Whole-brain Homogenates Research Articles

Ageing-Related Changes to H3K4me3, H3K27ac, and H3K27me3 in Purified Mouse Neurons.

Neurons are central to lifelong learning and memory, but ageing disrupts their morphology and function, leading to cognitive decline. Although epigenetic mechanisms are known to play crucial roles in learning and memory, neuron-specific genome-wide epigenetic maps into old age remain scarce, often being limited to whole-brain homogenates and confounded by glial cells. Here, we mapped H3K4me3, H3K27ac, and H3K27me3 in mouse neurons across their lifespan. This revealed stable H3K4me3 and global losses of H3K27ac and H3K27me3 into old age. We observed patterns of synaptic function gene deactivation, regulated through the loss of the active mark H3K27ac, but not H3K4me3. Alongside this, embryonic development loci lost repressive H3K27me3 in old age. This suggests a loss of a highly refined neuronal cellular identity linked to global chromatin reconfiguration. Collectively, these findings indicate a key role for epigenetic regulation in neurons that is inextricably linked with ageing.

GABA content and an antioxidant profile positively correlated with the anticonvulsive activity of Microcos paniculata in acute seizure mice

This study evaluated the effects of different parts of M. paniculata (MP) extracts on convulsions and antioxidant activities in mice. Six polyphenolic compounds were identified, where epicatechin and quercetin have been identified in the highest amounts (23.01 and 32.23 mg/100 g of dry MP extract, respectively) in MP leaf and stem extracts, using Ultra Performance Liquid Chromatography. 7-day oral administration of MP at doses of 100, 200, and 400 mg/kg body weight (BW) significantly reduced convulsions and reduced mortality rates compared with seizure inducer groups. Antioxidant potentials were measured by superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS), and reduced glutathione (GSH) content in whole-brain homogenates. Gamma-aminobutyric acid (GABA) levels significantly increased in leaves and stem-treated groups, suggesting that MP leaves and stems have potent antioxidant properties that can attenuate convulsions by modulating the GABAergic system and antioxidant activities.

Endosomal Trafficking is disrupted in Neurodevelopmental Disorders

Endosomal trafficking has been implicated in several neurodevelopmental disorders including Rett Syndrome (RTT) and Schizophrenia (SZ). Endosomal proteins have been identified in genomic studies in both disorders, suggesting a common molecular mechanism. To characterize endosomal proteins in these disorders, we analyzed protein expression in the cortex and dentate gyrus of the hippocampus as well as in synaptosome preparations from whole-brain lysates. Immunofluorescent staining demonstrated alterations in key endosomal proteins in coronal brain sections from Mecp2 deficient mice and BLOC-1 deficient mice in the cortex and hippocampus. In addition, biochemical analysis of whole-brain homogenate and synaptosome fractions demonstrated an alteration in endosomal markers. Neurite outgrowth was also significantly decreased in PC12 cells lacking dysbindin and Mecp2. Lastly, to characterize alterations in endosomal trafficking, we used a pulse-chase assay in Mecp2 KD cells and observed altered kinetics of endosomal trafficking. Together, these data further characterize the role of endosomal trafficking mechanisms that underlie neurodevelopmental disorder pathogenesis, highlighting potential biomarkers or therapeutic targets for these disorders.

Effect of citrate stabilized gold nanoparticle on the biochemical and histological alterations of liver in an experimental animal model

Although the nanoparticles (NP) are seemed to have the immense potentiality for using in different biomedical fields the possible toxic health effects of these NPs associated with human exposure is a serious concern. In general, the toxicity of gold nanoparticles (AuNPs) depends on their physical dimensions and surface chemistry. In the present study, size-specific AuNPs were used to test its potential harmful effect in the biochemical and histological alterations of liver tissue in experimental animals (swiss albino mice). Animals were exposed to the AuNPs for seven days. Oxidative stress in whole tissue homogenates and in mitochondrial extracts and histological studies were studied in the liver. Significant deterioration in glutathione reductase (GR), malondialdehyde (MDA), and total thiol content were observed in the case of whole liver tissue homogenates; on the other hand, in case of whole-brain tissue homogenates, deterioration in glutathione S-transferase (GST), total thiol and MDA was observed. Almost all the lipid peroxidation product and oxidative stress marker enzymes were affected in the hepatic system were observed in AuNP treated animals. Anatomical differences in liver tissue were also observed in the AuNP treated animals. Minute loss of hepatic cords, occurrence of swallowing and atrophic cells is the major detoriation observed in AuNP treated animals. Surface negativity and the small size of the AuNPs may be assumed to affect the biochemical and cellular architecture of the organs. The further molecular level analysis will reveal the usability potential of AuNP in the body system for different purposes.

