Cardiovascular activity of milk casein-derived tripeptides and plant sterols in spontaneously hypertensive rats.

The effect of chronic treatment with fermented milk products containing bioactive tripeptides and plant sterols on blood pressure and vascular function was investigated in spontaneously hypertensive rats (SHR). Six-weeks old male SHR (n=36) were randomized into 4 groups by body weight and blood pressure to receive either Lactobacillus helveticus fermented standard milk product (containing tripeptides Ile-Pro-Pro, Val-Pro-Pro and Leu-Pro-Pro), test product with enzymatically produced tripeptides without or with plant sterols or control product without the active constituents for 8 weeks. Systolic blood pressure (SBP) was measured weekly using the tail-cuff method. Thoracic aorta and mesenteric artery were excised for vascular response measurements. At the end, SBP values vs. control product group were: standard product group -14 mmHg (P<0.05), test product group -12 mmHg and test product +sterols group -7 mmHg. The average daily tripeptide dose was 2.8-5.2 mg/kg. Total serum cholesterol in the test product +sterols group tended to be lower than in the test product group (P=0.10) whereas serum plant sterol (campesterol, sitosterol) concentrations were higher (P<0.001). In conclusion, bioactive tripeptide-containing milk products attenuated the blood pressure development in SHR. The plant sterols did not improve this effect. Vascular responses did not markedly differ between the groups, except that endothelium-derived hyperpolarizing factor (EDHF) -related aortic relaxation was demonstrated in the test product +sterols group.

Molecular genetic analysis of the alpha-synuclein and the parkin gene in Parkinson's disease in Finland.

Two mutations in the alpha-synuclein gene and various mutations in the parkin gene are associated with familial Parkinson's disease (PD). The present study was performed to analyse if mutations in these genes could be detected in Finnish patients with familial PD. The subjects comprised 22 unrelated patients with familial PD. The molecular genetic analysis consisted of sequence analysis of the non-coding and coding exons of the alpha-synuclein gene and screening of eight point mutations in the parkin gene. In addition, a total of 67 controls and 45 patients with sporadic PD were included in the association analysis on polymorphism of the alpha-synuclein gene. Screened point mutations in the parkin gene were not detected. Sequencing of the coding exons 2-6 of the alpha-synuclein gene did not reveal any mutations or polymorphisms. However, three novel alterations in the T10A7 sequence at the 5' end of the non-coding exon 1' of the alpha-synuclein gene were found. The frequencies of the exon 1' polymorphic genotypes or alleles between familial PD patients and control subjects revealed no statistically significant differences. No association for sporadic PD was observed. The results do not support a role for the alpha-synuclein gene or point mutations of the parkin gene in familial PD in our sample.

The solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease.

Intracellular inclusions containing alpha-synuclein (alpha SN) are pathognomonic features of several neurodegenerative disorders. Inclusions occur in oligodendrocytes in multiple system atrophy (MSA) and in neurons in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). In order to identify disease-associated changes of alpha SN, this study compared the levels, solubility and molecular weight species of alpha SN in brain homogenates from MSA, DLB, PD and normal aged controls. In DLB and PD, substantial amounts of detergent-soluble and detergent-insoluble alpha SN were detected compared with controls in grey matter homogenate. Compared with controls, MSA cases had significantly higher levels of alpha SN in the detergent-soluble fraction of brain samples from pons and white matter but detergent-insoluble alpha SN was not detected. There was an inverse correlation between buffered saline-soluble and detergent-soluble levels of alpha SN in individual MSA cases suggesting a transition towards insolubility in disease. The differences in solubility of alpha SN between grey and white matter in disease may result from different processing of alpha SN in neurons compared with oligodendrocytes. Highly insoluble alpha SN is not involved in the pathogenesis of MSA. It is therefore possible that buffered saline-soluble or detergent-soluble forms of alpha SN are involved in the pathogenesis of other alpha SN-related diseases.

Accumulation of insoluble alpha-synuclein in dementia with Lewy bodies.

