Ivan Bednar

Chronic nicotine treatment reduces β-amyloidosis in the brain of a mouse model of Alzheimer's disease (APPsw)

Alzheimers disease neuropathology is characterised by β‐amyloid plaques and neurofibrillary tangles. Inhibition of β‐amyloid accumulation may be essential for effective therapy in Alzheimers disease. In this study we have treated transgenic mice carrying the Swedish mutation of human amyloid precursor protein [Tg(Hu.APP695.K670N‐M671L)2576], which develop brain β‐amyloid deposits, with nicotine in drinking fluid (200 µg/mL) from 9–14.5 months of age (5.5 months). A significant reduction in amyloid β peptide 1–42 positive plaques by more than 80% (p < 0.03) was observed in the brains of nicotine treated compared to sucrose treated transgenic mice. In addition, there was a selective reduction in extractable amyloid β peptides in nicotine treated mice; cortical insoluble 1–40 and 1–42 peptide levels were lower by 48 and 60%, respectively (p < 0.005), whilst there was no significant change in soluble 1–40 or 1–42 levels. The expression of glial fibrillary acidic protein was not affected by nicotine treatment. These results indicate that nicotine may effectively reduce amyloid β peptide aggregation in brain and that nicotinic drug treatment may be a novel protective therapy in Alzheimers disease.

Up-regulation of the inflammatory cytokines IFN-γ and IL-12 and down-regulation of IL-4 in cerebral cortex regions of APPSWE transgenic mice

Alzheimers disease (AD) is a progressive neurodegenerative disorder, of which the pathogenesis is thought to involve increased beta-amyloid (Abeta) deposition and abnormal immunological responses. To elucidate the mechanisms involved in Abeta-mediated inflammation, we used immunocytochemistry and in situ hybridization to study the potential role of the cytokines interferon-gamma (IFN-gamma), interleukin (IL)-12 and IL-4 in transgenic mice APP(SWE) (Tg2576) that overexpress the human beta-amyloid precursor protein gene. Cytokine and cytokine mRNA expression was detected in brain sections from cortical regions at various postnatal ages ranging from 3 to 19 months. High levels of IFN-gamma and IL-12 mRNA expression, as well as their protein production, appeared early at 9 months and peaked at 17-19 months in Tg2576 mice. Significantly increased transcripts of IFN-gamma and IL-12 genes were found in the reactive microglia and astrocytes surrounding beta-amyloid deposits. In accordance with the kinetics of mRNA levels, the expression of IFN-gamma and IL-12 at the protein level was positively correlated with age and reached a maximum in 17-19-month-old mice. Both findings suggest a role for the pro-inflammatory cytokines IFN-gamma and IL-12 in early disease development and are consistent with microglial activation related to beta-amyloid formation. In contrast, transcription and production of IL-4 in brain sections was almost undetectable in transgenic mice up to post-natal ages of 17-19 months. These results suggest a major pro-inflammatory role for IL-12 and IFN-gamma in Tg2576 transgenic mice that may provide the association between beta-amyloid plaque formation and microglial and astrocyte activation in these animals. These observations call for further studies on the potential role of anti-inflammatory therapeutic strategies for AD.

Nicotine reduces Aβ in the brain and cerebral vessels of APPsw mice

Ten days treatment with nicotine reduced insoluble amyloid Aβ1–40 and Αβ1–42 peptides by 80% in the cortex of 9‐month‐old APPsw mice, which is more than that observed in 14.5‐month‐old mice following nicotine treatment for 5.5 months. A reduction in Aβ associated with cerebral vessels was observed in addition to that deposited as parenchymal plaques after 5.5 months treatment. The diminution in Aβ peptides observed was not accompanied by changes in brain α, β or γ secretase‐like activities, NGF or BDNF protein expression measured in brain homogenates. A significant increase in sAPP was observed after nicotine treatment of SH‐SY5Yneuroblastoma cells that could be blocked by the nicotinic antagonist mecamylamine. Attenuation of elevated [125I]‐αbungarotoxin binding (α7) in APPsw mice was observed after 5.5 months nicotine treatment. Both these observations suggest that the reduction in insoluble Aβ by nicotine might be in part mediated via the α7 nicotinic receptor. Further studies are required to identify potential mechanisms of the nicotines amyloid‐reducing effect.

