Immunohistochemical quantification of the synapse-related protein synaptophysin in Alzheimer disease.

Alzheimer disease (AD) as well as other dementing disorders are characterized by a continuous loss of neurons in cortical and subcortical areas and probably by an extensive synaptic loss. In order to substantiate and localize the loss of synapses in AD, we quantified by microdensitometry the neuropil immunoreactivity to an antibody that labels the protein synaptophysin (p38), which is localized in the presynaptic terminals. We found in the AD cases an average 50% decrease in the density of the granular neuropil immunoreaction in parietal, temporal and midfrontal cortex. In contrast, Pick disease cases presented close to normal values in parietal cortex, but major losses in temporal and frontal cortex. Our data strongly suggest an important role of synapse loss in dementia.

Alterations of the central noradrenergic system in MPTP-induced monkey parkinsonism.

Alterations of the coeruleo-cortical adrenergic system in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced monkey parkinsonism were examined through neuropathological observation and radioreceptor assay. Neuronal degeneration was found not only in the substantia nigra but also in the locus coeruleus of all the MPTP-treated monkeys. From radioreceptor assay, decreases of alpha 1 and alpha 2 receptors in the parietal and temporal cortices were observed in parkinsonian monkeys. In addition, beta receptors increased in the temporal cortex. These changes in monkey parkinsonism are very similar to those in human Parkinson's disease.

Effects of injury on the indoleamines in cerebral cortex.

It was shown previously that focal cortical freezing lesions in rats cause widespread depression of local cerebral glucose utilization (LCGU) in cortical areas of the lesioned hemisphere. This was interpreted as reflecting functional depression. The underlying mechanisms were postulated to involve alterations of biogenic amine systems. Accordingly, levels of serotonin (5-HT), its metabolite 5-hydroxyindoleacetic acid (5-HIAA), and its precursor tryptophan were determined by an HPLC method with electrochemical detection in frontoparietal cortical areas of both hemispheres at 4 h and 1, 3, 6, 8, and 10 days after a unilateral cortical freezing lesion. The 5-HT content was significantly lower than normal in the lesioned hemisphere only at 24 h, whereas the 5-HIAA level peaked at 24 h but was significantly elevated above normal values between 4 h and 6 days after lesioning. No changes were noted in 5-HT and 5-HIAA contents in the hemisphere contralateral to the lesion. These results indicate that cortical 5-HT metabolism is increased throughout the lesioned hemisphere of a focally injured brain. The increase in tryptophan content of the lesioned brain appeared to have a time course more closely related to previously demonstrated changes in cortical LCGU than to the increase in 5-HIAA content.

Monoaminergic fibers form conventional synapses in the cerebral cortex.

The morphological characteristics of the monoaminergic axon-terminals in the mammalian cerebral cortex have been the subject of controversy in recent years. Systematic analysis of serial ultrathin sections, immunocytochemically stained with antibodies against noradrenaline and serotonin, has shown that nearly all stained terminals form synapses characterized by specialized junctional appositions. These results suggest that, contrary to the widely held view, monoamines in the cortex are released at specialized junctions.

Brain cortical amino acids measured by intracerebral dialysis in portacaval shunted rats.

The extracellular amino acid content was measured in the parietal cortex in portacaval and sham operated rats, using the brain dialysis technique. The amino acid content of the perfusate was determined for 10 min before and during stimulation with potassium chloride. Basal levels of aspartate, glutamine, glycine, methionie, valine, phenylalanine and leucine were 2-to 6-fold higher in the PC-shunted as compared to the sham operated rats. For glutamate, taurine, and GABA no differences were observed between the two groups. After KCl stimulation the release of glutamate and GABA increased significantly in both groups. For GABA this rise was approximately twice as high in the PC-shunted rats (+300%, P less than 0.01) as in the sham operated rats (+150%, P less than 0.01 as compared to basal). In the sham operated, but not in the PC-shunted rats, methionine and valine levels rose significantly (+200%, P less than 0.05) and glutamine release decreased (-50%, P less than 0.05). These findings suggest that the brain metabolism of amino acids is altered after a portacaval shunt. This could in turn alter the neurotransmission and partly explain the low spontaneous motor activity seen in these animals.