Role of Nrf2 in Regulation of Expression of CYP 2C11 and CYP 4X1 Epoxygenases and Production of Epoxyeicosatrienoic Acids (EETs) in the Rat Brain

Nuclear factor (erythroid‐derived 2)‐like‐2 (Nrf2) is the master antioxidant and cell‐protective transcription factor native in the rat brain. The CYP epoxygenases 2C11 and 4X1 are expressed in the brain and metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) that are known to induce cerebral vasodilation and increase nutritive blood flow to areas of heightened neuronal activity and also provide antioxidant and anti‐inflammatory actions. The influence of Nrf2 gene deletion on CYP 2C11 and 4X1 epoxygenase expression and activity in the rat brain is not known. In this study we used isoform specific antibodies and Western blotting to determine the effect of Nrf2 gene knockout (KO) on the expression of proteins for CYP 2C11 and CYP 4X1 epoxygenases in whole brain and isolated cerebral arteries. We also measured the production of epoxyeicosatrienoic acids (EETs) in homogenates of whole brain and isolated cerebral arteries of Nrf2 wild type (WT) and Nrf2 KO rats. CYP epoxygenase expression and EETs production were reduced in whole brain and arteries of the NRF2 KO rats compared to WT controls. These findings indicate that knockout of the Nrf2 gene leads to a marked reduction in the expression of proteins for CYP 2C11 and CYP 4X1 epoxygenases and in the production of 8, 9‐, 11,12‐ and 14,15‐EETs compared to those measured in the Nrf2 WT rat brain issues and cerebral arteries. The results of the current study suggest that CYP 2C11 and 4X1 epoxygenases may be components of the Nrf2‐induced phase 2 genes that are known to limit oxidative stress and provide cellular defense in the brain. We postulate that Nrf2 may regulate functions of the cerebral circulation and provide protection against oxidative stress, in part, via modulation of the expression level of CYP 2C11 and 4X1 epoxygenase proteins and the production of EETs.Support or Funding InformationNIH R01 #HL‐128242 and NIH #R21‐OD018309

Nardostachys jatamansi Targets BDNF-TrkB to Alleviate Ketamine-Induced Schizophrenia-Like Symptoms in Rats

Schizophrenia, a common neurological disorder appearing in the late teens or early adulthood, is characterized by disorganized thinking, behaviour, and perception of emotions. Aberrant N-methyl-D-aspartate (NMDA) receptor-mediated synaptic plasticity is a major pathological event here due to dysfunction of dopamine and glutamate transmission at NMDA receptors. De-regulated brain-derived neurotrophic factor (BDNF), i.e., its signalling through the tropomyosin receptor kinase B (TrkB) receptor, is a major feature of schizophrenia. With recent global awareness of traditional plant medicines in reducing side effects, the aim of our study was to evaluate the efficacy of the ethanolic root extract of a herb belonging to the Valerianacea family, Nardostachys jatamansi, against ketamine-induced schizophrenia-like model in rats. The effect of the N. jatamansi drug (oral dosage of 500 mg/kg body weight for 14 days) in ketamine-administered male Wistar albino rats (30 mg/kg body weight for 5 days) on modulating behaviour and the level of neurotransmitters like dopamine and glutamate was studied in whole-brain homogenates, and its influence on BDNF and TrkB levels in 2 relevant brain regions, the hippocampus and prefrontal cortex, was assessed. We observed that N. jatamansi treatment exhibited encouraging results in the modulation of ketamine-induced schizophrenia-like behaviours, principally the positive symptoms. Our drug both significantly upregulated the glutamate level and downregulated the dopamine level in whole-brain homogenates and retained the normal levels of BDNF (in the hippocampus but not in the prefrontal cortex) and TrkB (in both hippocampus and prefrontal cortex) induced by ketamine in rats. These findings suggest a neuroprotective effect of the ethanolic root extract of N. jatamansi against ketamine-induced schizophrenia-like symptoms in rats; possibly, regarding its effect on TrkB signalling. Further research is warranted in the treatment of schizophrenic symptoms.

