Regionally specific age-dependent decline in alpha 2-adrenoceptors: an autoradiographic study in human brain.

The effect of sex, postmortem delay and aging on alpha 2-adrenoceptor binding was studied in tissue sections from several representative regions of the human brain from 21 subjects using [3H]UK-14304 as a ligand. Sex and postmortem delay did not influence the density of alpha 2-receptors. Aging resulted in clear decreases in most forebrain areas examined (n. basalis greater than basal ganglia greater than hypothalamus greater than fronto-temporal cortex greater than hippocampus greater than visual cortex), whereas alpha 2-receptors did not significantly change with age in the amygdala and several infratentorial areas. We conclude that age-related, regionally specific decreases in the density of alpha 2-receptors occur in the human brain. The implications of these findings for age-dependent noradrenergic degeneration are discussed.     

Detection of interleukin-6 and alpha 2-macroglobulin immunoreactivity in cortex and hippocampus of Alzheimer's disease patients.

We examined brains from Alzheimer's disease (AD) patients by immunohistochemistry for the presence of protease inhibitors. Immunoreactivity for alpha 2-macroglobulin (alpha 2-M), the most potent of the known human protease inhibitors, was found in a subgroup of cortical and hippocampal AD senile plaques. In addition, large hippocampal neurons in AD brains displayed intracellular alpha 2-M immunoreactivity which was consistently stronger than in normal aged brains. In cultured human cells of neurogenic origin (SH-SY5Y neuroblastoma cells), alpha 2-M synthesis could be strongly induced by the inflammatory cytokine interleukin-6 (IL-6) indicating that human alpha 2-M behaves as an acute-phase protein in the nervous system. Therefore, we also examined AD brains for the presence of IL-6 and found strong immunostaining in and around a subgroup of senile plaques as well as around large cortical neurons. Only very few senile plaques also stained for C-reactive protein, an acute phase protein known to be inducible by IL-6. We propose that the presence of IL-6 and alpha 2-M immunoreactivity in AD brains is functionally linked and that a sequence of immunological events is part of the pathology of AD.     

Peripheral benzodiazepine binding sites in Alzheimer's disease frontal and temporal cortex.

Much evidence has accumulated to suggest that the peripheral type benzodiazepine (PBZ) binding site has a predominantly glial localization. Elevated PBZ binding densities have been reported in various models of brain damage, apparently reflecting glial proliferation in response to neurodegeneration. In the present study, PBZ receptor densities were examined in frontal and temporal cortex of Alzheimer's disease (AD) patients using the ligand [3H]PK 11195. There was a highly significant (p less than 0.01) increase in PBZ binding sites in the temporal cortex from AD patients. In the frontal cortex, a moderate increase was observed that approached statistical significance (p = 0.07). Decreased choline acetyltransferase activity was observed in both regions. These findings offer support for the potential use of the PBZ binding site as a marker for gliosis associated with neuronal cell death.     

Up-regulation of adenosine receptors in the frontal cortex in Alzheimer's disease

Adenosine receptors are G-protein coupled receptors which modulate neurotransmitter release, mainly glutamate. Adenosine A(1) and A(2A) receptors were studied in post-mortem human cortex in Alzheimer's disease (AD) and age-matched controls. Total adenosine A(1) receptor number, determined by radioligand binding assay, using [(3)H]DPCPX, was significantly increased in AD cases in early and advanced stages without differences with the progression of the disease. A significant increase of A(1)R (37 kDa) levels was also observed by Western blot in early and advanced stages of AD. In addition, increased numbers of adenosine A(2A) receptors were observed in AD samples as determined by a binding assay using [(3)H]ZM 241385 as a radioligand and by Western blot. Increased binding and protein expression levels of adenosine receptors were not associated with increased mRNA levels coding A(1) and A(2A) receptors. Finally, increased A(1) and A(2A) receptor-mediated response was observed. These results show up-regulation of adenosine A(1) and A(2A) receptors in frontal cortex in AD, associated with sensitization of the corresponding transduction pathways.     

