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266 Senktide attenuates scopolamine-induced impairment of spontaneous alternation performance in mice
FIFTH INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE rain after carbachol injection completely antagonized this learning impairment. In the microdialysis experiment, a probe was perfused with Krebs-Ringer's solution (contained 0.1 gM eserine) at the rate of 2 gl/min. Samples were collected at 20rain intervals and assayed for acetylcholine and choline by HPLC with electrochemical detection. Peffusion of earbaehol 300 glVl for 40 rain through a dialysis probe significantly decreased acetylcholine release in the hippocampus. However, carbachol did not decrease acetylcholinerelease from the frontal cortex. Administration of dynorphin A (1-13) 0.5 nmol/rat (i.c.v.) at the beginning of the perfusiou period, completely blocked the decrease in extracellular acetylcholine concentration induced by carbachol in the hippocampus. This reversal effect of dynorphin A (1-13) was abolished by pre-treatment with nor-binaltorphimine 5.44 nmol/rat (i.c.v.), a selective kappa-opioid receptor antagonist. Co-perfusion of atropine (0.1 mM) with carbachol abolished the decrease in acetylcholine release induced by earbachol. Scopolamine (1 mg/kg, s.c.), a muscarinic acetylcholine receptor antagonist, markedly increased the acetylcholine concentration in the hippocampus. Dynorphin A (1-13) did not affect this increase induced by scopolamine. Dynorphin A (1-13) (0.5 - 5.0 nmol) had no effect on the acetylcholine concentration in the hippocampus and the frontal cortex. These findings suggest that the dynorphin A (1-13) improves the carbachol-indueed impairment of learning and memory accompanied by abolishment of reductions in acetylcholine release via kappa opioid receptors. (This study was supported in part by a grant from the Kowa Life Science Foundation and Grants-in-Aid for Scientific Research (Nos. 07457560 and 07557303) from the Ministry of Education, Science and Culture, Japan)
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unrelated to this study. CSF was analyzed by Western blots using the monoclonal antibody 22C11 from Boehringer Mannheim and developed by enhanced chemiluminescence (ECL) solution and exposed to film for quantification by image analysis. Brain tissues and cervical CSF were removed within a few minutes of euthanasia and fixed in 10% formalin and then embedded in paraffin. After sectioning the tissues were deparaffinized and stained by immunohistochemistry using the monoclonal antibody 10D5 from Athena Neuroscience. The quantification of plaque formation in tissue was determined by image analysis from the slide tissues. A Pearson correlation was calculated between the surface area of densest plaque formation on the tissues and the levels of sAPP found in CSF. The result showed a positive correlation of r = 0.7 (p = 0.0008). These data are somewhat in contrast to those reported for AD patients. Human studies have reported an inverse relationship to CSF sAPP levels and individuals with AD symptoms. It may be that in AD patients sufficient brain cell loss has occurred by the time a patient is diagnosed with AD to reduce the amount ofsAPP seen the CSF. If this is the case, a high sAPP level in early AD may be associated with a higher probability of developing the disease.
268 266 Senktide Attenuates Scopolamine-Induced Impairment of Spontaneous Alternation Performance in Mice M. Ukai*, N. Shinkal andT. Kameyama Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, Nagoya 468, Japan The effects of the neurokinin-3 (NK-3) receptor agonist senktide on the scopolamine-induced amnesia were investigated by using spontaneous alternation performance associated with spatial working memory in mice. Male mice of the ddY strain weighing between 30 and 40 g were used. Spontaneous alternation performance was assessed in a black-painted Y-maze. The testing procedure was based on that of Sarter et al. (Psychopharmacology 94, 491-495, 1988). Following vehicle or drug injections, each of the mice was placed at the end of one arm and allowed to move freely through the maze for an 8-min test session. The sequence of arm entries was recorded manually. An alternation was defined as the entry into all three arms on consecutive choices. Senktide and [Trp 7, fl-Alas] neurokinin A-(4-10)(R-486) were administered into the lateral ventricle of the brain (i.c.v.) in an injection volume of 5 At l/mouse over 1 min through injection needle under brief ether anesthesia. Senktide, R-486 and scopolamine were injected 10, 20 and 30 min before measurements, respectively. Senktide (0.0003-0.03 At g) alone did not influence either spontaneous alternation performance or total arm entries, although scopolamine (1 mg/kg) inhibited spontaneous alternation performance, and contrary increased total arm enu'ies. In contrast, senktide (0.003 and 0.03 At g) inhibited the scopolamine (1 mg/kg)-indueed impairment of spontaneous alternation performance without affecting the scopolamine (1 mg/kg)-induced increase in total ann enu'ies. In addition, senktide (0.0003 At g) significantly inhibited the scopolamine-induced increase in total arm entries. R-486 (1 and 5.6 At g), an NK-3-selective receptor antagonist, alone did not influence either spontaneous alternation performance or total arm entries, whereas the beneficial effects of senktide (0.003 At g) were not influenced by pretreatment with R-486. These findings suggest that senktide attenuates the scopolamine-induced impairment of spontaneous alternation performance associated with spatial working memory, resulting from some pharmacological action of senktide other than its action on NK-3 receptors.
