Analysis of the GPR17 receptor in NG2-glia under physiological conditions unravels a new subset of oligodendrocyte progenitor cells with distinct functions.

NG2-glia comprise a heterogeneous population of cycling cells that give rise to mature, myelinating oligodendrocytes. The mechanisms that regulate the process of differentiation from NG2-glia into oligodendrocytes are still not fully understood but over the last years the G Protein-coupled Receptor 17 (GPR17) has been on the spotlight as a possible key regulator. Interestingly, GPR17-expressing NG2-glia show under physiological conditions a slower and lower level of differentiation compared to NG2-glia without GPR17. In contrast, after a CNS insult these react with proliferation and differentiation in a high rate, pointing towards a role in repair processes. However, the role of GPR17+ NG2-glia under healthy conditions in adulthood has not been addressed yet. Therefore, we aimed here to characterize the GPR17-expressing NG2-glia. Using transgenic mouse models, we showed restricted GPR17 expression in only some NG2-glia. Furthermore, we found that these cells constitute a distinct subset within the NG2-glia population, which shows a different gene expression profile and behavior when compared to the total NG2-glia population. Genetic depletion of GPR17+ cells showed that these are not contributing to the dynamic and continuous generation of new oligodendrocytes in the adult brain. Taken together, GPR17+ NG2-glia seem to play a distinct role under physiological conditions that goes beyond their classic differentiation control, that needs to be further elucidated. These results open new avenues for using the GPR17 receptor as a target to change oligodendrogenesis under physiological and pathological conditions, highlighting the importance of further characterization of this protein for future pharmacological studies.

Impact of the COVID-19 Pandemic on Healthcare Activity in the Regional Hospitals of Andalusia (Spain).

(1) Background: The large global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has overloaded the public health systems and reduced the regular healthcare activity, leading to a major health crisis. The main objective of this study was to carry out a comparative evaluation of the healthcare activities in the hospitals of Eastern Andalusia, Spain. (2) Methods: In this study, an observational, multicentered, and retrospective approach was adopted to compare the healthcare activities of the Poniente Hospital (PH) and the Alto Guadalquivir Health Agency (AGHA). Data was collected over a period of 24 months, i.e., from 1 January 2019 to 31 December 2020, and the variables evaluated were: patients seen in the hospital emergency service (HES), X-ray tests performed, patients cited in outpatient consultations, surgical interventions performed, and patients included in the waiting list. (3) Results: The analysis of the above-mentioned variables revealed a significant reduction in the number of patients registered in 2020 at HES as compared to that in 2019 for both PH (p = 0.002) and AGHA (p < 0.001). Moreover, the number of surgical interventions in 2020 was significantly reduced from that in 2019 for both PH (p = 0.001) and AGHA (p = 0.009). Moreover, for PH (p < 0.001), a significant reduction was observed in the waiting list admissions in 2020 compared to that in 2019; however, no significant difference in the waiting list admissions between the years 2020 and 2019 was observed for AGHA (p = 0.446). In 2020, the number of teleconsultations was significantly increased from that in 2019 for both PH (p < 0.001) and AGHA (p = 0.006). (4) Conclusion: The analysis carried out indicates that in 2020, compared to 2019, healthcare activity was significantly reduced in most of the parameters included in this study.

Tissue plasminogen activator worsens experimental autoimmune encephalomyelitis by complementary actions on lymphoid and myeloid cell responses.

BACKGROUND: Tissue plasminogen activator (tPA) is a serine protease involved in fibrinolysis. It is released by endothelial cells, but also expressed by neurons and glial cells in the central nervous system (CNS). Interestingly, this enzyme also contributes to pathological processes in the CNS such as neuroinflammation by activating microglia and increasing blood-brain barrier permeability. Nevertheless, its role in the control of adaptive and innate immune response remains poorly understood. METHODS: tPA effects on myeloid and lymphoid cell response were studied in vivo in the mouse model of multiple sclerosis experimental autoimmune encephalomyelitis and in vitro in splenocytes. RESULTS: tPA-/- animals exhibited less severe experimental autoimmune encephalomyelitis than their wild-type counterparts. This was accompanied by a reduction in both lymphoid and myeloid cell populations in the spinal cord parenchyma. In parallel, tPA increased T cell activation and proliferation, as well as cytokine production by a protease-dependent mechanism and via plasmin generation. In addition, tPA directly raised the expression of MHC-II and the co-stimulatory molecules CD80 and CD86 at the surface of dendritic cells and macrophages by a direct action dependent of the activation of epidermal growth factor receptor. CONCLUSIONS: Our study provides new insights into the mechanisms responsible for the harmful functions of tPA in multiple sclerosis and its animal models: tPA promotes the proliferation and activation of both lymphoid and myeloid populations by distinct, though complementary, mechanisms.