CB2 receptor and amyloid pathology in frontal cortex of Alzheimer's disease patients

The cannabinoid system seems to play an important role in various neurodegenerative diseases including Alzheimer's disease (AD). The relationship of cannabinoid receptors (CB₁R and CB₂R) to cognitive function and neuropathological markers in AD remains unclear. In the present study, postmortem cortical brain tissues (Brodmann area 10) from a cohort of neuropathologically confirmed AD patients and age-matched controls were used to measure CB₁R and CB₂R by immunoblotting. Correlational analyses were performed for the neurochemical and cognitive data. CB₁R expression was significantly decreased in AD. Levels of CB₁R correlated with hypophagia, but not with any AD molecular marker or cognitive status (Mini Mental State Examination score). The level of CB₂R was significantly higher (40%) in AD. Increases in the expression of the glial marker glial fibrillar acidic protein were also found. CB₂R expression did not correlate with cognitive status. Interestingly, expression levels of CB₂R correlated with two relevant AD molecular markers, Aβ₄₂ levels and senile plaque score. These results may constitute the basis of CB₂R-based therapies and/or diagnostic approaches.     

Oxidative modification of proteins in the frontal cortex of Alzheimer's disease brain

There is a large body of evidence highlighting the importance of oxidative stress in the pathogenesis of Alzheimer's disease (AD). We have previously standardised a method that can be applied to study oxidative changes in individual brain proteins by using two-dimensional oxyblots (Korolainen MA, Goldsteins G, Alafuzoff I, Koistinaho J, Pirttil? T. Proteomic analysis of protein oxidation in Alzheimer's disease brain. Electrophoresis 2002;23(19):3428-33). Here we have identified proteins that exhibited oxidative changes in AD when compared to age-matched controls and these protein changes have been further examined in relation to the neuropathological data. Indeed, several Tris-HCl soluble proteins tended to be less oxidised in AD when compared to controls. Two enzymes, mitochondrial glutamate dehydrogenase and cytosolic malate dehydrogenase, were increased in amount but showed significantly decreased degree of oxidation in AD brains when compared to controls. Furthermore, some changes related to the amounts or oxidation statuses of proteins were associated with the duration of the clinical impairment and also with the neuropathology. These results do not contradict the hypothesis of increased oxidative stress in AD but may represent co-existing compensatory changes in response to oxidative stress.     

Glutamatergic receptor expression changes in the Alzheimer's disease hippocampus and entorhinal cortex

Alzheimer''s Disease (AD) is the leading form of dementia worldwide. Currently, the pathological mechanisms underlying AD are not well understood. Although the glutamatergic system is extensively implicated in its pathophysiology, there is a gap in knowledge regarding the expression of glutamate receptors in the AD brain. This study aimed to characterize the expression of specific glutamate receptor subunits in post‐mortem human brain tissue using immunohistochemistry and confocal microscopy. Free‐floating immunohistochemistry and confocal laser scanning microscopy were used to quantify the density of glutamate receptor subunits GluA2, GluN1, and GluN2A in specific cell layers of the hippocampal sub‐regions, subiculum, entorhinal cortex, and superior temporal gyrus. Quantification of GluA2 expression in human post‐mortem hippocampus revealed a significant increase in the stratum (str.) moleculare of the dentate gyrus (DG) in AD compared with control. Increased GluN1 receptor expression was found in the str. moleculare and hilus of the DG, str. oriens of the CA2 and CA3, str. pyramidale of the CA2, and str. radiatum of the CA1, CA2, and CA3 subregions and the entorhinal cortex. GluN2A expression was significantly increased in AD compared with control in the str. oriens, str. pyramidale, and str. radiatum of the CA1 subregion. These findings indicate that the expression of glutamatergic receptor subunits shows brain region‐specific changes in AD, suggesting possible pathological receptor functioning. These results provide evidence of specific glutamatergic receptor subunit changes in the AD hippocampus and entorhinal cortex, indicating the requirement for further research to elucidate the pathophysiological mechanisms it entails, and further highlight the potential of glutamatergic receptor subunits as therapeutic targets.     