Long-term consumption of fermented rooibos herbal tea offers neuroprotection against ischemic brain injury in rats.

Stroke is the second leading cause of death worldwide, affecting about 240 people a day in South Africa and leaving survivors with residual disabilities. At the moment, there is no clinically approved neuroprotective product for stroke but the consumption of plant polyphenols has been suggested to offer some protection against stroke. In this study, we investigated the effects of long-term consumption of fermented rooibos herbal tea (FRHT) on ischemia/reperfusion (I/R)-induced brain injury in adult male Wistar rats. FRHT was administered to the animals ad libitum for 7 weeks prior to the induction of ischemic injury via a 20-minute bilateral occlusion of the common carotid arteries (BCCAO) followed by reperfusion for 24, 96 and 168 hours respectively. Neurobehavioural deficits, brain oedema, blood-brain barrier (BBB) damage, apoptosis, lipid peroxidation and total antioxidant capacity were subsequently evaluated using standard methods. Our results showed that long-term consumption of FRHT by Wistar rats signi icantly reduced brain oedema and neuronal apoptosis, but did not attenuate BBB damage following cerebral ischemia. Analysis of whole-brain homogenates showed significantly reduced lipid peroxidation levels, increased total antioxidant capacity and resulted in improved neurobehavioural outcomes in FRHT-treated rats when compared with untreated animals. Taken together, our results tend to suggest that continuous consumption of FRHT could confer some protection against ischemic brain injury (IBI) and is therefore highly recommended for patients with stroke-predisposing conditions.

Nurr1 expression is modified by inflammation in microglia.

Advances in neonatal care have allowed premature infants to survive at earlier gestational ages, but they are often afflicted with neurological delays or deficits. Maternal inflammation has been identified as a major risk factor for premature birth and once born, infants often require supplemental oxygen for survival. Nurr1 (NR4A2) is an orphan nuclear receptor with no known binding site and is essential for the growth of midbrain dopamine neurons. Others have reported that Nurr1 can act as an anti-inflammatory transcription factor in microglia and astrocytes and respond lipopolysaccharide (LPS). We have previously reported decreased numbers of oligodendrocytes and increased numbers of microglia in the mice exposed to both maternal inflammation and neonatal hyperoxia in the perinatal period. These studies tested the hypothesis that the combined exposures to inflammation and hyperoxia would increase Nurr1 expression in microglia in our mouse model and in an immortalized microglia cell line, BV2 cells. Our data indicate that Nurr1 protein expression is increased at postnatal day 0 and postnatal day 28 in whole-brain homogenates from mice exposed to LPS and hyperoxia. Alternatively, Nurr1 message is decreased at postnatal day 60 in isolated microglia, indicating that the increases in whole-brain homogenates may be due to other cell types. In BV2 cells, Nurr1 message in increased by exposure to hyperoxia, but this increase is attenuated in cells exposed to both LPS and hyperoxia. Although Nurr1 regulation is not straightforward, these data indicate that Nurr1 expression is increased in whole-brain homogenates in response to inflammation, but is decreased in isolated primary microglia and BV2 cells in response to similar inflammation. Our data support the hypothesis that Nurr1 expression may play a significant role in regulating inflammation in the brain and understanding the complex regulation of Nurr1 could lead to new therapeutic strategies.

Basal brain oxidative and nitrative stress levels are finely regulated by the interplay between superoxide dismutase 2 and p53.

