebral structures, while melatonin administration significantly reduced the LPO amounts in these brain regions. The level of NCAM (180 kDa) decreased significantly in the hippocampus and cortex of thinner-exposed rats. Furthermore, thinner-exposed rats showed cognitive deficits in the passive avoidance and Morris water maze tasks; these negative effects were considerably compensated in rats additionally chronically treated with melatonin. It is concluded that treatment with melatonin prevents the development of learning and memory deficits caused by thinner exposure, possibly by reducing oxidative stress and normalizing the neural plasticity.     

Lycopene attenuates diabetes-associated cognitive decline in rats

Diabetes-induced learning and memory impairment, characterized by impaired cognitive functions and neurochemical and structural abnormalities, involve direct neuronal damage caused by intracellular glucose. The present study was designed to investigate the effect of lycopene, a potent anti-oxidant and anti-inflammatory molecule, on cognitive functions, oxidative stress and inflammation in streptozotocin (STZ)-induced diabetic rats. Cognitive functions were investigated using a spatial version of the Morris water maze test. Acetylcholinesterase activity, a marker of cholinergic dysfunction, was increased by 1.8 fold in the cerebral cortex of diabetic rats. There was about 2 fold and 2.2 fold rise in thiobarbituric acid-reactive substance levels in cerebral cortex and hippocampus of diabetic rats, respectively. Non-protein thiol levels and enzymatic activities of superoxide dismutase and catalase were decreased in both cerebral cortex and hippocampal regions of diabetic rat brain. Total nitric oxide levels in cerebral cortex and hippocampus was increased by 2.4 fold and 2 fold respectively. Serum tumor necrosis factor-alpha, an inflammatory marker, was found to increase by 8 fold in diabetic rats. Chronic treatment with lycopene (1, 2 and 4 mg/kg; p.o.) significantly and dose dependently attenuated cognitive deficit, increased acetylcholinesterase activity, oxidative-nitrosative stress and inflammation in diabetic rats. The results emphasize the involvement of oxidative-nitrosative stress and peripheral inflammation in the development of cognitive impairment in diabetic animals and point towards the therapeutic potential of lycopene in diabetes-induced learning and memory impairment.     

Oxidative stress in head trauma in aging

Oxidative damage is proposed as a key mediator of exacerbated morphological responses and deficits in behavioral recovery in aged subjects with traumatic brain injury (TBI). In the present study, we show exacerbated loss of tissue in middle aged (12 months) and aged (22 months) Fisher-344 rats compared to young animals (3 months) subjected to moderate TBI. Analysis of 4-hydroxynonenal (4-HNE) and acrolein, neurotoxic by-products of lipid peroxidation, shows significant (P < 0.05) age-dependent increases in ipsilateral (IP) hippocampus 1 and 7 days post injury. In IP cortex, 4-HNE was significantly elevated 1 day post injury in all age groups, and both 4-HNE and acrolein were elevated in middle aged and aged animals 7 days post injury. Comparison of antioxidant enzyme activities shows significant (P < 0.05) age-dependent decreases of manganese superoxide dismutase in IP hippocampus and cortex 1 and 7 days post injury. Glutathione reductase activity also showed an age-dependent decrease. Overall, our data show increased levels of oxidative damage, diminished antioxidant capacities, and increased tissue loss in TBI in aging.     

CHANGES IN CENTRAL NORADRENALINE NEURONSADMINISTRATION AFTER SYSTEMIC 6-HYDROXYDOPAMINE ADMINISTRATION

—Intravenous injection of a large dose of 6-hydroxydopamine (100 mg/kg) to adult rats caused a significant and long-lasting reduction (about 30 per cent) of the in oirro uptake of [³H]NA in the cerebral cortex and spinal cord, while no changes were seen in the hypothalamus. The endogenous NA in whole brain was similarly reduced (about 20 per cent). Fluorescence histochemistry revealed catecholamine accumulations which are degenerative signs, induced by 6-hydroxydopamine, in axons of the dorsal NA bundle innervating the cerebral cortex. It is concluded that the blood–brain barrier in adult rats is not completely protective with respect to the neurotoxic action of systemically injected 6-hydroxydopamine, which can produce degeneration of a significant number of NA nerve terminals in the cerebral cortex and spinal cord. Previous studies have shown that 6-hydroxydopamine caused a permanent and selective degeneration of a large number of central NA nerve terminals when injected systemically up to 1 week after birth, due to an incompletely developed blood-brain barrier. This barrier for 6-hydroxydopamine develops between the 7th and 9th day after birth (Sachs , 1973). In the present study 6-hydroxydopamine was found to cause a small transient reduction in [³H]NA uptake in cerebral cortex of rats between 9 and 28 days of age, while in older rats the damage produced by 6-hydroxydopamine was long-lasting. Thus, the NA nerves ascending to the cerebral cortex seem to possess a regenerative capacity to a 6-hydroxydopamine-induced degeneration up to about 28 days postnatally, but which later disappears or is markedly retarded.     