The alpha-synuclein (alpha SN) protein is thought to play a central role in the pathogenesis of neurodegenerative diseases where it aggregates to form intracellular inclusions. We have used Western blotting to examine the expression levels and solubility of alpha SN in brain homogenates from dementia with Lewy bodies (DLB), Parkinson's disease (PD), Alzheimer's disease (AD), and normal controls using samples from the parahippocampus/transentorhinal cortex. Compared to controls, DLB brains accumulate significantly greater amounts of sodium dodecyl sulfate (SDS)-soluble and SDS-insoluble alpha SN but levels of TBS-soluble alpha SN did not change. Levels of synaptophysin, a marker of synaptic integrity, were significantly lower in DLB cases than in normal aged controls regardless of whether concurrent changes of AD were present. This limbic synaptic dysfunction may contribute to cognitive impairment in DLB. Whether aggregated alpha SN is a cause or effect of the disease process in DLB and PD remains to be determined, but the presence of aggregated alpha SN is consistent with a pathogenesis similar to that associated with aggregates of Abeta amyloid in AD.

Neural expression profile of alpha-synuclein in developing human cortex.

Alpha-synuclein is a predominantly neuronal presynaptic protein that may play an important role during synaptogenesis and CNS development. In order to elucidate the human developmental expression profile, we used a polyclonal antiserum against the NAC domain of alpha-synuclein. In normal fetal cortex neuroectodermal precursor cells elicited staining in the soma, whereas, in adult cortex, we observed a staining pattern compatible with synaptic function. The same developmental intraneuronal redistribution was found in neurodegenerative disorders, i.e. somatic staining in neuroectodermal precursors in fetal (trisomy 21) and a synaptic pattern in adult (Down's syndrome, Alzheimer's disease) brains. RT-PCR and Western blot analysis revealed expression at all time points studied (4-7.5 months) during human brain development.

Non-Abeta component of Alzheimer's disease amyloid (NAC) revisited. NAC and alpha-synuclein are not associated with Abeta amyloid.

alpha-Synuclein (alphaSN), also termed the precursor of the non-Abeta component of Alzheimer's disease (AD) amyloid (NACP), is a major component of Lewy bodies and Lewy neurites pathognomonic of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). A fragment of alphaSN termed the non-Abeta component of AD amyloid (NAC) had previously been identified as a constituent of AD amyloid plaques. To clarify the relationship of NAC and alphaSN with Abeta plaques, antibodies were raised to three domains of alphaSN. All antibodies produced punctate labeling of human cortex and strong labeling of Lewy bodies. Using antibodies to alphaSN(75-91) to label cortical and hippocampal sections of pathologically proven AD cases, we found no evidence for NAC in Abeta amyloid plaques. Double labeling of tissue sections in mixed DLB/AD cases revealed alphaSN in dystrophic neuritic processes, some of which were in close association with Abeta plaques restricted to the CA1 hippocampal region. In brain homogenates alphaSN was predominantly recovered in the cytosolic fraction as a 16-kd protein on Western analysis; however, significant amounts of aggregated and alphaSN fragments were also found in urea extracts of SDS-insoluble material from DLB and PD cases. NAC antibodies identified an endogenous fragment of 6 kd in the cytosolic and urea-soluble brain fractions. This fragment may be produced as a consequence of alphaSN aggregation or alternatively may accelerate aggregation of the full-length alphaSN.

Clonidine, but not guanfacine, impairs choice reaction time performance in young healthy volunteers.

The present study compares the effects of two alpha 2-adrenoceptor agonists, clonidine (0.5, 2, and 5 micrograms/kg, p.o.), and guanfacine (7 and 29 micrograms/kg, p.o.), in young healthy volunteers on attentional performance. A placebo-controlled double-blind cross-over design (one drug dose/group) was employed. Neither of the drugs affected measures of motor performance or performance at easy levels in an attentional test. However, at the most difficult level in the attentional test, the highest dose of clonidine (5 micrograms/kg), but not guanfacine, decreased the number of correct responses and increased reaction latency. Clonidine 5 and guanfacine 29 micrograms/kg equally increased subjective feelings of sedation and reduced systolic and diastolic blood pressures. Thus, the effects of the drugs on attentional performance could be dissociated from their sedative effects. The results demonstrate that clonidine, but not guanfacine, disrupts performance in an attentional task requiring effortful processing, while leaving performance intact in tests requiring more automatic processing. The lower alpha 2A-vs. alpha 2C-adrenoceptor selectivity ratio of clonidine and the affinity for alpha 1-adrenoceptors of clonidine may be responsible for the different action of these drugs on attention.