Selective Nicotinic Receptor Consequences in APPSWE Transgenic Mice

The nicotinic (nAChRs) and muscarinic (mAChRs) acetylcholine receptors and acetylcholinesterase (AChE) activity were studied in the brains of APP(SWE) transgenic mice (Tg+) and age-matched nontransgenic controls (Tg-) that were between 4 and 19 months of age. A significant increase in the binding of 125I-labeled alpha-bungarotoxin (alpha7 nAChRs) was observed in most brain regions analyzed in 4-month-old Tg+ mice, preceding learning and memory impairments and amyloid-beta (Abeta) pathology. The enhanced alpha7 receptor binding was still detectable at 17-19 months of age. Increase in [3H]cytisine binding (alpha4beta2 nAChRs) was measured at 17-19 months of age in Tg+ mice, at the same age when the animals showed heavy Abeta pathology. No significant changes in [3H]pirenzepine (M1 mAChRs) or [3H]AFDX 384 (M2 mAChRs) binding sites were found at any age studied. The upregulation of the nAChRs probably reflects compensatory mechanisms in response to Abeta burden in the brains of Tg+ mice.

Involvement of Cholecystokinin in Food Intake: III. Oestradiol Potentiates the Inhibitory Effect of Cholecystokinin Octapeptide on Food Intake in Ovariectomized Rats.

The role of Cholecystokinin in a model of hypophagia, oestradiol‐treated Ovariectomized rats, was investigated. Implantation of oestradiol‐filled constant‐release implants in rats made obese by ovariectomy potentiated the inhibitory effect of intraperitoneal injection of Cholecystokinin octapeptide on food intake after 24 h of food deprivation. The alterations in the concentration of Cholecystokinin in pjasma and of cholecystokinin‐like immunoreactivity in cerebrospinal fluid produced by deprivation of food for 24 h and subsequent food intake for 1 h were unaffected by the oestradiol treatment as was the amount of food consumed during 1 h. Oestradiol‐treated rats deprived of food for 6 h, however, consumed less food during a 15‐min test than controls. Treatment with oestradiol blunted the decrease in the concentration of cholecystokinin‐like immunoreactivity in the cerebrospinal fluid in response to 6 h of food deprivation. No alterations in the concentration of Cholecystokinin in plasma occurred after this period of food deprivation and subsequent feeding during 15 min in either oestradiol‐treated or control rats. Thus, treatment with oestradiol enhances responsivity to exogenous Cholecystokinin octapeptide and changes the response of endogenous levels of cholecystokinin‐like immunoreactivity in the cerebrospinal fluid to a short period of food deprivation. It is suggested that these effects are caused by an action of oestradiol on Cholecystokinin pathways in the brain. The results support the suggestion that hunger in the rat is inversely related to the decrease in the concentration of cholecystokinin‐like immunoreactivity in the cerebrospinal fluid.

Expression of nicotinic acetylcholine receptors in human and rat adrenal medulla.

Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent.

Involvement of Dopamine in Inhibition of Food Intake by Cholecystokinin Octapeptide in Male Rats

Deprivation of food reduced the level of dopamine in the cerebrospinal fluid of male rats and subsequent ingestion of food or intraperitoneal injection of Cholecystokinin octapeptide restored the level. Injection of a dopamine receptor agonist (apomorphine) or Cholecystokinin octapeptide inhibited food intake and these effects were reversed by pretreatment with a dopamine receptor antagonist (cis‐flupentixol). Blockade of cholecystokinin‐A receptors, by treatment with L‐364,718, but not cholecystokinin‐B receptors, by treatment with L‐365,260, blocked the inhibitory effect of Cholecystokinin octapeptide on food intake but did not affect the inhibitory effect of apomorphine. It is suggested that Cholecystokinin interacts with dopamine in the control of food intake.