Subpopulations of Rat Congo Red-Staining Neurons Display Distinct Morphological Features and Altered Distribution of fl-Amyloid Precursor Protein Immunoreactivity. J.J. Miguel-Hidalgo* and R. Cacabelos Department of Basic Neuroscienees, EuroEspes Biomedical Research Center, Santa Marta de Babio s/n, Bergondo, 15166 La Comfia, Spain We have developed a technique based on Congo Red staining that permits to sort out a subpopulation of neurons with characteristics suggesting inactivity or a reversible degenerative state. The neurons labeled by Congo Red (tentatively called CR neurons) show shrinked somata, and smaller and darker nuclei than negative cells (Non-CR ceils). In many instances, their main dendritic shafts are also labeled with Congo Red. With the Bielschowsky silver impregnation method and the Cresyl Violet Nissl staining method, two comparable subpnpulations of cells could be distinguished when using the same morphometrical criteria as those used for CR and Non-CR cells. We found that CR neurons are located preferentially in some brain regions while some areas and nuclei are virtually devoid of them. The distribution in some of the regions where these cells can be seen (i.e. hippocampus, neocortex, red nucleus) and their proportion as compared to non-RC cells varies greatly from animal to animal and also after particular lesions in the hippocampus. Unilateral injections of water that neatly damaged the hippocampal dentate gyms, large cortical lesions or eye-enucleatinn produced a decrease in the number of CR-eells in the CA1 area of the hippocampus, that appears to represent a shift of the CR staining subclass to the Non-CR subclass. Since morphological variations in the soma and nucleus might be paralleled by alterations in membrane proteins related to plasticity, a combination of the Congo Red staining technique with immunohistochemical demonstration of the B-amyluid precursor protein (APP) was applied to brain sections from experimental animals. We observed that Congo Red-positive neurons from various encephalic regions contained APP immunoreactivity distributed in relatively large patches irregularly interspersed with non-immunoreactive spaces. By contrast, APP-immunoreactivity appeared in CR-negative cells as small granules uniformly distributed in the soma. The present results suggests that plastic morphological changes in neurons are concomitant with alterations in the distribution of the 13-amyloid precursor protein.
Supported by EUROESPESFoundation.