Alpha-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease beta-amyloid plaque cores.

A growing body of evidence suggests that the non-Abeta component of Alzheimer's disease amyloid precursor protein (NACP) or alpha-synuclein contributes to the neurodegenerative processes in Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In the present study antisera to the N terminus and the NAC domain of the alpha-synuclein protein were employed to elucidate the expression pattern in brains of patients with AD, PD, DLB and control specimen. Alpha-synuclein exhibited an overall punctuate expression profile compatible with a synaptic function. Interestingly, while Lewy bodies were strongly immunoreactive, none of the alpha-synuclein antisera revealed staining in mature beta-amyloid plaques in AD. These observations suggest that alpha-synuclein does not contribute to late neurodegenerative processes in AD brains.

Clonidine impairs sustained attention and memory in Alzheimer's disease.

We investigated the effect of the alpha2-agonist, clonidine (orally: 0.5 and 2 microg/kg), administration on parameters assessing attention and short-term recognition memory in Alzheimer's disease patients. Clonidine 2 microg/kg, but not 0.5 microg/kg, disrupted memory accuracy in delayed matching to sample test delay-dependently in nine out of 28 patients. The volumes of the hippocampus and the entorhinal cortex of those Alzheimer's disease patients who were sensitive to clonidine administration were larger than those whose performance was unaffected by clonidine. These two groups of Alzheimer's disease patients performed equally in measures of attention after placebo or clonidine administration. Clonidine 2 microg/kg disrupted attention only at levels of testing that were demanding for the individual patients. Our results suggest that the disruptive effect of clonidine on short-term memory in Alzheimer's disease patients may be mediated via the hippocampus and the entorhinal cortex. Furthermore, the deleterious effect of clonidine on effortful attention is mediated via different brain systems from those involved in the modulation of memory function.

The alpha2 agonist, clonidine, improves spatial working performance in Parkinson's disease.

Previous work has shown that the dopaminergic defect in Parkinson's disease is involved, to some extent, in the "frontal"-like impairment in spatial working memory and attentional set-shifting functions. We investigated whether an alpha2 agonist, clonidine (0.5 and 2 microg/kg, per os), could alleviate spatial working memory and attentional set-shifting defect in Parkinson's disease patients. We observed that 2 microg/kg clonidine stimulated spatial working memory accuracy, but had no effect on attentional set shifting or visual recognition memory. Clonidine was also effective in stimulating spatial working memory after withdrawal of dopaminergic drugs, and when this was done, its effect was greater in severe Parkinson's disease patients. In contrast, clonidine failed to stimulate visual recognition memory. These results suggest that disrupted activation of alpha2 adrenoceptors may contribute to the impairment of spatial working memory in Parkinson's disease.

Guanfacine, but not clonidine, improves planning and working memory performance in humans.

The present study compares, using a double-blind, placebo controlled design the effects of two alpha 2-agonists, clonidine (0.5, 2, and 5 micrograms/kg) and guanfacine (7 and 29 micrograms/kg) on spatial working memory, planning and attentional set-shifting, functions thought to be dependent on the "central executive" of the prefrontal cortex. Blood pressure and the subjective feeling of sedation were affected equally by clonidine and guanfacine. The 0.5 microgram/kg and 5 micrograms/kg doses of clonidine disrupted spatial working memory, but the medium dose had no effect. The 0.5 and 2 micrograms/kg doses of clonidine increased impulsive responding in the planning test. The 5 micrograms/kg dose of clonidine slowed responding at effortful levels of planning and attentional set-shifting tests. The 29 micrograms/kg dose of guanfacine improved spatial working memory and planning. Guanfacine had no effect on attentional set-shifting. These data indicate that guanfacine improved planning and spatial working memory, but clonidine dose-dependently disrupted performance. It is possible that the greater selectivity of guanfacine for alpha 2A-adrenoceptor subtype may underlie its differences from clonidine.