Inhibition of sexual behavior in female rats by intracerebral injections of Met-enkephalin in combination with an inhibitor of enkephalin degrading enzymes

Injection of Met-enkephalin, either subcutaneously (10 or 100 microgram, s.c.), intracerebroventricularly (100 ng or 1 microgram, i.c.v.) or into the mesencephalic central gray (100 ng), had no inhibitory effect on sexual behavior in female rats. If combined with [(R)-3-(N-hydroxy)-carboxamido-2-benzylpropanoyl]-L-alanine (kelatorphan, 5 micrograms i.c.v.; 1.5 micrograms in the central gray; 200 micrograms s.c.), an inhibitor of enkephalin degrading enzymes, however, i.c.v. or central gray (100 ng), but not s.c. (100 micrograms), injection of Met-enkephalin suppressed the behavior. Injection of Leu-enkephalin, s.c. or i.c.v. alone or in combination with kelatorphan, had no inhibitory effect. Peptidases may rapidly inactivate Met-enkephalin after intracerebral injection and prevent the behavioral effect. Inhibition of sexual behavior by Met-enkephalin may occur during lactation, a physiological state when the behavior is suppressed by an opioid peptide.

Simultaneous display of sexual and ingestive behaviour by rats

Intraoral infusion of sucrose activates consummatory ingestive behaviour in rats selectively, i.e. the rat only emits the responses used to ingest food. Activation of consummatory ingestive behaviour in this way had no effect on the subsequent display of sexual behaviour by male or female rats and vice versa. Rats infused intraorally with sucrose and presented with a sexual partner showed ingestive and sexual behaviour simultaneously. Pretreatment with cholecystokinin octapeptide inhibited the ingestion of sucrose in both males and females but had no effect on the simultaneous display of sexual behaviour. Ingestion of sucrose from a drinking spout, a test in which the rat has to emit responses to obtain food, i.e. show appetitive ingestive behaviour, was inhibited by the presentation of a sexual partner in rats of both sexes. These results show that the mechanisms controlling consummatory sexual and ingestive behaviour operate independently and that the presentation of a sexual partner inhibits appetitive ingestive behaviour.

Involvement of Cholecystokinin in Food Intake: II. Lactational Hyperphagia in the Rat

The role of Cholecystokinin in the hyperphagia of lactation was studied by measuring the concentration of this hormone in plasma and cerebrospinal fluid in relation to food intake in lactating rats. Cholecystokinin was measured by high‐performance liquid chromatography and radioimmunoassay in plasma and by radioimmunoassay in cerebrospinal fluid. Plasma concentrations of Cholecystokinin were increased in freely‐fed lactating rats compared with non‐lactating, regularly cycling rats. However, after 24 h of food deprivation the concentration of plasma Cholecystokinin was markedly decreased in the lactating rats to levels which were lower than those of non‐lactating animals. Furthermore, plasma levels of Cholecystokinin did not increase in response to 1 h of feeding in lactating rats, whereas in non‐lactating rats they did. In contrast, the concentration of cholecystokinin‐like immunoreactivity in the cerebrospinal fluid was the same in freely‐fed lactating and non‐lactating rats. As in plasma, food deprivation markedly decreased the levels of cholecystokinin‐like immunoreactivity in the cerebrospinal fluid of lactating rats but unlike in plasma, the levels were restored by feeding. The levels of cholecystokinin‐like immunoreactivity were not changed under these conditions in the non‐lactating rats. These results show that there is no correlation between the concentration of Cholecystokinin in plasma and cerebrospinal fluid, which supports the suggestion that the cholecystokinin‐like immunoreactivity in the cerebrospinal fluid is derived from the brain. Removal of the litter from lactating rats deprived of food for 24 h reduced food intake and increased the concentration of cholecystokinin‐like immunoreactivity in the cerebrospinal fluid, but not in plasma. The inhibition of food intake caused by an intraperitoneal injection of Cholecystokinin octapeptide increased after litter removal. It is suggested that hunger in the lactating rat is reflected by a decrease in the levels of cholecystokinin‐like immunoreactivity in the cerebrospinal fluid and satiety by the restoration of these levels.