Telemedicine in Times of the Pandemic Produced by COVID-19: Implementation of a Teleconsultation Protocol in a Hospital Emergency Department.

Since the first case of COVID-19 was reported in Spain, almost 22% of healthcare professionals have been infected. Among the main causes are exposure during the care of suspected patients and asymptomatic patients, which caused a greater lack of protection in some cases, and to the global shortage of personal protective equipment due to the strong demand for it. The main objective of this study was to evaluate the effectiveness of a teleconsultation protocol with patients who had respiratory symptoms in the reduction of the consumption of personal protective equipment (PPE) in a hospital emergency service (HES) during the COVID-19 pandemic. This is a descriptive and retrospective study that analyzes the implementation of a teleconsultation protocol with patients with respiratory problems treated in the HES at the Hospital de Poniente (Almeria), between 18 March and 30 April 2020. In the selected study period, 5353 patients were treated in the HES of the Hospital de Poniente; of these, 15.43% showed respiratory symptoms and were referred to the Respiratory Circuit, of which 42.2% did so via teleconsultation. Sixty-six cases of COVID-19 were diagnosed, 57.6% were male, and the median age was 71 years old. The main disease related was pneumonia (89.4%), symptoms more frequent were cough (77.3%), fever (77.3%), and dyspnea (60.6%). Lastly, 56.1% of the patients that attended had one or more comorbidities, high blood pressure (53%), and diabetes (36.4%), which became the main risk factors. The results showed that the implementation of teleconsultation in the HES reduced the possibility of infection and allowed for a more efficient consumption of personal protective equipment.

Improved age-related deficits in cognitive performance and affective-like behavior following acute, but not repeated, 8-OH-DPAT treatments in rats: regulation of hippocampal FADD.

The aims of this study were (1) to behaviorally phenotype rats at different ages for both cognitive performance and affect, (2) to evaluate the possible beneficial effects of 8-OH-DPAT (a 5-HT1A receptor agonist) treatments on improving age-related behavioral deficits, and (3) to uncover putative key brain targets (e.g., Fas-associated protein with death domain [FADD] and related partners) that might contribute to the observed age-related behavioral changes. The principal results showed that acute, but not repeated, 8-OH-DPAT treatments improved age-related deficits in cognitive performance and affect while induced hypothermia. Moreover, multifunctional FADD protein decreased with age specifically in the hippocampus (as compared to the prefrontal cortex) and was further decreased following acute 8-OH-DPAT. The major conclusions indicate a parallelism between the beneficial effects observed following acute 8-OH-DPAT on improving the negative consequences of aging on cognition and affect, together with the acute induction of hypothermia and hippocampal FADD regulation. Because these effects were not observed following repeated treatment (i.e., observed tolerance to acute hypothermia), the results suggest 5-HT1A receptors desensitization and/or the activation of compensatory adaptive mechanisms.

Repeated treatment with the α2-adrenoceptor agonist UK-14304 improves cognitive performance in middle-age rats: Role of hippocampal Fas-associated death domain.