Effect of cysteamine on somatostatin and neuropeptide Y in rat striatum and cortical synaptosomes.

Local injection of cysteamine into rat striatum results in a rapid but reversible reduction in somatostatin-like immunoreactivity (SLI). Since somatostatin and neuropeptide Y are co-localized in striatal and cortical neurons, we examined the effects of cysteamine in these areas. SLI and neuropeptide Y-like immunoreactivity (NPYLI) were measured following local injection of cysteamine into the striatum. In addition, we examined the effects of cysteamine on SLI and NPYLI in cortical synaptosomes. SLI was significantly reduced in both experiments, but NPYLI was unaffected. These results suggest that the mechanism by which somatostatin is depleted by cysteamine is one of specific biochemical modification, probably affecting the somatostatin disulfide bond, rather than one affecting neuronal metabolism.

Decreased somatostatin immunoreactivity but not neuropeptide Y immunoreactivity in cerebral cortex in senile dementia of Alzheimer type.

The content of two neuropeptides, somatostatin (SRIF) and neuropeptide Y (NPY) has been determined in two cerebral cortical areas of Alzheimer's disease brain and in age-matched control brains. The content of SRIF-like immunoreactivity (SRIF-LI) was found to be decreased in Alzheimer temporal cortex (Brodmann area 21) compared to control temporal cortex. The decreased content of SRIF was significantly correlated with the observed number of neuritic plaques and neurofibrillary tangles. No difference was observed in NPY-LI between Alzheimer cerebral cortex and control cortex. Furthermore, no correlations were observed between NPY content and plaque count, neurofibrillary tangle estimate or SRIF content despite widespread reports of NPY/SRIF coexistence.

Effect of 5,7-dihydroxytryptamine on the concentration of individual proteins in different areas of the rat brain.

Proteins which are apparently regulated in concentration in two different areas of the rat brain by the indole neurotransmitter serotonin were identified using two-dimensional gel electrophoresis combined with computerized scanning densitometry. Reduction in central serotonin levels produced a decrease in the concentration of 3 different proteins (2 in the parietal cortex, 1 in the hippocampus). Two proteins, both in the hippocampus, were elevated in concentration following serotonin depletion. These results demonstrate that there exist in the brain a limited number of proteins whose concentration is influenced by serotonin.

Effects of bilateral lesion of the locus coeruleus and of neonatal administration of 6-hydroxydopamine on the concentration of individual proteins in rat brain.

The role that norepinephrine plays in regulating the concentration of different proteins in the parietal cortex, hippocampus and cerebellum was assessed by investigating the effects of either a bilateral lesion of the locus coeruleus or neonatal administration of 6-hydroxydopamine. Two weeks after lesioning the locus coeruleus, the concentration of two different proteins was elevated in the hippocampus; a third protein was reduced in concentration in this brain area as a result of the lesion. Three proteins were affected in concentration in the cerebellum after the locus coeruleus lesion--two were elevated in concentration and one was reduced in concentration. No proteins were altered in concentration in the parietal cortex as a result of the lesion. Seventy days after neonatal treatment with 6-hydroxydopamine, a total of 6 proteins were found to be changed. Four of these (one in the hippocampus and 3 in the parietal cortex) were reduced in concentration while two proteins (both in the cerebellum) were elevated in concentration after neonatal treatment with the catecholamine neurotoxin. There was little overlap between those proteins affected in concentration by the bilateral lesion of the locus coeruleus and those changed by neonatal treatment with 6-hydroxydopamine. These results suggest that the concentration of a number of different proteins may, under normal physiological conditions, be regulated in vivo by norepinephrine in the brain.

