Efficient clearance of Aß protofibrils in AßPP-transgenic mice treated with a brain-penetrating bifunctional antibody.

BACKGROUND: Amyloid-ß (Aß) immunotherapy is one of the most promising disease-modifying strategies for Alzheimer's disease (AD). Despite recent progress targeting aggregated forms of Aß, low antibody brain penetrance remains a challenge. In the present study, we used transferrin receptor (TfR)-mediated transcytosis to facilitate brain uptake of our previously developed Aß protofibril-selective mAb158, with the aim of increasing the efficacy of immunotherapy directed toward soluble Aß protofibrils. METHODS: Aß protein precursor (AßPP)-transgenic mice (tg-ArcSwe) were given a single dose of mAb158, modified for TfR-mediated transcytosis (RmAb158-scFv8D3), in comparison with an equimolar dose or a tenfold higher dose of unmodified recombinant mAb158 (RmAb158). Soluble Aß protofibrils and total Aß in the brain were measured by enzyme-linked immunosorbent assay (ELISA). Brain distribution of radiolabeled antibodies was visualized by positron emission tomography (PET) and ex vivo autoradiography. RESULTS: ELISA analysis of Tris-buffered saline brain extracts demonstrated a 40% reduction of soluble Aß protofibrils in both RmAb158-scFv8D3- and high-dose RmAb158-treated mice, whereas there was no Aß protofibril reduction in mice treated with a low dose of RmAb158. Further, ex vivo autoradiography and PET imaging revealed different brain distribution patterns of RmAb158-scFv8D3 and RmAb158, suggesting that these antibodies may affect Aß levels by different mechanisms. CONCLUSIONS: With a combination of biochemical and imaging analyses, this study demonstrates that antibodies engineered to be transported across the blood-brain barrier can be used to increase the efficacy of Aß immunotherapy. This strategy may allow for decreased antibody doses and thereby reduced side effects and treatment costs.

Posterior cerebral artery stenosis and posterior circulation revascularization surgery in pediatric patients with moyamoya disease.

OBJECTIVE Some pediatric patients with moyamoya disease (MMD) present with posterior cerebral artery (PCA) stenosis before and after anterior circulation revascularization surgery and require posterior circulation revascularization surgery. This study evaluated the factors associated with PCA stenosis and assessed the efficacy of posterior circulation revascularization surgery, including occipital artery (OA)-PCA bypass, in pediatric patients with MMD. METHODS The presence of PCA stenosis before and after anterior circulation revascularization surgery and its clinical characteristics were investigated in 62 pediatric patients (< 16 years of age) with MMD. RESULTS Twenty-three pediatric patients (37%) with MMD presented with PCA stenosis at the time of the initial diagnosis. A strong correlation between the presence of infarction and PCA stenosis before anterior revascularization was observed (p < 0.001). In addition, progressive PCA stenosis was observed in 12 patients (19.4%) after anterior revascularization. The presence of infarction and a younger age at the time of initial diagnosis were risk factors for progressive PCA stenosis after anterior revascularization (p < 0.001 and p = 0.002, respectively). Posterior circulation revascularization surgery, including OA-PCA bypass, was performed in 9 of the 12 patients with progressive PCA stenosis, all of whom showed symptomatic and/or radiological improvement. CONCLUSIONS PCA stenosis is an important clinical factor related to poor prognosis in pediatric MMD. One should be aware of the possibility of progressive PCA stenosis during the postoperative follow-up period and consider performing posterior circulation revascularization surgery.

Shaker-related voltage-gated potassium channels Kv1 in human hippocampus.

In this study, we investigated the tissue expression levels, alpha subunit composition and distribution of Shaker-related voltage-dependent potassium Kv1 channels in human hippocampus by combining western blotting experiments, toxin autoradiography, in vivo radioligand binding studies, immunoprecipitation and immunohistochemistry. Tissue expression of Kv1.1 and Kv1.2 α-subunits in human post-mortem brain tissue was confirmed in immunoblot analysis using a panel of specific monoclonal and polyclonal antibodies. Immunoprecipitation experiments using toxin-prelabeled Kv1 channels revealed that all toxin-sensitive Kv1 channels in human hippocampus contained either a Kv1.1 or Kv1.2 α-subunit with the majority being composed of Kv1.1/Kv1.2 heterotetramers. Receptor autoradiography suggested Kv1.1/Kv1.2 channel expression in the molecular layer of dentate gyrus. In accordance, immunohistochemical experiments also observed Kv1.1 and Kv1.2 α-subunits in the molecular layer of the dentate gyrus, in addition to the CA3 stratum lucidum and the CA1 stratum oriens. These findings indicate expression in axons and terminals of hippocampal pathways, namely the perforant path, the mossy fiber pathway and the Schaffer collaterals. Herein we present the first direct demonstration that Kv1.1 and Kv1.2 channel proteins are targeted to distinct compartments of the human hippocampal formation and that this expression pattern largely reflects their distribution profile in murine brain.

Distribution of corticotropin-releasing factor (CRF) receptor binding in the mouse brain using a new, high-affinity radioligand, [125 I]-PD-Sauvagine.