Superoxide dismutases (SODs) are the primary reactive oxygen species (ROS)-scavenging enzymes of the cell and catalyze the dismutation of superoxide radicals O2- to H2O2 and molecular oxygen (O2). Among the three forms of SOD identified, manganese-containing SOD (MnSOD, SOD2) is a homotetramer located wholly in the mitochondrial matrix. Because of the SOD2 strategic location, it represents the first mechanism of defense against the augmentation of ROS/reactive nitrogen species levels in the mitochondria for preventing further damage. This study seeks to understand the effects that the partial lack (SOD2(-/+) ) or the overexpression (TgSOD2) of MnSOD produces on oxidative/nitrative stress basal levels in different brain isolated cellular fractions (i.e., mitochondrial, nuclear, cytosolic) as well as in the whole-brain homogenate. Furthermore, because of the known interaction between SOD2 and p53 protein, this study seeks to clarify the impact that the double mutation has on oxidative/nitrative stress levels in the brain of mice carrying the double mutation (p53(-/-) × SOD2(-/+) and p53(-/-) × TgSOD2). We show that each mutation affects mitochondrial, nuclear, and cytosolic oxidative/nitrative stress basal levels differently, but, overall, no change or reduction of oxidative/nitrative stress levels was found in the whole-brain homogenate. The analysis of well-known antioxidant systems such as thioredoxin-1 and Nrf2/HO-1/BVR-A suggests their potential role in the maintenance of the cellular redox homeostasis in the presence of changes of SOD2 and/or p53 protein levels.

In vitro inhibition of plasma and brain cholinesterases of growing chicks by chlorpyrifos and dichlorvos

This study assessed in vitro inhibition of plasma and brain cholinesterases (ChE) by the organophosphate insecticides chlorpyrifos and dichlorvos in chicks at ages of 2, 10, 20 and 30 days. Plasma samples and whole-brain homogenates of each age group were pooled and ChE activity was determined by an electrometric method with or without the organophosphate (0.5 and 1.0 µM). ChE inhibitions were the highest in 2- and 10-day-old chicks (20–83%), followed by 5–73% inhibitions at ages of 20 and 30 days. Cholrpyrifos decreased plasma ChE activity only at the ages of 2 and 10 days by 21–75%. Dichlorvos decreased plasma ChE activity at all the ages tested (2–30 days) by 39–83%. The highest percentage of plasma ChE inhibition by chlorpyrifos and dichlorvos was in 2-day-old chicks. In the brain homogenate, chlorpyrifos decreased the ChE activity between ages 2 and 20 days by 20–62%. The highest percentage of brain ChE inhibition occurred at the age of 10 days and the least was at 20 and 30 days. Dichlorvos decreased brain ChE activity between ages 2 and 20 days by 24–74%. At the age of 30 days, only the higher concentration of dichlorvos (1 µM) decreased brain ChE activity by 27%. The data suggest an age-related differential in vitro inhibition of plasma and brain ChE s in chicks. In vitro ChE inhibition, though it does not exclude interfering factors, could be useful for initial screening of the inhibitory actions of organophosphate insecticides in chickens.

Treatment of Congenital Neurotransmitter Deficiencies by Intracerebral Ventricular Injection of an Adeno-Associated Virus Serotype 9 Vector

Dopamine and serotonin are produced by distinct groups of neurons in the brain, and gene therapies other than direct injection have not been attempted to correct congenital deficiencies in such neurotransmitters. In this study, we performed gene therapy to treat knock-in mice with dopamine and serotonin deficiencies caused by a mutation in the aromatic L-amino acid decarboxylase (AADC) gene (Ddc(KI) mice). Intracerebral ventricular injection of neonatal mice with an adeno-associated virus (AAV) serotype 9 (AAV9) vector expressing the human AADC gene (AAV9-hAADC) resulted in widespread AADC expression in the brain. Without treatment, 4-week-old Ddc(KI) mice exhibited whole-brain homogenate dopamine and serotonin levels of 25% and 15% of normal, respectively. After gene therapy, the levels rose to 100% and 40% of normal, respectively. The gene therapy improved the growth rate and survival of Ddc(KI) mice and normalized their hindlimb clasping and cardiovascular dysfunctions. The behavioral abnormalities of the Ddc(KI) mice were partially corrected, and the treated Ddc(KI) mice were slightly more active than normal mice. No immune reactions resulted from the treatment. Therefore, a congenital neurotransmitter deficiency can be treated safely through inducing widespread expression of the deficient gene in neonatal mice.