Expression levels of adenosine receptors in hippocampus and frontal cortex in argyrophilic grain disease

Expression of adenosine receptors of the A1, A2A and A2B type has been examined in the post-mortem frontal cortex and hippocampus in argyrophilic grain disease (AGD), a tauopathy affecting the hippocampus but usually not the frontal cortex, in an attempt to learn about the modulation of the adenosine pathway in this disorder. Significant increased levels of A1, but not of A2A and A2B, have been observed in AGD in the hippocampus but not in the frontal cortex, when compared with age-matched controls. This is accompanied by increased levels of adenylyl cyclase (AC), an effector of A1, and by increased (although not significant) percentage of inhibition of forskolin-stimulated AC by the A1 agonist cyclohexyladenosine in the hippocampus in AGD. These findings indicate sensitization of A1/AC in the hippocampus in AGD, and support a putative activation of the A1/AC pathway that may facilitate protection of this preferentially involved region in AGD.     

Nicotinic cholinoceptive neurons of the frontal cortex are reduced in Alzheimer's disease

The cellular distribution of nicotinic acetylcholine receptors was studied in the frontal cortex (area 10) of 1) Alzheimer patients and compared to 2) age-matched and 3) middle-aged controls using the monoclonal antibody WF 6 and an immunoperoxidase protocol. Statistical analysis revealed significant differences between the number of labeled neurons among all three groups tested (middle-aged controls greater than aged controls greater than Alzheimer cases). No differences were seen for cresyl violet-stained samples. These findings underline that the nicotinic receptor decrease found with radioligand binding may reflect a postsynaptic in addition to a presynaptic component.     

GABAergic nerve terminals in rat hippocampus possess alpha 2-adrenoceptors regulating GABA release

Noradrenaline enhanced in a concentration-dependent way the basal release of endogenous GABA from superfused rat hippocampus synaptosomes. The alpha 2-adrenoceptor antagonist yohimbine prevented the releasing effect of noradrenaline while the alpha 1-adrenoceptor antagonist prazosin was ineffective. It is concluded that GABAergic nerve terminals in rat hippocampus possess adrenoceptors of the alpha 2-subtype whose activation causes enhancement of GABA release.     

Slowing of electroencephalogram and choline acetyltransferase activity in post mortem frontal cortex in definite Alzheimer's disease.

Twenty-five (96%) of 26 patients with histologically verified moderate to severe Alzheimer's disease had abnormal electroencephalograms. The patients with the slowest (5-6 Hz) dominant occipital rhythms had significantly lower choline acetyltransferase activity in the post mortem frontal cortex than the patients with highest rhythm (8-9 Hz) (analysis of covariance adjusted for the neuropsychological test score). Concentrations of dopamine, noradrenaline or serotonin in the frontal cortex did not differ in the patient groups with the slowest and highest rhythms. Neither did scores of senile plaques or neurofibrillary tangles differ between these groups. In Alzheimer patients, the frequency of the dominant occipital rhythm correlated with the total score of the neuropsychological test (r = 0.58, P less than 0.01) and with the subscales of praxic functions and expressive speech, memory and general reasoning. The results suggest that the cholinergic deficit may contribute to the slowing of the electroencephalogram found in patients with Alzheimer's disease.     

Correlation of alpha activity between the frontal and occipital cortex.

A method of modified coherence analysis was applied to the interpretation of EEG differences between the frontal and occipital cortex. This paper discusses a bidirectional communication model for the frontal and occipital cortex. Directed coherence method was used to analyze alpha activities over the scalp. The results show that both in the left and right hemisphere, slow waves in the alpha frequency band had high correlation in the frontal-occipital direction, and fast waves had high correlation in the occipital-frontal direction. It may suggest that the structure of information transmission is different for fast and slow waves of alpha rhythm.     

Autotransplantation of the adrenal cortex: a morphological and autoradiographic study.

