Infusion of beta-FNA into the thalamus attenuates morphine-induced c-Fos induction in the rat caudate putamen.

The medial thalamus contains mu opioid receptors and sends a glutamatergic projection to the caudate putamen (CPu) in rat. Morphine-induced c-Fos expression in the CPu has been shown to be blocked by pretreatment with antagonists to N-methyl-D-aspartate receptors, indicating the involvement of glutamate in this morphine-induced response. The importance of the glutamatergic projections from the thalamus was assessed by infusing the mu opioid receptor antagonist, beta-funaltrexamine (beta-FNA), prior to systemic morphine injection. Infusion of beta-FNA near specific medial thalamic nuclei attenuated morphine-induced c-Fos expression in the CPu.     

Role of the dorsal raphe nucleus in morphine-induced immediate early gene expression in the rat striatum

Serotonin (5-HT) is thought to be involved in morphine action in the brain. To determine if the periaqueductal gray (PAG) and the dorsal raphe nucleus (DRN) are involved in morphine-induced c-Fos and JunB expression in the caudate-putamen (CPu), the mu receptor antagonist, beta-funaltrexamine (beta-FNA), was unilaterally infused into the PAG adjacent to DRN prior to morphine. Behaviorally, beta-FNA prevented morphine-induced loss of righting and Straub tail. In the CPu of beta-FNA treated rats, morphine-induced c-Fos and JunB were attenuated compared to vehicle-infused rats. These results suggest that morphine acts within the PAG-DRN to exert rapid behavioral effects and to induce c-Fos and JunB in the striatum.     

Pro-opiocortin fragments in human and rat brain: β-endorphin and α-MSH are the predominant peptides

The ‘pro-opiocortin’ fragments, β-lipotropin, β-endorphin, ACTH and α-MSH, were estimated in discrete areas of rat and human brain and pituitaries by means of radioimmunoassay in combination with gelfiltration. These peptides exihibited parallel patterns of distribution, but with β-endorphin and α-MSH predominant in the brain of rat and man, and, in contrast, their respective precursors, β-LPH and ACTH predominant in the adenohypophysis of rat and man. These data may be indicative of important differences in post-translational processing of ‘pro-opiocortin’ between these contrasting tissues.     

Release of β-lipotropin and β-endorphin from rat hypothalami in vitro

Hypothalamic tissue extracts of rats were chromatographed and β-endorphin immunoreactivity (β-End_i) was measured. The two major peaks of β-End_i co-eluted with β-lipotropin (β-LPH) and β-End respectively. Hypophysectomy caused a local decrease of β-LPH and β-End concentrations in the mediobasal hypothalamus. During superfusion of hypothalamic tissue blocks in vitro, membrane depolarization by electric stimulation or 45 mM K⁺ induced a Ca²⁺-dependent release of both β-LPH and β-End.     

Androgenic–anabolic steroids blunt morphine-induced c-fos expression in the rat striatum: possible role of β-endorphin

Self-administration of large doses of androgenic–anabolic steroids (AAS) in a significant portion of the population suggests that these agents are drugs of abuse. However, acute administration of AAS did not induce striatal immediate-early genes (IEG) expression in male rats, indicating that AAS do not share a common mechanism of action with other drugs of abuse. Surveys have indicated that people who abuse AAS are more likely to self-administer other drugs of abuse than do people who do not take AAS. In the present study, chronic administration of AAS blunted the striatal c-fos response to morphine, indicating that AAS can alter the molecular responses to at least one drug of abuse. Chronic administration of AAS also increased the content of β-endorphin in the midline thalamus, suggesting a possible mechanism by which AAS may modulate the response to morphine through regulation of thalamo-striatal neurons.     

Regional distribution of β-lipotropin converting enzymes in rat pituitary and brain

Among the brain areas studied only pars distalis and pars intermedia are found to contain β-lipotropin activating enzyme indicating that these may be the exclusive organs for a physiologically significant conversion of β-lipotropin into β-endorphin. β-Endorphin inactivating enzyme is found to be rather uniformly distributed in all the pituitary and brain regions, α- and γ-endorphins are presumably formed by the action of this enzyme on β-endorphin.     