Guanfacine and clonidine, alpha 2-agonists, improve paired associates learning, but not delayed matching to sample, in humans.

The present study compares the effects of two alpha 2-agonists, clonidine (0.5, 2, and 5 micrograms/kg, p.o.) and guanfacine (7 and 29 micrograms/kg, p.o.) in young healthy volunteers on their performance in visual paired associates learning (PAL) and delayed matching to sample (DMTS) visual short-term recognition memory tests. In the PAL test, clonidine 2 and guanfacine 29 micrograms/kg improved the subjects' performance. In the DMTS test, clonidine at 5 micrograms/kg delay-dependently impaired performance accuracy, and at 2 and 5 micrograms/kg it also slowed responses. Guanfacine had no effect on DMTS test performance. Clonidine 5 and guanfacine 29 micrograms/kg equally increased subjective feelings of sedation and reduced blood pressure. The results suggest that both clonidine and guanfacine facilitated PAL learning by improving "frontal strategies," but only clonidine disrupted "mneomonic processing" decreasing DMTS accuracy. The greater selectivity of guanfacine for alpha 2A-adrenoceptor subtype may explain the different profile of action of the drugs.

Reduction of noradrenaline impairs attention and dopamine depletion slows responses in Parkinson's disease.

We investigated the role dopamine and noradrenaline in the modulation of attention in Parkinson's disease (PD) patients. We observed that PD patients with mild and moderate motor disability did not differ in their attentional accuracy in easy tests, but the severe PD group was slightly disrupted in a more arduous test of attention. Attentional accuracy was not affected by withdrawal of dopaminergic drugs in mild or severe PD patients. The movements of severe PD patients were slower, and withdrawal of dopaminergic drugs aggravated motor slowing more in severe PD patients. Clonidine (0.5 and 2 microg/kg) retarded accuracy of performance in the most difficult attention test in mild PD patients, but had no effect in the severe PD group. Clonidine had no effect on movement times. These data suggest that a defect in noradrenaline release may contribute to the impaired accuracy of attention in severe PD patients and that dopamine may be important for maintaining rapid motor responding.

Alpha2C-adrenoceptor-overexpressing mice are impaired in executing nonspatial and spatial escape strategies.

Drugs acting via alpha2-adrenoceptors modulate cognitive functions mediated via frontostriatothalamic feedback loops. The alpha2C-adrenoceptor subtype is expressed in the basal ganglia, hippocampus, and neocortex, areas that are involved in memory and other cognitive functions. alpha2C-Overexpressing (OE) mice were impaired in spatial or nonspatial water maze (WM) tests, and alpha2 antagonist treatment fully reversed the WM escape defect in OE mice. However, alpha2C-overexpression did not influence open field and passive avoidance behaviors or cortical EEG arousal or the actions of alpha2 agonist or antagonist drugs on these functions. Our results suggest that alpha2C-adrenoceptors can modulate navigation to a hidden or visible escape platform, whereas many other actions of alpha2-adrenergic agents, such as sedation, are not mediated via alpha2C-adrenoceptors. Therefore, alpha2-agonists lacking alpha2C-AR affinity or alpha2C-AR subtype-selective alpha2 antagonists could modulate functioning of frontostriatothalamic feedback loops more effectively than the current subtype-nonselective drugs.

Muscarinic M1 and M2 receptor subtype selective drugs modulate neocortical EEG via thalamus.

The present study was designed to investigate the effects of infusions into reticular nucleus of thalamus (NRT) or intracerebroventricular (i.c.v.) infusions of muscarinic M1 and M2 receptor subtype selective drugs on thalamocortically generated neocortical high voltage spindles (HVSs) in awake immobile rats. NRT administration of 2.0 and 20.0 microg McN-A-343, a muscarinic M1 agonist, and 20.0 microg methoctramine, a muscarinic M2 antagonist, suppressed HVSs. The results suggest that the blockade of presynaptic M2 receptors and activation of postsynaptic M1 receptors in the NRT suppress thalamocortical oscillations and increase neocortical electrical arousal.