A morphological and autoradiographic study was made of the adrenal gland of adult male rats after autotransplantation. The simple technique involved placement of pieces of the adrenal gland in a dorsal plane between the skin and muscle. Animals for morphological studies were sacrificed at 2, 3, 4, 7, 15, 30, 90, and 180 days after autotransplantation. Those for autoradiographic studies were sacrificed at 2, 3, 7, and 15 days after autotransplantation, with 3H-thymidine being administered intraperitoneally 2 h before sacrifice. Sham-operated animals were used as controls. The majority of glandular adrenal cells suffered necrosis in the first days (2 and 3) after autotransplantation. Up until 15 days and after revascularization, morphological features of the cells were compatible with protein synthesis exhibiting a developed RER, scarce SER, and mitochondria with tubular and lamellar cristae. These data may correspond to a proliferative phase of glandular cells. At day 15, cells showed morphological signs of steroidogenic activity (mitochondria with vesicular cristae, increase of SER), and at day 30, an increased number of microvilli were seen. Between 30 and 90 days zonation of the adrenal was evident with glomerulosa, fasciculata, and reticular zones readily apparent. The quantitative analysis showed a significant increase of the volumetric density of mitochondria and microvilli between the days 7 and 30. Autoradiographic studies showed an intense labelling of fibroblast-like cells at days 2 and 3 and of glandular cells at days 7 and 15, which was confirmed by the quantitative studies. Corticosterone in autotransplanted animals decreased during the first 15 days, but after 30 days the values were similar to controls. The model reported here seems to be good for study of the regeneration of the adrenal gland and can be a simple, useful, and reproducible method for adrenal transplantation.     

Regional and laminar distributions of alpha 1-adrenoceptors and their subtypes in human and rat hippocampus.

The distributions of the alpha 1-adrenoceptor and its subtypes (alpha 1A and alpha 1B) in human and rat hippocampus are analysed by quantitative receptor autoradiography. alpha 1-Adrenoceptors are labelled by [3H]prazosin. The alpha 1A subtype is visualized by [3H]prazosin after irreversible blockade of alpha 1B adrenoceptors with chloroethylclonidine or directly by [3H]5-methyl-urapidil. The alpha 1B subtype is investigated by [3H]prazosin binding in the presence of the alpha 1A antagonist 5-methyl-urapidil. Considerable differences in the regional and laminar patterns of alpha 1-adrenoceptors are found between rat and human hippocampi. The rat hippocampus is characterized by a low overall density and a rather homogeneous regional and laminar distribution. This is in contrast to the human pattern, which shows a much higher overall level of alpha 1 receptor density and a restriction of alpha 1 receptors to the CA3 region of Ammon's horn and the dentate gyrus. Moreover, alpha 1A and alpha 1B receptors of the human hippocampus are differentially distributed with the alpha 1A subtype concentrated in the hilus and lucidum layer of CA3, and the alpha 1B subtype concentrated in the molecular layer of the dentate gyrus. Additionally, the distribution of alpha 1 receptors is compared with the distribution of 5-hydroxytryptamine 1A receptors. The subtype specific pattern is correlated with the distribution of glutamatergic systems in the human (but not in the rat) hippocampus. alpha 1A Receptor localization coincides with the target area of the mossy fibre system, and alpha 1B receptors are preferentially localized in the target area of the hippocampal associational fibres and partly of the perforant pathway. This result points to possible interactions between noradrenaline- and glutamate-mediated neurotransmission differentiated by topographically segregated alpha 1-adrenoceptor subtypes.     

Adrenergic receptors in aging and Alzheimer's disease: decreased alpha 2-receptors demonstrated by [3H]p-aminoclonidine binding in prefrontal cortex.

Biochemical and pathological studies have described abnormalities in the brainstem locus coeruleus noradrenergic neurones in Alzheimer's disease (AD) and in aging. Loss of cortical noradrenergic fibers originating from the locus coeruleus may cause a decrease in presynaptic receptors or induce an increase in postsynaptic receptors, similar to "denervation supersensitivity" in animal models. Thus far it is unclear whether alpha 2-adrenergic receptors are affected in AD. In this study, we assessed the specific binding of [3H]p-aminoclonidine, an agonist at alpha 2-receptors and at imidazoline-preferring binding sites, to prefrontal cortex and other regions including hippocampus, temporal cortex, putamen and cerebellum from subjects with AD and aging controls. We particularly focused on the prefrontal cortex because of its relatively rich monoaminergic innervation and recent evidence suggesting involvement of noradrenergic mechanisms in cognition in aging nonhuman primates. The other regions, which are also innervated by noradrenergic fibers, were examined for comparison. Ligand binding to prefrontal cortex decreased with age of controls and was also significantly reduced by approximately 50% in AD subjects compared to age-matched controls. This change in AD was related to the maximum binding capacity (Bmax) rather than to an altered affinity of the ligand for the receptor. There were no significant changes in any of the other regions studied. Binding did not change with postmortem delay or with duration of tissue storage. We suggest that presynaptic alpha 2-receptors presumably labeled by [3H]p-aminoclonidine on noradrenergic synapses are those that are selectively decreased in the prefrontal cortex in AD and in aging.     