The cell fate regulator Fas-associated death domain (FADD) balances cell death with non-apoptotic actions via its phosphorylated form. A recent study associated loss of cortical FADD with cognitive decline and increased risk of clinical dementia. Since the activation of cortical α2A-adrenoceptors improved memory deficits in various animal models of working memory loss, the present study evaluated whether UK-14304, an α2-adrenoceptor agonist known to acutely regulate brain FADD forms, would improve cognitive function in middle-aged rats. Sprague-Dawley rats were treated with UK-14304 (0.3 or 1 mg/kg) or saline (1 mL/kg) for seven days. Cognitive performance was evaluated in the eight-arm radial maze. FADD protein content was measured in the prefrontal cortex and hippocampus by Western blot analysis. The results showed that UK-14304 (1 mg/kg) improved cognitive performance (less time: -310±45 s, p=0.025 and fewer errors: -2.75±1.06, p=0.043 to complete the maze) and increased FADD selectively in the hippocampus (+35±11%, p=0.029). Interestingly, hippocampal FADD content negatively correlated with the time ( r=-0.651, p<0.01) needed to complete the maze. Thus, better cognitive scores were associated with higher FADD hippocampal content. These results support a role for α2-adrenoceptors in ameliorating cognition and suggest FADD protein content as a possible correlate for cognitive performance.

Effects of Resveratrol and Other Polyphenols on the Most Common Brain Age-Related Diseases.

BACKGROUND: With global increase in elderly population, modern societies must find strategies to reduce the consequences of aging process; thereby decreasing the incidence of age-related neurodegenerative diseases. Oxidative stress and recently inflammation, have been pointed out as the leading causes of brain aging. Thereby, the consumption or administration of antioxidant and anti-inflammatory molecules, such as polyphenols, is a beneficial strategy recommended for preventing brain aging and several brain age-related diseases. METHODS AND RESULTS: Several studies suggest that long term consumption of dietary polyphenols offers protection against development of neurodegenerative diseases. These beneficial effects are in part due to their antioxidant and anti-inflammatory properties, together with their positive role in the modulation of processes involved in the physiopathology of several neurodegenerative diseases (e.g., epigenetic factors, amyloid deposition, cholinesterase inhibition, autophagy, and neurotrophic factors, among others). Altogether, these molecules open the door to the research of new neuroprotective strategies. This review summarizes the latest discoveries in how polyphenols can exert positive effects on brain health in aging, emphasizing those effects on the diseases that most commonly affect the brain during aging: Parkinson's Disease (PD), Alzheimer's disease (AD), dementia and depression. Moreover, within are addressed the epigenetic effects of polyphenols as possible mediators in their positive effects on brain health, and the future challenges of research in this topic Conclusion: In brief, this review presents a report of state-of the art knowledge regarding the positive influences of polyphenols on the most common brain age-related diseases as well as in healthy brain aging.

Chronic α-Tocopherol Increases Central Monoamines Synthesis and Improves Cognitive and Motor Abilities in Old Rats.

Limiting enzymes in the synthesis of brain monoamines seems to be susceptible to oxidative damage, one of the most important factors in aging. It has been suggested that the use of anti-oxidants can reduce the rate of free radical production related with aging and the associated damage. Therefore, this study aims to analyze the effects of the chronic treatments with the anti-oxidant α-tocopherol (vitamin E) on central monoamines (high-performance liquid chromatography [HPLC] analysis) mediating cognitive functions, as well as on the evaluation of memory and motor abilities in old rats measured by radial maze, Barnes maze, novel object recognition test, and rotarod test. Results show that α-tocopherol significantly increased in a dose- and/or time-dependent manner the synthesis rate and the levels of monoaminergic neurotransmitters (serotonin, dopamine, and noradrenaline) in the hippocampus and striatum, brain regions involved in memory processing and motor coordination. These positive neurochemical effects, largely due to an increased activity of the limiting enzymes in monoamines synthesis, tryptophan hydroxylase and tyrosine hydroxylase, were accompanied by an improvement in cognitive and motor abilities in old rats. Altogether these findings suggest that α-tocopherol exhibits neuroprotective actions in old rats; thus, diets with α-tocopherol might represent a promising strategy to mitigate or delay the cognitive and motor decline associate with aging and related-diseases.

Caloric restriction, resveratrol and melatonin: Role of SIRT1 and implications for aging and related-diseases.