267 Spinal Fhlid sAPP as an Index of ~-Amyloid in the Canine Brain M. J. Russell', Y. Hou, R. G. White, and J. S. Marks University of California, Davis, CA 95616, U.S.A. Some breeds of dogs spontaneously develop diffuse and occasionally neuritic plaques in 60-80% of the brains of aged individuals. However, it has not been possible to determine which aged dogs have developed amyloid plaques in their brains prior to tissue analysis. Human studies have suggested a negative correlation between individuals with Alzheimer's disease (AD) symptoms and soluble amyloid precursor protein (sAPP) levels in cerebral spinal fluid (CSF). Thus, the lower the sAPP levels the more likely an individual is to have AD. To determine if there is a relationship in dogs between CSF sAAP and [3-amy[old plaque formation, we analyzed brain tissue and CSF from 40 canines ranging from 1 to 16 years of age. Twenty of these dogs were colony raised beagles, and 20 were random source mixed breed animals. All of these animals were sacrificed for clinical reasons
269 Lack of Correlation Between Plaque Burden and Cognition in the Aging Monkey J.A. Sloane*, M. Pietropaolo, D.L. Rosene, M.B. Moss, A. Peters, T. Kemper and C.R. Abraham Departments of Pathology, Biochemistry, Medicine and Anatomy and Neurobiology, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118, USA, [email protected] In order to assess whether amyloid plaque accumulation in the monkey brain can account for age-related cognitive impairment that begin about 20 years of age, we measured plaque density in the brains of rhesus monkeys ages 5 to 31. We used immunohistochemistry employing the monoclonal antibody 6E10 which is specific to amino acids 1-17 of amyloid 13peptide to identify amyloid plaques in serial coronal sections of the forebrain. Amyloid plaques accumulate with age, starting at 25 and escalating at 30 years of age. From 25 to 29 years of age, plaques are found only in a few monkeys and are relatively sparse in number. Only at 30 years of age does plaque density increase dramatically. Results from
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unrelated to this study. CSF was analyzed by Western blots using the monoclonal antibody 22C11 from Boehringer Mannheim and developed by enhanced chemiluminescence (ECL) solution and exposed to film for quantification by image analysis. Brain tissues and cervical CSF were removed within a few minutes of euthanasia and fixed in 10% formalin and then embedded in paraffin. After sectioning the tissues were deparaffinized and stained by immunohistochemistry using the monoclonal antibody 10D5 from Athena Neuroscience. The quantification of plaque formation in tissue was determined by image analysis from the slide tissues. A Pearson correlation was calculated between the surface area of densest plaque formation on the tissues and the levels of sAPP found in CSF. The result showed a positive correlation of r = 0.7 (p = 0.0008). These data are somewhat in contrast to those reported for AD patients. Human studies have reported an inverse relationship to CSF sAPP levels and individuals with AD symptoms. It may be that in AD patients sufficient brain cell loss has occurred by the time a patient is diagnosed with AD to reduce the amount ofsAPP seen the CSF. If this is the case, a high sAPP level in early AD may be associated with a higher probability of developing the disease.
268 266 Senktide Attenuates Scopolamine-Induced Impairment of Spontaneous Alternation Performance in Mice M. Ukai*, N. Shinkal andT. Kameyama Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, Nagoya 468, Japan The effects of the neurokinin-3 (NK-3) receptor agonist senktide on the scopolamine-induced amnesia were investigated by using spontaneous alternation performance associated with spatial working memory in mice. Male mice of the ddY strain weighing between 30 and 40 g were used. Spontaneous alternation performance was assessed in a black-painted Y-maze. The testing procedure was based on that of Sarter et al. (Psychopharmacology 94, 491-495, 1988). Following vehicle or drug injections, each of the mice was placed at the end of one arm and allowed to move freely through the maze for an 8-min test session. The sequence of arm entries was recorded manually. An alternation was defined as the entry into all three arms on consecutive choices. Senktide and [Trp 7, fl-Alas] neurokinin A-(4-10)(R-486) were administered into the lateral ventricle of the brain (i.c.v.) in an injection volume of 5 At l/mouse over 1 min through injection needle under brief ether anesthesia. Senktide, R-486 and scopolamine were injected 10, 20 and 30 min before measurements, respectively. Senktide (0.0003-0.03 At g) alone did not influence either spontaneous alternation performance or total arm entries, although scopolamine (1 mg/kg) inhibited spontaneous alternation performance, and contrary increased total arm enu'ies. In contrast, senktide (0.003 and 0.03 At g) inhibited the scopolamine (1 mg/kg)-indueed impairment of spontaneous alternation performance without affecting the scopolamine (1 mg/kg)-induced increase in total ann enu'ies. In addition, senktide (0.0003 At g) significantly inhibited the scopolamine-induced increase in total arm entries. R-486 (1 and 5.6 At g), an NK-3-selective receptor antagonist, alone did not influence either spontaneous alternation performance or total arm entries, whereas the beneficial effects of senktide (0.003 At g) were not influenced by pretreatment with R-486. These findings suggest that senktide attenuates the scopolamine-induced impairment of spontaneous alternation performance associated with spatial working memory, resulting from some pharmacological action of senktide other than its action on NK-3 receptors.