Estrous cycle variations in cholecystokinin and substance P concentrations in discrete areas of the rat brain.

Cholecystokinin (CCK) and substance P (SP) were measured in discrete areas of the rat brain at different stages of the estrous cycle. Significantly higher levels of CCK were found in the lateral septum during diestrus as compared to proestrus. In the parietal cortex, CCK concentrations were significantly higher in diestrus than in proestrus. In the amygdala, estrous levels of CCK were significantly higher than proestrous levels. SP concentrations were significantly higher in diestrus than in proestrus in the medial and lateral septum, and the medial and lateral preoptic area. In the amygdala and ventral tegmental area, SP concentrations were significantly higher in estrus than in proestrus. These data suggest that certain CCK and SP neuronal systems may play a role in regulating the hypothalamo-pituitary-gonadal axis and/or be involved in steroid-dependent behavior.

In vivo modulation of norepinephrine-induced cerebral oxygenation states by hypoxia and hyperoxia.

The effect of intravenous norepinephrine (NE) administration on three O2-dependent parameters of cerebral oxygenation was studied in the parietal cortex of skull intact anesthetized rats. Reflectance spectrophotometry was used to measure in vivo changes in cortical hemoglobin saturation (Hb/HbO2), blood volume (BV), and cytochrome c oxidase (cyt. a,a3) oxidation-reduction state. The influence of arterial pressure of oxygen (paO2) on norepinephrine-induced changes in cortical microcirculatory O2 delivery and cyt. a,a3 redox state was tested under conditions of normoxia, hypoxia, and hyperoxia. Norepinephrine produced cyt. a,a3 redox changes which were independent of compensatory alterations in cortical blood volume and changes in systemic blood pressure at the tested physiological extremes. During normoxia, NE caused dose-dependent systemic pressure-related increases in the oxidation level of cyt. a,a3. Conversely, in hypoxia NE caused a reduction. Microcirculatory and cyt. a,a3 redox responses to low doses of NE during hyperoxia were similar to those obtained at high doses during normoxia. The kinetic pattern of changes in hemoglobin saturation, cyt. a,a3 redox state, and cortical blood volume during normoxia and hypoxia was consistent with direct alteration in oxygen delivery to the respiratory chain and possible modification of cerebral oxidative metabolism. Blood-brain barrier alterations and vascular smooth muscle resistance changes to NE under tested conditions of oxygenation are postulated to be responsible for the observed results.

Ontogeny of vasoactive intestinal peptide and somatostatin in different structures of the rat brain: effects of hypo- and hypercorticism.

The ontogeny of the vasoactive intestinal peptide (VIP) and somatostatin (SRIF) was studied in various structures of the rat central nervous system (CNS), using specific radioimmunoassays. The effect of adrenal corticoids on the concentration of both peptides was investigated during the development of the rat from 3 days before birth to 2 months after birth. The evolution of both peptides was different since SRIF was found before birth in each structure tested while VIP appeared only after birth in the same structures. However, after birth the ontogeny of VIP and SRIF was quite similar and the maximum concentration of both peptides occurred between day 14 and day 21. Hypercorticism (implant of corticosterone) and hypocorticism (Metyrapone injections) modified the postnatal evolution of both peptides, suggesting that corticoids play an important role in the brain developmental patterns of VIP and SRIF.

Cerebral cortex and amino acid neurotransmitters: higher levels of aspartic acid but not GABA in the frontal cortex of the rat.

Endogenous levels of Aspartic acid, GABA and Glutamic acid plus Glutamine were measured in the frontal, occipital, temporal and parietal cortex. Aspartic acid levels were found higher in the frontal cortex than in the rest of the cortical areas studied. GABA, however, had a homogenous distribution among all cortical areas.

Effect of desmethylimipramine and reserpine on the concentration of specific proteins in the parietal cortex and the hippocampus of rats as analyzed by two-dimensional gel electrophoresis.