Effects of sulpiride treatment on the levels of immunoreactive β-endorphin in rat hypothalamic nuclei

The effects of sulpiride, a specific dopamine receptor antagonist, on the levels of immunoreactive β-endorphin (ir-β-END) in discrete hypothalamic nuclei of rats were investigated. Single injections of sulpiride significantly decreased the levels of ir-β-END in the nucleus arcuatus, nucleus paraventricularis and median eminence. Repeated injections of sulpiride significantly increased the levels of ir-β-END in the nucleus arcuatus, nucleus periventricularis and median eminence. These findings suggest that β-endorphinergic neurons are under dopaminergic control.     

Regional distribution of α- and γ-type endorphins in rat brain

The regional distribution of Met-enkephalin, beta-endorphin and alpha- and gamma-type endorphins in rat brain was investigated. To that end, brains were dissected into anatomically defined areas. Acetic acid tissue extracts were fractionated using an HPLC system suitable for separation of endorphins and peptide concentrations were subsequently measured by specific radioimmunoassay systems. The distribution of Met-enkephalin and beta-endorphin through the brain was fairly uneven and in accordance with results obtained by others. The peptides alpha-endorphin, gamma-endorphin, des-Tyr-alpha-endorphin (DT alpha E) and des-Tyr-gamma-endorphin (DT gamma E) were detectable in almost all brain areas. Their distribution, however, appeared to be uneven. Hypothalamus and septum showed the highest levels of alpha- and gamma-type endorphins. These regions also contained high amounts of beta-endorphin, underscoring a precursor function of this peptide in the formation of alpha- and gamma-type fragments. In general, levels of alpha-endorphin were higher than those of des-Try-alpha-endorphin, whereas the opposite was found for gamma- and des-Tyr-gamma-endorphin.     

α-Melanotropin and β-endorphin-like immunoreactivities are contained within neurons and nerve fibers of the rat duodenum

Both α-melanotropin and β-endorphin were revealed by immunofluorescence microscopy studies within neurons and nerve fibers of the rat duodenum. An immunohistochemical staining for α-melanotropin was seen within neuronal cell bodies and nerve fibers bundles of the myenteric and submucous plexus. A β-endorphin immunofluorescence was visualized within perikarya and nerve fibers of both the myenteric and submucous plexus. α-Melanotropin as well as β-endorphin immunoreactivities were strictly localized to structures of the enteric nervous system. In crypts and epithelial cells only a non-specific staining was observed.     

Circadian variations in β-endorphin concentrations in pituitary and in some brain nuclei of the adult male rat

Using both the ‘punch’ microdissection and a radioimmunological technique, circadian variations in β-endorphin concentrations can be observed in the pituitary and in some discrete brain regions of the male rat (Wistar CFY). Animals were synchronized with light from 06.00 to 18.00 h, then darkness. Water and food were available ad libitum. Very well marked circadian rhythms were in evidence in the anterior lobe of the pituitary, the septum, the pons, the medulla obblongata and the cerebellum. There crest time locations were situated between 20.00 and 24.00 h. No significant circadian rhythms but biphasic variations were observed in the intermidiate lobe of the pituitary, the POA, the thalamus, the central gray and the caudatus. There crest time locations were synchronized around 08.00 and 20.00 h.The most striking finding was that, regardless of the brain area investigated so far, maximal vaalue were observed a short time after the beginning of the activity period of rats. This fact is identical with the which has been observed for substance P and LH-RH contents in brain areas where these peptides are mostly present in nerve terminals in high concentrations.     

δ‐Catenin/NPRAP: A new member of the glycogen synthase kinase‐3β signaling complex that promotes β‐catenin turnover in neurons

Through a multiprotein complex, glycogen synthase kinase‐3β (GSK‐3β) phosphorylates and destabilizes β‐catenin, an important signaling event for neuronal growth and proper synaptic function. δ‐Catenin, or NPRAP (CTNND2), is a neural enriched member of the β‐catenin superfamily and is also known to modulate neurite outgrowth and synaptic activity. In this study, we investigated the possibility that δ‐catenin expression is also affected by GSK‐3β signaling and participates in the molecular complex regulating β‐catenin turnover in neurons. Immunofluorescent light microscopy revealed colocalization of δ‐catenin with members of the molecular destruction complex: GSK‐3β, β‐catenin, and adenomatous polyposis coli proteins in rat primary neurons. GSK‐3β formed a complex with δ‐catenin, and its inhibition resulted in increased δ‐catenin and β‐catenin expression levels. LY294002 and amyloid peptide, known activators of GSK‐3β signaling, reduced δ‐catenin expression levels. Furthermore, δ‐catenin immunoreactivity increased and protein turnover decreased when neurons were treated with proteasome inhibitors, suggesting that the stability of δ‐catenin, like that of β‐catenin, is regulated by proteasome‐mediated degradation. Coimmunoprecipitation experiments showed that δ‐catenin overexpression promoted GSK‐3β and β‐catenin interactions. Primary cortical neurons and PC12 cells expressing δ‐catenin treated with proteasome inhibitors showed increased ubiquitinated β‐catenin forms. Consistent with the hypothesis that δ‐catenin promotes the interaction of the destruction complex molecules, cycloheximide treatment of cells overexpressing δ‐catenin showed enhanced β‐catenin turnover. These studies identify δ‐catenin as a new member of the GSK‐3β signaling pathway and further suggest that δ‐catenin is potentially involved in facilitating the interaction, ubiquitination, and subsequent turnover of β‐catenin in neuronal cells. © 2010 Wiley‐Liss, Inc.     