Loss of connexin36 in rat hippocampus and cerebellar cortex in persistent Borna disease virus infection

Neonatal Borna disease virus (BDV) infection of the Lewis rat leads to progressive degeneration of dentate gyrus granule cells, and cerebellar Purkinje neurons. Our aim here was to clarify whether BDV interfered with the formation of electrical synapses, and we, therefore, analysed expression of the neuronal gap junction protein connexin36 (Cx36) in the Lewis rat hippocampal formation, and cerebellar cortex, 4 and 8 weeks after neonatal infection. Semiquantitative RT-PCR, revealed a BDV-dependent decrease in Cx36 mRNA in the hippocampal formation 4 and 8 weeks post-infection (p.i.), and in the cerebellar cortex 8 weeks p.i. Correspondingly, immunofluorescent staining revealed reduced Cx36 immunoreactivity in both dentate gyrus, and ammons horn CA3 region, 4 and 8 weeks post-infection. In the cerebellar cortex, Cx36 immunoreactivity was detected only 8 weeks post-infection in the molecular layer, where it was down regulated by BDV. Our findings demonstrate, for the first time, distinct BDV-dependent reductions in Cx36 mRNA and protein in the rat hippocampal formation and cerebellar cortex, suggesting altered neuronal network properties to be an important feature of persistent viral brain infections.     

Galanin receptor over-expression within the amygdala in early Alzheimer's disease: an in vitro autoradiographic analysis

Galanin receptor (GALR) expression is increased in various areas of the limbic system in end stage Alzheimer's disease (AD). The amygdaloid complex is a key component of the limbic circuit, is involved in homeostatic and cognitive functions, is impacted in AD and contains the peptide and receptor for galanin. Although GALR expression occurs in the amygdala in end stage AD, it remains to be determined whether a plasticity response occurs early or late in the disease. Therefore, we analyzed the distribution and associated changes in GALR binding in the amygdala during the progression of AD using an in vitro receptor autoradiographic method. Human galanin ([125I]hGAL) receptor binding was performed on brain sections from early and late AD cases, as well as normal age-matched control subjects. In aged controls, densities of [125I]hGAL binding sites were found in the central and the corticomedial nuclei. Relative to controls, possible/early AD cases displayed significantly greater numbers of [125I]hGAL binding sites in the central nucleus and cortico-amygdaloid transition area. In contrast, we found a decrease in the number of binding sites for [125I]hGAL in late as compared to early AD cases. The over-expression of GALRs in subfields of the amygdaloid early in AD suggests that galaninergic systems play a key role in limbic related behavioral changes during the disease process.     

Ultrastructural changes in the frontal cortex and hippocampus in the ageing marmoset

The ultrastructure of the cells and capillaries of the frontal cortex and hippocampus was studied in marmosets to assess age-related changes. The appearances in the brains of four marmosets, more than 12 years of age, were compared with those of two young marmosets aged 26 and 21 months. There was widespread accumulation of lipofuscin in neurons, glial cells, perivascular macrophages and pericytes, but not in endothelial cells. Many of the nerve cell nuclei showed marked membrane infolding, alteration of nuclear morphology and occasional inclusions. A few degenerated and dystrophic axons containing abnormal organelles were seen. The capillaries displayed irregularly thickened and split basal laminae. No neurofibrillary tangles, neuritic plaques, amyloid deposits or granulovacuoles were present. These changes are compared to those occurring in other animal species and in man during ageing.