Oxotremorine suppresses thalamocortical oscillations via thalamic muscarinic acetylcholine receptors.

We investigated whether the local intrathalamic infusion of a muscarinic acetylcholine receptor agonist (oxotremorine) at either the reticular nucleus of thalamus (NRT) or the ventroposteromedial nucleus of thalamus (VPM) suppresses thalamocortically generated neocortical high-voltage spindles (HVSs). In addition, we studied whether the intracerebroventricular (ICV) infusion of a selective muscarinic M2 acetylcholine receptor antagonist (methoctramine) could block the suppression of HVSs induced by either systemic (IP) administration of an anticholinesterase drug [tetrahydroaminoacridine (THA)] or ICV infusion of oxotremorine in rats. Intrathalamic administration of oxotremorine at 3 and 15 microg in the NRT, and at 15 microg in the VPM suppressed HVSs. ICV oxotremorine at 30 and 100 microg and IP THA at 3 mg/kg decreased HVSs. ICV methoctramine at 100 microg increased HVSs and completely blocked the decrease in HVSs produced by oxotremorine 100 microg and THA 3 mg/kg. The results suggest that activation of muscarinic M2 acetylcholine receptors in thalamic nuclei (NRT and VPM) can suppress thalamocortical oscillations and that ICV or systemically administered drugs that activate either directly (oxotremorine and methoctramine) or indirectly (THA) the muscarinic M2 acetylcholine receptors may modulate neocortical HVSs via the thalamus.

Activation of acetylcholine receptors and 5-HT2 receptors have additive effects in the suppression of neocortical high-voltage spindles in aged rats.

We investigated if activation of the muscarinic or nicotinic acetylcholine receptors and serotonin (5-hydroxytryptamine; 5-HT) subtype 2 receptors would have additive or synergistic effects on the suppression of thalamocortically generated rhythmic neocortical high-voltage spindles (HVSs) in aged rats. The 5-HT2 receptor antagonist, ketanserin, at a moderate dose (5 mg/kg) prevented the ability of a muscarinic acetylcholine receptor agonist, (oxotremorine 0.1 mg/kg), and a nicotinic acetylcholine receptor agonist (nicotine 0.1 mg/kg), to decrease HVSs. At a higher dose (20 mg/kg), ketanserin completely blocked the decrease in HVSs produced by moderate doses of muscarinic acetylcholine receptor agonists (pilocarpine 1 mg/kg and oxotremorine 0.1 mg/kg), and by a high dose of nicotine (0.3 mg/kg), though not that produced by high doses of pilocarpine (3 mg/kg) and oxotremorine (0.9 mg/kg). The ability of a 5-HT2 receptor agonist, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.1-1.0 mg/kg), to suppress HVSs was non-significantly modulated by the nicotinic acetylcholine receptor antagonist, mecamylamine (1-15 mg/kg), and the muscarinic acetylcholine receptor antagonist, scopolamine (0.03-0.3 mg/kg). The effects of the drugs on behavioral activity could be separated from their effects on HVSs. The results suggest that activation of the muscarinic or nicotinic acetylcholine receptors plus 5-HT2 receptors has additive effects in the suppression of thalamocortical oscillations in aged rats.

Fragile-X: neuropsychological test performance, CGG triplet repeat lengths, and hippocampal volumes.

We compared cognitive performance and hippocampal volumes using magnetic resonance imaging (MRI) in adult fragile-X [fra(X)] males and females with either premutation (pM) or full mutation (fM) (n = 10 in all groups). Cognitive performance of fM males in the Wechsler Adult Intelligence Scale-Revised was worse than that of pM males, and the deficits in fM females were qualitatively similar, but less severe. In a visual memory test, both fM groups were impaired. In a list learning test, fM males were impaired in the learning phase and in delayed recognition. In a logical memory test, fM males and females were not significantly different from pM subjects. Hippocampal volumes normalized for intracranial or brain area did not significantly differ between fM and pM groups. However, positive correlations between left normalized hippocampal volumes and performance in many delayed memory tests observed in pM subjects were absent in fM subjects. Furthermore, in > 50% of the fM subjects, nonspecific changes, such as enlargement of ventricles and perivascular spaces, focal hyperintensities in temporal pole white matter, and/or subjectively assessed atypical appearance of hippocampal morphology, were observed in MRI. The data suggest minor abnormalities in temporal lobe structures in adult fra(X) subjects with fM.