Metabolites of the β-amyloid precursor protein generated by β-secretase localise to the trans-golgi network and late endosome in 293 cells

Deposition of β-amyloid occurs in the brains of all sufferers of Alzheimer's disease. β-amyloid is proteolytically derived from the β-amyloid precursor protein by as yet unidentified enzymes termed secretases. We have generated and characterised antisera to the carboxy-terminal domain and β-secretase cleavage site of the Alzheimer's amyloid precursor protein. The β-secretase cleavage event occurs at the extreme N-terminus of the β-amyloid peptide. Our antiserum to the N-terminus of the β-amyloid peptide (NTβ4) specifically recognises β-secretase cleaved species as opposed to intact βAPP. NTβ4 specifically immunoprecipitates a 13 kDa fragment of βAPP (p13) which is potentially amyloidogenic. We have used these anti-sera in confocal laser scanning immunofluorescence microscopy to localise the intracellular location of potentially amyloidogenic βAPP processing fragments such as p13. Using a number of marker antisera of known intracellular location, we have defined the major location of βAPP fragments possessing the Asp-1 N-terminus of β-amyloid as the trans-Golgi network or late endosome on the basis of colocalisation with a monoclonal antibody to the cation-independent mannose-6-phosphate receptor. The colocalisation was further investigated using brefeldin A which demonstrated that the p13 fragment and mannose-6-phosphate receptor are trafficked by alternative pathways from the trans-Golgi network. © 1996 Wiley-Liss, Inc.     

Electrophysiological investigation of β, β′-iminodipropionitrile neuropathy: Intracellular recordings in spinal cord

β, β′-Iminodipropionitrile (IDPN) was given to cats (50 mg/kg/week for 5 weeks) to induce giant axonal swellings in the proximal internodes of motor axons. Conventional intracellular recording techniques were used to investigate the influence of the axon swellings on axonal impulse conduction and generation of action potentials in unidentified lumbosacral motoneurons (MN).Action potentials recorded from axon swellings, verified by lack of orthodromically or antidromically elicited EPSPs or IPSPs, afterhyperpolarization potentials or initial segment-somaldendritic (IS-SD) inflections, were variable in shape. Some were indistinguishable from recordings in normal axons. Component or extra potentials occurred in 45% of recordings from axon swellings; their position on the action potential depended on the direction of impulse invasion into the swelling. Many action potentials were broad, with amplitudes less than 30 mV. Impulse conduction was estimated to be blocked in 19% of motor axons tested.Action potentials recorded in MN of IDPN treated cats resembled in many aspects those recorded in chromatolytic MN, with increased latencies upon antidromic stimulation and decreased IS conduction times and SD thresholds; other parameters did not differ significantly. The minimal interval between two stimuli which each evoked action potentials increased from3.3 ± 0.1to5.8 ± 0.5ms. IS-SD portions of the action potentials could not be fractionated in 49% of cells regardless of interpulse interval. Many MN failed to follow frequencies as low as 10 Hz. Delayed depolarizations were observed in 14% of MN recordings. Repetitive action potentials were elicited by single stimuli in 14% of MN and more frequently by orthodromic than antidromic stimulation. Action potentials could often be elicited in the same MN by stimulation of more than one ventral root filament. The incidence of this ephaptic transmission or crosstalk was estimated to be 12%. The findings are discussed in terms of the influence of proximal axon swellings on action potential generation in MN, propagation along non-homogeneous regions of axons and functional chromatolysis.     