The effect of desmethylimipramine (DMI) and reserpine on the concentration of specific proteins in the parietal cortex and the hippocampus of rats was assessed using two-dimensional gel electrophoresis combined with computer-assisted scanning densitometry. Chronic administration of DMI for 3 weeks was found to produce a significant reduction in the concentration of two proteins in both brain regions examined. Both of these proteins have a molecular weight of approximately 57,000 daltons and isoelectric point of 6.2 to 6.3. A third, smaller protein (MW 28,000 daltons, isoelectric point 5.9) was increased in concentration in rats treated repeatedly with DMI. Acute drug treatment was, in all three cases, found to be without effect. In contrast, chronic treatment of rats with reserpine produced effects on these three proteins in the hippocampus which were quantitatively opposite to those obtained after chronic DMI administration. Again, acute drug treatment was without effect. These results demonstrate that chronic, but not acute, administration of agents affecting noradrenergic reactivity can also have an effect on the concentration of specific proteins within the central nervous system and are of interest in view of the known effects of these drugs on neurotransmitter and enzyme systems in the central nervous system.

Sex differences in specific proteins in the preoptic medial nucleus of the rat hypothalamus.

Using two-dimensional gel electrophoresis and a highly sensitive silver stain, specific proteins in adult male and female rat brain were examined. Based on previous studies, the preoptic medial nucleus (POM) of the hypothalamus served as the area of interest, with the parietal cortex acting as control. A significant difference between the sexes was found in the concentration of two proteins in the POM, a difference which was not found in the parietal cortex.

Progesterone, androstenedione, testosterone, 5 alpha-dihydrotestosterone and androsterone concentrations in specific regions of the human brain.

The concentrations of progesterone, androstenedione, testosterone, 5 alpha-dihydrotestosterone and androsterone were determined in tissue samples from the human hypothalamus, anterior pituitary, pineal, amygdala and parietal cortex, taken at autopsy from male (n = 4) and female cadavers (n = 4) of various ages. The measurements were performed using radioimmunoassays for the individual steroids after the chromatographic purification of solvent extracts of tissue samples on Lipidex-5000TM. Preliminary qualitative analyses of the chromatographic profiles of various steroids by radioimmunoassay demonstrated the presence of these steroids in various regions of the brain, but an immunoreactive peak corresponding to 17-hydroxyprogesterone was not found. The concentrations (ng/g tissue wet wt.) of all steroids measured were either very low or below the limit of detection in brain tissues taken from male and female infants. In the adult brain, there was no difference in the distribution of steroids between the various regions studied. There was no sex difference in the brain tissue steroid concentrations, with the exception of testosterone which was clearly much higher in brain tissues from men as compared to women. Although testosterone was undetectable in most samples taken from adult women. 5 alpha-dihydrotestosterone could be measured in almost all samples, which suggests that this is the most important androgen in the human brain. When brain tissue steroid levels are compared with serum concentrations, it can be postulated that a state of equilibrium exists between the fraction of serum steroids which are not bound to high-affinity binding proteins and the amount of steroids in brain tissues.

The evaluation of autofluorescence emission spectra derived from neuronal lipopigment.

A method for measuring the emission spectra from regions of neuronal lipopigment in tissue sections is described and illustrated. Each emission spectrum was derived from the means of six sets of readings, from either six regions of lipopigment from six neurones which were presumed to be from a homogeneous cell population, or from one region of one neurone. Characteristics of the emission spectra from lipopigment in various forms of neuronal ceroid-lipofusinoses (NCLs) and in brains without evidence of NCL are presented and discussed. The results indicate that the classification of lipopigments should not be restricted to the two categories of 'lipofuscin' and 'ceroid'. This method may aid the identification of various pathogenic mechanisms in neurones, and provide another means of investigating the effects of certain drugs on cerebral function.