Aging is an inevitable and multifactorial biological process. Free radicals have been implicated in aging processes; it is hypothesized that they cause cumulative oxidative damage to crucial macromolecules and are responsible for failure of multiple physiological mechanisms. However, recent investigations have also suggested that free radicals can act as modulators of several signaling pathways such as those related to sirtuins. Caloric restriction is a non-genetic manipulation that extends lifespan of several species and improves healthspan; the belief that many of these benefits are due to the induction of sirtuins has led to the search for sirtuin activators, especially sirtuin 1, the most studied. Resveratrol, a polyphenol found in red grapes, was first known for its antioxidant and antifungal properties, and subsequently has been reported several biological effects, including the activation of sirtuins. Endogenously-produced melatonin, a powerful free radical scavenger, declines with age and its loss contributes to degenerative conditions of aging. Recently, it was reported that melatonin also activates sirtuins, in addition to other functions, such as regulator of circadian rhythms or anti-inflammatory properties. The fact that melatonin and resveratrol are present in various foods, exhibiting possible synergistic effects, suggests the use of dietary ingredients to promote health and longevity.

Cognitive improvement by acute growth hormone is mediated by NMDA and AMPA receptors and MEK pathway.

It has been reported that Growth hormone (GH) has an immediate effect enhancing excitatory postsynaptic potentials mediated by AMPA and NMDA receptors in hippocampal area CA1. As GH plays a role in adult memory processing, this work aims to study the acute effects of GH on working memory tasks in rodents and the possible involvement of NMDA and AMPA receptors and also the MEK/ERK signalling pathway. To evaluate memory processes, two different tests were used, the spatial working memory 8-arm radial maze, and the novel object recognition as a form of non-spatial working memory test. Acute GH treatment (1mg/kg i.p., 1h) improved spatial learning in the radial maze respect to the control group either in young rats (reduction of 46% in the performance trial time and 61% in the number of errors), old rats (reduction of 38% in trial time and 48% in the number of errors), and adult mice (reduction of 32% in the performance time and 34% in the number of errors). GH treatment also increased the time spent exploring the novel object respect to the familiar object compared to the control group in young rats (from 63% to 79%), old rats (from 53% to 70%), and adult mice (from 61 to 68%). The improving effects of GH on working memory tests were blocked by the NMDA antagonist MK801 dizocilpine (0.025 mg/kg i.p.) injected 10 min before the administration of GH, in both young and old rats. In addition, the AMPA antagonist DNQX (1mg/kg i.p.) injected 10 min before the administration of GH to young rats, blocked the positive effect of GH. Moreover, in mice, the MEK inhibitor SL 327 (20mg/kg i.p.) injected 30 min before the administration of GH, blocked the positive effect of GH on radial maze and the novel object recognition. In conclusion, GH improved working memory processes through both glutamatergic receptors NMDA and AMPA and it required the activation of extracellular MEK/ERK signalling pathway. These effects could be related to the enhancement of excitatory synaptic transmission in the hippocampus reported by GH.

Chronic treatment with selective I2-imidazoline receptor ligands decreases the content of pro-apoptotic markers in rat brain.

Selective I(2)-imidazoline receptor ligands induce neuroprotection through various molecular mechanisms including blockade of N-methyl-D-aspartate (NMDA) receptors. To investigate new neuroprotective mechanisms associated with I(2)-imidazoline receptors, the effects of selective (2-styryl-2-imidazoline (LSL 61122), 2-(2-benzofuranyl)-2-imidazoline (2-BFI), 2-(4,5-dihydroimidazol-2-yl) quinoline hydrochloride (BU-224)) and non-selective (idazoxan) I(2)-drugs on canonical apoptotic pathways were assessed in rat brain cortex. The acute treatment with LSL 61122 (10 mg/kg) reduced the content of mitochondrial (pro-apoptotic) Bax (-33%) and cytochrome c (-31%), which was prevented by idazoxan, an I(2)-receptor antagonist. The sustained stimulation of I(2)-imidazoline receptors with selective drugs (10 mg/kg, every 12 h for seven days) was associated with down-regulation of key components of the extrinsic (Fas receptor: -20%; Fas associated protein with death domain (FADD) adaptor: -47-54%) and/or intrinsic (Bax: -20-23%; cytochrome c: -22-28%) apoptotic signalling and/or up-regulation of survival anti-apoptotic factors (p-Ser194 FADD/FADD ratio: +1.6-2.5-fold; and/or Bcl-2/Bax ratio: +1.5-fold), which in the long-term could dampen cell death in the brain. Similar chronic treatments with LSL 60101 (the imidazole analogue of 2-BFI) and idazoxan (a mixed I(2)/α(2)-ligand) did not induce significant alterations of pro- or anti-apoptotic proteins. The disclosed anti-apoptotic mechanisms of selective I(2)-imidazoline drugs may work in concert with other molecular mechanisms of neuroprotection (e.g. blockade of NMDA receptors) that are engaged by I(2)-ligands.