The influence of heparin and indol on the catalytic properties of α- and β/δ -thrombins

Heparin was shown to form an equimolar complex with α- and β/δ -forms of thrombin. The formation of the complex resulted in inhibition of the TAME esterase activity of thrombin ( by 40% form α- and 17% for β/δ-form ) and in stimulation of its BAME esterase activity ( by 50% for α- and 64% for β/δ-form ). Heparin caused the 70% inhibition of the activity of both forms of the enzyme towards the synthetic amid substrate Bz-Phe-Val-Arg-pNA; at the same time it had little if any effect on the enzyme activity towards Tos-Gly-Pro-Arg-pNA and stimulated the α- and β/δ-thrombins activities towards H-D-Phe-Pip-Arg-pNA by 16% and 57% respectively. In the case of both ester and amid substrates heparin exerted its effect on kcat, but had no effect on Km(app).Indol was shown to activate the TAME hydrolysis catalyzed by α- and β/δ-thrombins. The identity of the binding site for indol and for the additional TAME molecule in the effect of substrate activation was demonstrated. Heparin did not prevent the effects of indol and substrate activation of the thrombin-catalyzed hydrolysis of ester substrates. Moreover the kinetic parameters of indol activation are similar for the free enzyme and its complex with heparin indicating the different localization of the binding sites for indol and heparin in the molecule of thrombin.     

Effects of α-endorphin, β-endorphin and (des-tyr)-γ-endorphin on α-MPT-induced catecholamine disappearance in discrete regions of the rat brain

Following the intracerebroventricular administration of α-endorphin, β-endorphin and (des-tyrosine¹)-γ-endorphin in a dose of 100 ng, the α-MPT-induced catecholamine disappearance was found to be altered in discrete regions of the rat brain. In the regions in which α-endorphin exerted an effect, it without exception caused a decrease in catecholamine disappearance. Thus, in rats treated with α-endorphin the disappearance of noradrenaline was decreased in the medial septal nucleus, dorsomedial nucleus, central amygdaloid nucleus, subiculum, the ventral part of the nucleus reticularis medullae oblongatae and the A1 region, and that of dopamine in the caudate nucleus, globus pallidus, medial septal nucleus, nucleus interstitialis striae terminalis, paraventricular nucleus, zona incerta and central amygdaloids nucleus. β-endorphin was found to decrease noradrenaline disappearance in the ventral part of the nucleus reticularis medullae oblongatae, dopamine disappearance in the lateral septal nucleus and the disappearance of both amines in the rostral part of the nucleus tractus solitarii. Dopamine disappearance was increased in the medial septal nucleus and the zona incerta following β-endorphin treatment. Following treatment with (des-tyrosine¹)-γ-endorphin, noradrenaline disappearance was enhanced in the anterior hypothalamic nucleus, whereas dopamine disappearance was increased in the paraventricular nucleus, the zona incerta and the rostral part of the nucleus tractus solitarii. In addition to this the latter peptide also caused a decreased noradrenaline disappearance in the periventricular thalamus and the A7 region. The results fit well with the suggestion that endorphins act as modulators of catecholamine neurotransmission in particular brain regions. The pattern of effects of the endorphins differ from that previously observed following intracerebroventricular administration of methionine-enkephalin. This is in keeping with the notion that the enkephalin containing network in the brain and that containing β-LPH represent two independent systems with distinct differences in their projections to various brain regions.     

Attenuation of β‐amyloid‐induced tauopathy via activation of CK2α/SIRT1: Targeting for cilostazol

β‐Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2‐coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac‐tau) and tau phosphorylation (P‐tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac‐tau and P‐tau levels. Concomitantly, these cells showed increased P300 and GSK3β P‐Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3–30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP‐dependent protein kinase‐linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau‐related neurodegeneration in the AD brain. © 2013 Wiley Periodicals, Inc.     

Testosterone attenuates β-amyloid toxicity in cultured hippocampal neurons

Accumulating evidence suggests that testosterone has neurotrophic and perhaps neuroprotective actions. Thus, age-related depletion of testosterone may increase the brain’s vulnerability to Alzheimer’s disease and related disorders. To begin investigating this issue, cultured neurons were exposed to the Alzheimer-related insult β-amyloid in the presence of testosterone. β-Amyloid neurotoxicity was significantly reduced by testosterone via a rapid, estrogen-independent mechanism. These data may provide additional insight into the treatment of age-related neurodegenerative disorders.