Multiple reaction monitoring for the detection of disease-related synaptic proteins.

Multiple reaction monitoring for the detection of disease-related synaptic proteins.

Differential Control of Opioid Antinociception to Thermal Stimuli in a Knock-In Mouse Expressing Regulator of G-Protein Signaling-Insensitive GαoProtein

Regulator of G-protein signaling (RGS) proteins classically function as negative modulators of G-protein-coupled receptor signaling. In vitro, RGS proteins have been shown to inhibit signaling by agonists at the μ-opioid receptor, including morphine. The goal of the present study was to evaluate the contribution of endogenous RGS proteins to the antinociceptive effects of morphine and other opioid agonists. To do this, a knock-in mouse that expresses an RGS-insensitive (RGSi) mutant Gαo protein, Gαo(G184S) (Gαo RGSi), was evaluated for morphine or methadone antinociception in response to noxious thermal stimuli. Mice expressing Gαo RGSi subunits exhibited a naltrexone-sensitive enhancement of baseline latency in both the hot-plate and warm-water tail-withdrawal tests. In the hot-plate test, a measure of supraspinal nociception, morphine antinociception was increased, and this was associated with an increased ability of opioids to inhibit presynaptic GABA neurotransmission in the periaqueductal gray. In contrast, antinociception produced by either morphine or methadone was reduced in the tail-withdrawal test, a measure of spinal nociception. In whole-brain and spinal cord homogenates from mice expressing Gαo RGSi subunits, there was a small loss of Gαo expression and an accompanying decrease in basal G-protein activity. Our results strongly support a role for RGS proteins as negative regulators of opioid supraspinal antinociception and also reveal a potential novel function of RGS proteins as positive regulators of opioid spinal antinociceptive pathways.

Hippocampal Adult Neurogenesis Is Maintained by Neil3-Dependent Repair of Oxidative DNA Lesions in Neural Progenitor Cells

Accumulation of oxidative DNA damage has been proposed as a potential cause of age-related cognitive decline. The major pathway for removal of oxidative DNA base lesions is base excision repair, which is initiated by DNA glycosylases. In mice, Neil3 is the main DNA glycosylase for repair of hydantoin lesions in single-stranded DNA of neural stem/progenitor cells, promoting neurogenesis. Adult neurogenesis is crucial for maintenance of hippocampus-dependent functions involved in behavior. Herein, behavioral studies reveal learning and memory deficits and reduced anxiety-like behavior in Neil3(-/-) mice. Neural stem/progenitor cells from aged Neil3(-/-) mice show impaired proliferative capacity and reduced DNA repair activity. Furthermore, hippocampal neurons in Neil3(-/-) mice display synaptic irregularities. It appears that Neil3-dependent repair of oxidative DNA damage in neural stem/progenitor cells is required for maintenance of adult neurogenesis to counteract the age-associated deterioration of cognitive performance.

Visual social preferences of lone zebrafish in a novel environment: strain and anxiolytic effects

Zebrafish (Danio rerio) have an innate tendency to join shoals. Based on this, we refined visual choice tests to focus on social interaction and novelty preference. Our design follows mouse three-chamber sociability protocols, except testing is conducted under 940 Lux fluorescent lighting. Initially, we compared performance among zebrafish strains: inbred (AB) or wild-crossbred (WIK) from Zebrafish International Resource Center, to golden and short-fin from Petco stores. AB fish exhibited a preference for shoaling; they dwelled longest near transparent boxes containing zebrafish, while short fin favored blue boxes without fish. AB and golden exhibited a strong preference for social novelty, not evident in short-fin or WIK fish. Serotonin and cannabinoids shape mammalian social behavior, and equivalents of both receptor types are expressed in the zebrafish brain. We examined the effects of the cannabinoid receptor agonist WIN 55,212-2 (1 mg/l), or serotonin 5-HT(1A) receptor agonist buspirone (10 mg/l) on Petco short-fin social choice. Fish were bath exposed to test compounds for 10 min, under these conditions [(3) H]CP55,940 (4 nm) bound to brain with a concentration of 1.9-6.4 fmol/mg 5-30 min afterward. Social approach was measured 20 min after acclimation to the test arena. WIN 55,212-2 and buspirone increased dwelling near boxed zebrafish. In zebrafish whole-brain homogenates, buspirone displaced [(3) H] 8-hydroxy-N,N-dipropylaminotetralin (dissociation constant, K(D) = 16 ± 1.2 nm) with an inhibition constant (K(i) ) of 1.8 ± 1.0 nm lower than that of WAY 100,635 (K(i) ∼1000 nm). These fish social choice tests may enhance social behavior research, and are useful for studying the effects of genetic manipulations, pharmaceuticals or environmental toxins.