Correlation of rat cortical Fas-associated death domain (FADD) protein phosphorylation with the severity of spontaneous morphine abstinence syndrome: role of α(2)-adrenoceptors and extracellular signal-regulated kinases.

Fas-associated death domain (FADD) phosphorylation was recently implicated in opiate-induced neuroplasticity. To further explore the role of FADD in the mechanisms of morphine-induced physical dependence, the regulation of cortical p-FADD (and their interactions with α(2)-adrenoceptors and other signalling pathways) was assessed during spontaneous opiate withdrawal (SW) in morphine-dependent rats (10-100 mg/kg for 6 days). The main results indicated that oligomeric p-FADD in the cerebral cortex mirrored the time course of morphine SW (12-96 h), which resulted in a striking correlation between p-FADD and the intensity (behavioural scores) of morphine abstinence (Spearman correlation coefficient: 0.59, n = 39, p < 0.0001). The inactivation of brain α(2)-adrenoceptors (EEDQ at SW 12 h) further enhanced morphine abstinence intensity and cortical p-FADD content at SW 24 h. The disruption of ERK1/2 signalling (SL 327 at SW 4 h and SW 8 h) did not alter morphine abstinence at SW 12 h, but it attenuated the behavioural syndrome at SW 24 h. This inhibition of ERK1/2, however, did not prevent the up-regulation of oligomeric p-FADD at SW 12 h and 24 h. These data indicate that cortical oligomeric p-FADD, mainly through an interaction with inhibitory α(2)-adrenoceptors, plays a functional role in the behavioural expression of morphine abstinence in rats.

Opioid receptor agonists enhance the phosphorylation state of Fas-associated death domain (FADD) protein in the rat brain: functional interactions with casein kinase Ialpha, Galpha(i) proteins, and ERK1/2 signaling.

Opioid drugs have been proposed to promote anti-apoptotic signals in brain through inhibition of FADD protein [García-Fuster et al., 2007. Effects of opiate drugs on Fas-associated protein with death domain (FADD) and effector caspases in the rat brain: Regulation by the ERK1/2 MAP kinase pathway. Neuropsychopharmacology 32, 399-411]. FADD phosphorylation by casein kinase Ialpha (CKIalpha) appears to regulate its non-apoptotic activity. This study investigated the effects of opioids on p-FADD in rat brain, as well as various mechanisms that could link opioid receptors with p-FADD, including the modulation of CKIalpha, Galpha(i) proteins and ERK1/2 signaling. In rat, mouse and human brains, various anti-p-FADD antibodies immunodetected the monomeric and oligomeric forms of this protein, irrespective of the antibody origin and specific Ser191 or Ser194 phosphorylation site. Acute mu- and delta-agonists increased, through specific opioid receptor mechanisms, the content of oligomeric and monomeric p-FADD forms in rat cortical homogenates (25-61%) and subcellular compartments, with most relevant effects for sufentanil in membrane (239%) and nucleus (136%). p-FADD induction vanished with repeated (5days) morphine but not SNC-80, and opioid withdrawal induced a new (morphine) or sustained (SNC-80) stimulatory effect (32-33%). The kappa-agonist (-)-U-50488H failed to stimulate p-FADD. Sufentanil reduced CKI protein and kinase activity in the cytosol (30-37%). Morphine, but not SNC-80, augmented CKIalpha in cytosol, membrane and nucleus (36-104%). In contrast to FADD, the ability of SNC-80 to stimulate p-FADD was not sensitive to ERK1/2 blockade. Pertussis toxin did not prevent the opposite effects of SNC-80 on p-FADD and FADD because the toxin by itself markedly altered their basal contents, indicating that FADD could be a novel toxin target. The upregulation of p-FADD induced by mu/delta-agonists could play a relevant role in the anti-apoptotic and/or neuroplastic effects of opioids.