Differential effects of three 5-HT receptor antagonists on the performance of rats in attentional and working memory tasks.

The effects of three different serotonin (5-HT) receptor antagonists (ketanserin, methysegide, methiothepin) in the modulation of attention, working memory and behavioural activity were investigated in this study by assessing the performance of rats in two separate cognitive models; the 5-choice serial reaction time (5-CSRT) task, which measures attention, and the delayed non-matching to position (DNMTP) task, which measures working memory. Methysergide and methiothepin bind at the 5-HT1 and 5-HT2 as well as the 5-HT5-7 receptors, with varying degrees of selectivity, and ketanserin binds at the 5-HT2A receptors rather selectively. None of these agents bind to any significant extent to 5-HT3 or 5-HT4 receptors. In the 5-CSRT task, neither methiothepin (0.15 mg/kg) nor ketanserin (1.0 and 3.0 mg/kg) impaired the choice accuracy of rats, although they induced sedation. The low doses of methysergide (1.5 and 3.0 mg/kg) slightly increased the behavioural activity of rats, whereas the high dose of methysergide (15.0 mg/kg) reduced behavioural activity and slightly reduced choice accuracy of the rats in the attentional task (monitoring of visual stimuli) under the baseline conditions or curtailed stimulus duration. This effect was not augmented at the reduced stimulus intensity. These findings suggest that the high dose of methysergide did not interfere with the visual discrimination of rats. Furthermore, methysergide did not reduce motivation for this task, since it did not increase food collection latencies. In the DNMTP task, methiothepin (0.15 mg/kg) induced a delay non-dependent deficit in choice accuracy. This could be due to an impaired alternation ability or akinesia, which increases an actual delay between sample and choice. Methiothepin (0.15 mg/kg) also interfered with behavioural activity of rats. Interestingly, ketanserin (1.0 mg/kg and 3.0 mg/kg) and methysergide (3.0-15.0 mg/kg) neither impaired the choice accuracy nor reduced the behavioural activity of rats in the DNMTP task. These results suggest that the blockade of 5-HT2A receptors does not interfere with attention and working memory per se. However, all three serotonin receptor antagonists interfered with behavioural activity of rats in the 5-CSRT task more severely than in the DNMTP task. The possible role of serotonin and non-serotonin receptors underlying the influence of these antagonists on behavioural activity will be discussed.

Combined cholinergic and 5-HT2 receptor activation suppresses thalamocortical oscillations in aged rats.

The present study investigated whether combined stimulation of the cholinergic system and 5-hydroxytryptamine (5-HT) subtype 2 receptors can suppress neocortical high-voltage spindles (HVSs) reflecting thalamocortical oscillations in aged rats. Cholinesterase inhibitors-tetrahydro-aminoacridine (THA: 1.0 and 3.0 mg/kg i.p.) and physostigmine (0.36 mg/kg i.p.)- and a 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 0.3 and 1.0 mg/kg SC)-suppressed HVSs in aged rats. A combination of subthreshold doses of THA (0.3 mg/kg i.p.) and DOI (0.1 mg/kg s.c.) suppressed HVSs more effectively than either drug alone. Furthermore, a 5-HT2 receptor antagonist, ketanserin (5.0 and 20.0 mg/kg s.c.), reduced the efficacy of THA (1.0 and 3.0 mg/kg i.p.) and physostigmine (0.12 and 0.36 mg/kg i.p.) in decreasing HVSs. THA and ketanserin slightly decreased, physostigmine tended to increase, and DOI significantly increased behavioral activity of the rats, demonstrating that the effects of the drugs on behavioral activity may be separated from their effects on generation of thalamocortical oscillations. The results suggest that activation of the cholinergic system and 5-HT2 receptors has additive effects in the suppression of thalamocortical oscillations in aged rats.