Brain antioxidant markers, cognitive performance and acetylcholinesterase activity of rats: efficiency of Sonchus asper.

BackgroundSonchus asper (SA) is traditionally used as a folk medicine to treat mental disorders in Pakistan. The aim of this study was to investigate the effect of polyphenolic rich methanolic fraction of SA on cognitive performance, brain antioxidant activities and acetylcholinesterase activity in male rats.Methods30 male Sprague–Dawley rats were equally divided into three groups in this study. Animals of group I (control) received saline (vehicle), group II received SA (50 mg/kg) body weight (b.w.), and group III treated with SA (100 mg/kg b.w.,) orally in dimethyl sulphoxide (DMSO) for 7 days. The effect of SA was checked on rat cognitive performance, brain antioxidatant and acetylcholinesterase activities. Evaluation of learning and memory was assessed by a step-through a passive avoidance test on day 6 after two habituation trials and an initial acquisition trial on day 5. Antioxidant potential was determined by measuring activities of superoxide dismutase (SOD), catalase (CAT), contents of thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) in whole-brain homogenates. Acetylcholinesterase (AChE) activity was determined by the colorimetric method.ResultsResults showed that 100 mg/kg b.w., SA treated rats exhibited a significant improvement in learning and memory (step-through latency time). SA administration reduced lipid peroxidation products and elevated glutathione levels in the SA100-treated group. Furthermore, salt and detergent soluble AChE activity was significantly decreased in both SA-treated groups. Short-term orally supplementation of SA showed significant cognitive enhancement as well as elevated brain antioxidant enzymes and inhibited AChE activity.ConclusionThese findings stress the critical impact of Sonchus asper bioactive components on brain function.

Temperature elevation increases GABAA-mediated cortical inhibition in a mouse model of genetic epilepsy

A missense mutation (R43Q) in the γ2 subunit of the γ-aminobutyric acid (GABA)(A) receptor is associated with generalized (genetic) epilepsy with febrile seizures plus (GEFS+). Heterozygous GABA(A) γ2(R43Q) mice displayed a lower temperature threshold for thermal seizures as compared to wild-type littermates. Temperature-dependent internalization of GABA(A) γ2(R43Q)-containing receptors has been proposed as a mechanism underlying febrile seizure genesis in patients with this mutation. We tested this idea using the GABA(A) γ2(R43Q) knockin mouse model and analyzed GABAergic miniature postsynaptic inhibitory currents (mIPSCs) in acute brain slices after exposure to varying temperatures. Incubation of slices at an elevated temperature increased mIPSC amplitude in neurons from heterozygous mice, with no change seen in wild-type controls. [³H]Flumazenil binding measured in whole-brain homogenates from mutant and control mice following elevation of body temperature showed no temperature-dependent differences in γ2-containing receptor density. Therefore, in vivo mouse data do not support earlier in vitro observations that proposed temperature-dependent internalization of γ2 R43Q containing GABA(A) receptors as the cellular mechanism underlying febrile seizure genesis in patients with the GABA(A) γ2(R43Q) mutation.