Effects of opiate drugs on Fas-associated protein with death domain (FADD) and effector caspases in the rat brain: regulation by the ERK1/2 MAP kinase pathway.

This study was designed to assess the effects of opiate treatment on the expression of Fas-associated protein with death domain (FADD) in the rat brain. FADD is involved in the transmission of Fas-death signals that have been suggested to contribute to the development of opiate tolerance and addiction. Acute treatments with high doses of sufentanil and morphine (mu-agonists), SNC-80 (delta-agonist), and U50488H (kappa-agonist) induced significant decreases (30-60%) in FADD immunodensity in the cerebral cortex, through specific opioid receptor mechanisms (effects antagonized by naloxone, naltrindole, or nor-binaltorphimine). The cannabinoid CB1 receptor agonist WIN 55,212-2 did not alter FADD content in the brain. Chronic (5 days) morphine (10-100 mg/kg), SNC-80 (10 mg/kg), or U50488H (10 mg/kg) was associated with the induction of tachyphylaxis to the acute effects. In morphine- and SNC-80-tolerant rats, antagonist-precipitated (2 h) or spontaneous withdrawal (24-48 h) induced a new and sustained inhibition of FADD (13-50%). None of these treatments altered the densities of caspases 8/3 (including the active cleaved forms) in the brain. Pretreatment of rats with SL 327 (a selective MEK1/2 inhibitor that blocks ERK activation) fully prevented the reduction of FADD content induced by SNC-80 in the cerebral cortex (43%) and corpus striatum (29%), demonstrating the direct involvement of ERK1/2 signaling in the regulation of FADD by the opiate agonist. The results indicate that mu- and delta-opioid receptors have a prominent role in the modulation of FADD (opposite to that of Fas) shortly after initiating treatment. Opiate drugs (and specifically the delta-agonists) could promote survival signals in the brain through inhibition of FADD, which in turn is dependent on the activation of the antiapoptotic ERK1/2 signaling pathway.

Stimulation of mitogen-activated protein kinase kinases (MEK1/2) by mu-, delta- and kappa-opioid receptor agonists in the rat brain: regulation by chronic morphine and opioid withdrawal.

Opioid addiction modulates the extracellular signal-regulated kinase (ERK) leading to synaptic plasticity in the brain. ERK1/2 are stimulated by mitogen-activated protein kinase kinases (MEK1/2), but little is known about the regulation of MEK activity by opioid drugs. This study was designed to assess the acute effects of selective mu-, delta-, and kappa-opioid receptor agonists, as well as those induced by chronic morphine and opioid withdrawal, on the content of phosphorylated MEK1/2 in the rat brain. Sufentanil (1-30 microg/kg, 30-120 min) induced dose- and time-dependent increases in MEK1/2 phosphorylation in the cerebral cortex and corpus striatum (30-177%) through a naloxone-sensitive mechanism. Morphine (100 mg/kg, 2 h) also augmented MEK1/2 phosphorylation in the both brain regions (50-70%). Similarly, the selective delta-opioid receptor agonist SNC-80 (10 mg/kg, 30 min) increased MEK1/2 activity in the cortex (60%) that was antagonized by naltrindole. In contrast, the selective kappa-opioid receptor agonist (-)-U50488H (10 mg/kg, 30-120 min) did not modify significantly MEK1/2 phosphorylation in the cortex. Chronic morphine (10-100 mg/kg, 5 days) was not associated with alterations in the content of phosphorylated MEK1/2 in the brain (induction of tachyphylaxis to the acute effects). In morphine-dependent rats, however, naloxone (2 mg/kg)-precipitated withdrawal (2-6 h) induced robust increases in MEK1/2 phosphorylation in cortex (27-49%) and striatum (83-123%). Spontaneous opioid withdrawal (24 h) in morphine-dependent rats did not alter MEK1/2 activity in the brain. The findings may be relevant in the context of the pivotal role played by the MEK/ERK pathway in various long-lasting forms of synaptic plasticity associated with opioid addiction.