Promoting positive states: the effect of early human handling on play and exploratory behaviour in pigs

It is known that tactile stimulation (TS) during ontogeny modifies brain plasticity and enhances the motor and cognitive skills. Our hypothesis was that early handling including TS would increase play and exploratory behaviour in commercial pigs under standardized test conditions. Piglets from 13 litters were subjected to three handling treatments from 5 to 35 days of age: all the piglets were handled (H), none of the piglets were handled (NH) or half of the piglets in the litter were handled (50/50). At 42 days of age, the pigs’ behaviour was observed in pairs in a novel pen with a ‘toy’ (tug rope). The main results were that more locomotor play was performed by pigs from litters where all or half of them had been handled, whereas social exploratory behaviour was more pronounced in pigs from litters where half of them had been handled. Although behaviour was affected by the interaction of treatment with sex or with weight category, we propose that the handling procedure does seem to have acted to increase locomotor skills and that handling half of the piglets in the litter may have triggered a series of socio-emotional interactions that were beneficial for the whole group.     

Resveratrol prevents alcohol-induced cognitive deficits and brain damage by blocking inflammatory signaling and cell death cascade in neonatal rat brain

Human prenatal ethanol exposure that occurs during a period of increased synaptogenesis known as the 'brain growth spurt' has been associated with significant impairments in attention, learning and memory. Recent studies have shown that administration of ethanol to infant rats during the synaptogenesis period (first 2 weeks after birth) triggers extensive apoptotic neurodegeneration throughout many regions of the developing brain and results in cognitive dysfunctions as the animal matures. The present study was designed with an aim to investigate the effect of resveratrol, a polyphenolic phytoalexin (trans-3,5,4-trihydroxy stilbene) present in red wine on alcohol-induced cognitive deficits and neuronal apoptosis in rat pups postnatally exposed to ethanol. Pups were administered ethanol (5 g/kg, 12% v/v) by intragastric intubation on postnatal days 7, 8, and 9. Ethanol-exposed pups showed impaired memory performance in both Morris water maze elevated plus maze task recorded by using computer tracking with EthoVision software. Behavioral deficit in ethanol-exposed pups was associated with enhanced acetylcholinesterase activity, increased oxidative-nitrosative stress, cytokine (TNF-α, IL-1β and TGF-β), nuclear factor kappa beta and caspase 3 levels in both cerebral cortex and hippocampus. Chronic treatment with resveratrol (10 and 20 mg/kg) significantly attenuated all the behavioral, biochemical and molecular changes in different brain regions of ethanol administered pups. The major finding of the study is that resveratrol blocks activation of nuclear factor kappa beta pathway and apoptotic signaling and prevents cognitive deficits in rats postnatally exposed to ethanol.     

The effects of nerve growth factor upon the neuropeptide content of the suprachiasmatic nucleus of rats withdrawn from ethanol are mediated by the nucleus basalis magnocellularis

It has been previously shown that withdrawal from alcohol decreases the synthesis and expression of vasopressin (VP) and vasoactive intestinal polypeptide (VIP) in the suprachiasmatic nucleus (SCN), and that the infusion of NGF over 1 month completely restores these changes. Because SCN neurons do not express TrkA, NGF might have exerted its effects either through direct signalling of the neurons via p75^NTR or by enhancing the activity of the cholinergic afferents to the SCN, which arise from the nucleus basalis magnocellularis (NBM). The observation that the infusion of NT-3 to withdrawn rats does not elicit any change in neuropeptide expression in the SCN suggests that ACh might be implicated in this process, a hypothesis that we have attempted to clarify in this study. For this purpose we destroyed, with quinolinic acid, the NBM of rats withdrawn from ethanol and later infused them with NGF over a period of 13 days. The total number and the somatic volume of SCN neurons immunoreactive for VP and VIP were stereologically estimated. No differences were found in the total number of neurons between quinolinic-injected NGF-treated withdrawn animals and intact withdrawn rats. However, the somatic volume of SCN neurons from quinolinic-injected animals was significantly reduced relative to control and withdrawn rats. The present results unequivocally demonstrate that the trophic effects exerted by NGF upon SCN neurons do not depend on direct neuronal signalling. Instead, they are indirect and, according to our results, NBM neurons, whose axons give rise to a cholinergic projection to the SCN, seem to be essential for eliciting those effects.