Depletion of glutathione does not affect electron transport chain complex activity in brain mitochondria: Implications for Parkinson disease and postmortem studies

Glutathione is an important antioxidant in the brain that appears to be decreased, in conjunction with mitochondrial complex I activity, in Parkinson disease patients. In postmortem analysis, measurement of glutathione levels and complex I activity can be delayed up to 20h. We investigated whether depletion of glutathione in the preweanling rat induces a reduction in complex I activity in brain mitochondria and the effects that postmortem delay has on glutathione levels and electron transport chain activity. After injection with the glutamate-cysteine ligase inhibitor, buthionine sulfoximine (L-BSO), glutathione levels were decreased by 53% compared to the control values in whole-brain homogenates. During postmortem delay of 24h, in which animals were kept at 4°C, the levels of glutathione decreased in the control group by 58% and in the L-BSO-treated group by 79%. However, during this period, there were no changes in mitochondrial electron transport chain complex I, II–III, or IV activity in either group. These results suggest that a preexisting deficiency of glutathione or a loss of glutathione during postmortem delay does not influence mitochondrial respiratory chain activity in the brain.

The Brain Renin-Angiotensin System Controls Divergent Efferent Mechanisms to Regulate Fluid and Energy Balance

The renin-angiotensin system (RAS), in addition to its endocrine functions, plays a role within individual tissues such as the brain. The brain RAS is thought to control blood pressure through effects on fluid intake, vasopressin release, and sympathetic nerve activity (SNA), and may regulate metabolism through mechanisms which remain undefined. We used a double-transgenic mouse model that exhibits brain-specific RAS activity to examine mechanisms contributing to fluid and energy homeostasis. The mice exhibit high fluid turnover through increased adrenal steroids, which is corrected by adrenalectomy and attenuated by mineralocorticoid receptor blockade. They are also hyperphagic but lean because of a marked increase in body temperature and metabolic rate, mediated by increased SNA and suppression of the circulating RAS. β-adrenergic blockade or restoration of circulating angiotensin-II, but not adrenalectomy, normalized metabolic rate. Our data point to contrasting mechanisms by which the brain RAS regulates fluid intake and energy expenditure.

(R,S)-2-Iodo-reboxetine: Development as a novel SPECT radiotracer for imaging the noradrenaline transporter in brain

Introduction: Dysregulation of noradrenergic function has been implicated in multiple brain disorders, including: depression, post-traumatic stress, anxiety, attention-deficit/hyperactivity disorder, Alzheimer's disease and Parkinson's disease. Despite this, few attempts have been made to develop radiotracers for imaging the noradrenaline transporter (NAT) in vivo using either positron emission tomography or single photon emission computerised tomography (SPECT). We set out to develop a novel SPECT radiotracer, based on the selective noradrenaline re-uptake inhibitor, reboxetine, for imaging the NAT in brain. Methods: The lead candidate, (R,S)-2-iodo-reboxetine [1], was radiolabelled via electrophillic iododestannylation of the corresponding tin precursor using peracetic acid as an oxidant. In vitro binding assays, in vitro and ex vivo brain autoradiography, in vivo biodistribution and pharmacokinetics studies using whole body dynamic planar imaging and ex vivo displacement studies, were performed in rats in order to evaluate radiolabelled (R,S)-2-iodo-reboxetine as a NAT SPECT radiotracer. Results: (R,S)-2-Iodo-reboxetine against 3H-Nisoxetine has nanomolar affinity for the NAT in homogenates of whole rat brain (Ki = 8.4 ± 1.7 nM, mean ± SEM, n = 5). 123I and 125I radiolabelling of (R,S)-2-iodo-reboxetine was successfully accomplished. The percentage non specific binding (of total binding), as measured by in vitro autoradiography with 123I-(R,S)-2-iodo-reboxetine in rat brain sections (mean ± SEM, n = 3), was 26 ± 2% in the locus coeruleus, 97 ± 3% in the corpus callosum, 98 ± 2% in the caudate putamen, 97 ± 2% in the hippocampus, 87 ± 6% in the anterior thalamus, 75 ± 2% in the raphe and 85 ± 8% in the cerebral cortex. Ex vivo autoradiography showed a specific to non-specific binding ratio (locus coeruleus:striatum) of 2.7. In vivo brain uptake was 2.96 ± 0.33% (mean ± SEM, n = 4) of the injected dose, as measured by whole body dynamic planar imaging. The elimination route of 123I-(R,S)-2-iodo-reboxetine, determined by both in vivo whole body dynamic planar imaging and ex vivo studies, was mainly intestinal. Conclusion: The data indicate that 123I-(R,S)-2-iodo-reboxetine may have potential as a radiotracer for imaging the NAT in brain.