High-affinity binding of beta-carbolines to imidazoline I2B receptors and MAO-A in rat tissues: norharman blocks the effect of morphine withdrawal on DOPA/noradrenaline synthesis in the brain.

This study was designed to determine the affinity and binding profile of beta-carbolines for imidazoline I2 receptors and catalytic sites of monoamine oxidase (MAO)-A/B in rat brain and liver. The aim was also directed to assess the in vivo effects of norharman (beta-carboline) and LSL 60101 (I2 ligand) on brain 3,4-dihydroxyphenylalanine (DOPA) synthesis in morphine-dependent rats. Competition experiments against [3H]2-BFI revealed that beta-carbolines recognize the high- and low-affinity components of the brain imidazoline I2 receptor with the rank order of potency (K(iH) in nM): noreleagnine (12)>norharman (20)>harmalol (82)>harmaline (177)>>harmine (630)>harman (700)>>FG-7142 (>100,000). In liver, this rank was different: harmine (51)>harmaline (103)=noreleagnine (103)>>harmalol (1290)>harman (2000)>>norharman (12,382)>>FG-7142 (>100,000). In brain and liver, competition curves for beta-carbolines against [3H]Ro41-1049 (MAO-A) and [3H]Ro19-6327 (MAO-B) were monophasic and resulted in different drug potencies for the two MAO isozymes (higher affinities for MAO-A) and in similar pharmacological profiles in both tissues. In morphine-dependent rats, naloxone (2 mg/kg, 2 h)-precipitated withdrawal increased the synthesis of DOPA in the cerebral cortex and hippocampus (50%). Pretreatment with norharman (20 mg/kg) or LSL 60101 (20 mg/kg) (30 min before naloxone) fully prevented the stimulatory effect of opiate withdrawal on DOPA synthesis. Norharman and LSL 60101 also attenuated the severity of the withdrawal syndrome. The results indicate that beta-carbolines bind with high affinity to imidazoline I2B receptors, and similarly to I2 ligands (LSL 60101) can block the behavioural and biochemical effects of opiate withdrawal.

Deglycosylation of Fas receptor and chronic morphine treatment up-regulate high molecular mass Fas aggregates in the rat brain.

This study was designed to immunodetect and characterize Fas receptor aggregates (oligomerization) in the brain and to assess its possible modulation in opiate addiction. High molecular mass, sodium dodecyl sulfate (SDS)- and beta-mercaptoethanol-resistant Fas aggregates (approximately 110/120 and approximately 203 kDa specific peptides) were immunodetected with a cytoplasmic domain-specific antibody in brain tissue (rat, mouse and human) and SH-SY5Y cells by Western blot analysis. Preincubation of rat cortical membranes with N-ethylmaleimide (NEM; 1 mM for 1 h at 37 degrees C) reduced the immunodensity of approximately 203 kDa Fas aggregates (51%) and increased that of 35 kDa native Fas (172%) and 51/48 kDa glycosylated Fas (47%), indicating that disulfide bonds are involved in Fas dimerization. Enzymatic N-deglycosylation of Fas receptor increased the content of Fas aggregates (approximately 110/120 kDa: five- to sixfold, and approximately 203 kDa: two- to threefold), suggesting that Fas glycosylation is involved in regulating receptor dimerization. Chronic (10-100 mg/kg for 5 days), but not acute (30 mg/kg for 2 h), treatment with morphine (a micro-opioid peptide receptor agonist) induced up-regulation of Fas aggregates in the brain (approximately 110/120 kDa: 39%, and approximately 203 kDa: 89%). The acute and/or chronic treatments with delta- and kappa-opioid peptide receptor agonists and with a sigma1-receptor agonist did not readily alter the content of Fas aggregates in the rat brain. The results indicate that Fas aggregates are natively expressed in the brain and that its density is regulated by the state of Fas glycosylation. These forms of Fas (receptor homodimerization) are functionally relevant because they were up-regulated in the brain of morphine-dependent rats.