Subpopulations of Rat Congo Red-Staining Neurons Display Distinct Morphological Features and Altered Distribution of fl-Amyloid Precursor Protein Immunoreactivity. J.J. Miguel-Hidalgo* and R. Cacabelos Department of Basic Neuroscienees, EuroEspes Biomedical Research Center, Santa Marta de Babio s/n, Bergondo, 15166 La Comfia, Spain We have developed a technique based on Congo Red staining that permits to sort out a subpopulation of neurons with characteristics suggesting inactivity or a reversible degenerative state. The neurons labeled by Congo Red (tentatively called CR neurons) show shrinked somata, and smaller and darker nuclei than negative cells (Non-CR ceils). In many instances, their main dendritic shafts are also labeled with Congo Red. With the Bielschowsky silver impregnation method and the Cresyl Violet Nissl staining method, two comparable subpnpulations of cells could be distinguished when using the same morphometrical criteria as those used for CR and Non-CR cells. We found that CR neurons are located preferentially in some brain regions while some areas and nuclei are virtually devoid of them. The distribution in some of the regions where these cells can be seen (i.e. hippocampus, neocortex, red nucleus) and their proportion as compared to non-RC cells varies greatly from animal to animal and also after particular lesions in the hippocampus. Unilateral injections of water that neatly damaged the hippocampal dentate gyms, large cortical lesions or eye-enucleatinn produced a decrease in the number of CR-eells in the CA1 area of the hippocampus, that appears to represent a shift of the CR staining subclass to the Non-CR subclass. Since morphological variations in the soma and nucleus might be paralleled by alterations in membrane proteins related to plasticity, a combination of the Congo Red staining technique with immunohistochemical demonstration of the B-amyluid precursor protein (APP) was applied to brain sections from experimental animals. We observed that Congo Red-positive neurons from various encephalic regions contained APP immunoreactivity distributed in relatively large patches irregularly interspersed with non-immunoreactive spaces. By contrast, APP-immunoreactivity appeared in CR-negative cells as small granules uniformly distributed in the soma. The present results suggests that plastic morphological changes in neurons are concomitant with alterations in the distribution of the 13-amyloid precursor protein.
Supported by EUROESPESFoundation.
267 Spinal Fhlid sAPP as an Index of ~-Amyloid in the Canine Brain M. J. Russell', Y. Hou, R. G. White, and J. S. Marks University of California, Davis, CA 95616, U.S.A. Some breeds of dogs spontaneously develop diffuse and occasionally neuritic plaques in 60-80% of the brains of aged individuals. However, it has not been possible to determine which aged dogs have developed amyloid plaques in their brains prior to tissue analysis. Human studies have suggested a negative correlation between individuals with Alzheimer's disease (AD) symptoms and soluble amyloid precursor protein (sAPP) levels in cerebral spinal fluid (CSF). Thus, the lower the sAPP levels the more likely an individual is to have AD. To determine if there is a relationship in dogs between CSF sAAP and [3-amy[old plaque formation, we analyzed brain tissue and CSF from 40 canines ranging from 1 to 16 years of age. Twenty of these dogs were colony raised beagles, and 20 were random source mixed breed animals. All of these animals were sacrificed for clinical reasons
269 Lack of Correlation Between Plaque Burden and Cognition in the Aging Monkey J.A. Sloane*, M. Pietropaolo, D.L. Rosene, M.B. Moss, A. Peters, T. Kemper and C.R. Abraham Departments of Pathology, Biochemistry, Medicine and Anatomy and Neurobiology, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118, USA, [email protected] In order to assess whether amyloid plaque accumulation in the monkey brain can account for age-related cognitive impairment that begin about 20 years of age, we measured plaque density in the brains of rhesus monkeys ages 5 to 31. We used immunohistochemistry employing the monoclonal antibody 6E10 which is specific to amino acids 1-17 of amyloid 13peptide to identify amyloid plaques in serial coronal sections of the forebrain. Amyloid plaques accumulate with age, starting at 25 and escalating at 30 years of age. From 25 to 29 years of age, plaques are found only in a few monkeys and are relatively sparse in number. Only at 30 years of age does plaque density increase dramatically. Results from