Modulation of Fas receptor proteins and dynamin during opiate addiction and induction of opiate withdrawal in rat brain.

The Fas receptor is involved in the regulation of apoptosis but also can function as a non-apoptotic signal transducer. This study was mainly designed to quantitate Fas proteins in rat brain during heroin addiction and opiate withdrawal. In rat, mouse and human brains, and in SH-SY5Y cells, similar forms of Fas were immunodetected with different antibodies (i.e., 35 kDa native Fas and 48- and 51-kDa glycosylated Fas). Acute (2 h) treatments with the micro-opioid receptor agonists heroin (10 mg/kg) and morphine (30 mg/kg) increased the immunodensity of native Fas (124% and 36%) but not that of glycosylated Fas in the cerebral cortex. Chronic (5 days) heroin (5-30 mg/kg) and morphine (10-100 mg/kg) were also associated with increased native Fas (76% and 45%) and with different expressions of glycosylated Fas. In heroin-dependent rats, opiate withdrawal (48 h) resulted in a sustained increase in native Fas (107%) and in up-regulation of 51 kDa glycosylated Fas (51%). Acute treatments with selective delta-receptor (SNC-80, 10 mg/kg) or kappa-receptor (U 50488-H, 10 mg/kg) agonists did not alter the content of native or glycosylated Fas. Chronic pentazocine (10-80 mg/kg, 5 days), a mixed opiate drug and sigma(1) receptor agonist, decreased native (48%) and glycosylated (38-82%) Fas proteins. Similarly, the selective sigma(1) agonist (+)-SKF 10047 also decreased native Fas (37%) and the effect was blocked by the sigma(1) antagonist BD 1063. Brain dynamin was up-regulated by acute and/or chronic heroin (30-39%), morphine (47-85%), pentazocine (51%) and heroin withdrawal (74%). The main results indicate that chronic heroin/morphine treatment and heroin withdrawal are associated with up-regulation of 35 kDa native Fas (and with different expressions of glycosylated Fas), and also with concomitant increases of dynamin in rat brain.

Withdrawal from chronic ethanol increases the sensitivity of presynaptic 5-HT(1A) receptors modulating serotonin and dopamine synthesis in rat brain in vivo.

The in vivo sensitivity of presynaptic 5-HT(1A) receptors (autoreceptors and heteroreceptors) modulating the synthesis of 5-hydroxytryptophan/serotonin (5-HTP/5-HT) and 3,4-dihydroxyphenylalanine/dopamine (DOPA/DA) in rat brain was investigated after ethanol treatment and withdrawal. In saline-treated rats as well as in acute ethanol (2 g/kg, intraperitoneally (i.p.), 2 h)- and chronic ethanol (2 g/kg for 7 days)-treated rats, a low dose of the 5-HT(1A) receptor agonist 8-hydroxy-2-di-n-propylamino-tetralin (8-OH-DPAT; 0.1 mg/kg, i.p., 1 h) did not decrease the synthesis of 5-HTP in brain (except modestly in striatum; 20% after the chronic treatment) or that of DOPA in striatum. In contrast, in chronic ethanol-withdrawn rats (24 h), 8-OH-DPAT significantly decreased the synthesis of 5-HTP in the hippocampus (29%), cerebral cortex (41%) and striatum (33%) and that of DOPA in the striatum (28%). Similar effects were induced by the mixed 5-HT(1A) agonist/D(2) antagonist buspirone (1 mg/kg, i.p., 1 h) which also decreased 5-HTP synthesis in the hippocampus (24%), cerebral cortex (36%) and striatum (35%) of chronic ethanol-withdrawn rats. These results indicate that chronic ethanol and more clearly the spontaneous withdrawal from chronic ethanol induce supersensitivity of 5-HT(1A)-auto/heteroreceptors modulating the synthesis of 5-HT and DA in rat brain.