The corticotropin-releasing factor (CRF) family of peptides includes CRF and three urocortins, which signal through two distinct G-protein coupled receptors, CRF1 and CRF2 . Although the cellular distribution of CRF receptor expression has been well characterized at the mRNA level, the localization of receptor protein, and, by inference, of functional receptors, has been limited by a lack of reliable immunohistochemical evidence. Recently, a CRF-related peptide, termed PD-sauvagine, was isolated from the skin of the frog, Pachymedusa dacnicolor, and validated as a high-affinity ligand for CRF receptor studies. A radiolabeled analog, [125 I]-PD-sauvagine, with high signal-to-noise ratio, was used in autoradiographic studies to map the distribution of CRF receptor binding sites in the mouse brain. Through the use of receptor-deficient mice and subtype-specific antagonists, CRF1 and CRF2 binding sites were isolated, and found to be readily reconcilable with regional patterns of mRNA expression. Binding site distributions within a given structure sometimes differed from mRNA patterns, however, particularly in laminated structures of the isocortex, hippocampus, and cerebellum, presumably reflecting the trafficking of receptors to their operational homes on neuronal (mostly dendritic) processes. Binding patterns of [125 I]-PD-sauvagine provided independent assessments of controversial receptor localizations, failing to provide support for CRF1 expression in central autonomic components of the limbic forebrain, the locus coeruleus and cerebellar Purkinje cells, or for CRF2 in any aspect of the cerebellar cortex. Though lacking in ideal resolution, in vitro binding of the PD-sauvagine radioligand currently provides the most sensitive and accurate available tool for localizing CRF receptors in rodent brain.

Anterior cingulate serotonin 1B receptor binding is associated with emotional response inhibition.

Serotonin has a well-established role in emotional processing and is a key neurotransmitter in impulsive aggression, presumably by facilitating response inhibition and regulating subcortical reactivity to aversive stimuli. In this study 44 men, of whom 19 were violent offenders and 25 were non-offender controls, completed an emotional Go/NoGo task requiring inhibition of prepotent motor responses to emotional facial expressions. We also measured cerebral serotonin 1B receptor (5-HT1BR) binding with [11C]AZ10419369 positron emission tomography within regions of the frontal cortex. We hypothesized that 5-HT1BR would be positively associated with false alarms (failures to inhibit nogo responses) in the context of aversive (angry and fearful) facial expressions. Across groups, we found that frontal cortex 5-HT1BR binding was positively correlated with false alarms when angry faces were go stimuli and neutral faces were nogo stimuli (p = 0.05, corrected alpha = 0.0125), but not with false alarms for non-emotional stimuli (failures to inhibit geometric figures). A posthoc analysis revealed the strongest association in anterior cingulate cortex (p = 0.006). In summary, 5-HT1BRs in the anterior cingulate are involved in withholding a prepotent response in the context of angry faces. Our findings suggest that serotonin modulates response inhibition in the context of certain emotional stimuli.

Combined in vitro and in silico approaches to the assessment of stimulant properties of novel psychoactive substances - The case of the benzofuran 5-MAPB.

Novel psychoactive substances (NPS) are increasingly prevalent world-wide although their pharmacological characteristics are largely unknown; those with stimulant properties, due to interactions with the dopamine transporter (DAT), have addictive potential which their users may not realise. We evaluated the binding of 1-(1-benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) to rat striatal DAT by means of quantitative autoradiography with [125I]RTI-121, and the effects of 5-MAPB on electrically-evoked dopamine efflux by fast-cyclic voltammetry in rat brain slices. 5-MAPB displaced [125I]RTI-121 in a concentration-dependent manner, with significant effects at 10 and 30µM. The voltammetry data suggest that 5-MAPB reduces the rate of dopamine reuptake; while the peak dopamine efflux was not increased, the area under the curve was augmented. 5-MAPB can also cause reverse dopamine transport consistent with stimulant properties, more similar to amphetamine than cocaine. Molecular modelling and docking studies compared the binding site of DAT in complex with 5-MAPB to dopamine, amphetamine, 5-APB, MDMA, cocaine and RTI-121. This structural comparison reveals a binding mode for 5-MAPB found in the primary binding (S1) site, central to transmembrane domains 1, 3, 6 and 8, which overlaps with the binding modes of dopamine, cocaine and its analogues. Atomistic molecular dynamics simulations further show that, when in complex with 5-MAPB, DAT can exhibit conformational transitions that spontaneously isomerize the transporter into inward-facing state, similarly to that observed in dopamine-bound DAT. These novel insights, offered by the combination of computational methods of biophysics with neurobiological procedures, provide structural context for NPS at DAT and relate them with their functional properties at DAT as the molecular target of stimulants.

A Lack of Serotonin 1B Autoreceptors Results in Decreased Anxiety and Depression-Related Behaviors.

The effects of serotonin (5-HT) on anxiety and depression are mediated by a number of 5-HT receptors, including autoreceptors that act to inhibit 5-HT release. While the majority of anxiety and depression-related research has focused on the 5-HT1A receptor, the 5-HT1B receptor has a lesser known role in modulating emotional behavior. 5-HT1B receptors are inhibitory GPCRs located on the presynaptic terminal of both serotonin and non-serotonin neurons, where they act to inhibit neurotransmitter release. The autoreceptor population located on the axon terminals of 5-HT neurons is a difficult population to study due to their diffuse localization throughout the brain that overlaps with 5-HT1B heteroreceptors (receptors located on non-serotonergic neurons). In order to study the contribution of 5-HT1B autoreceptors to anxiety and depression-related behaviors, we developed a genetic mouse model that allows for selective ablation of 5-HT1B autoreceptors. Mice lacking 5-HT1B autoreceptors displayed the expected increases in extracellular serotonin levels in the ventral hippocampus following administration of a selective serotonin reuptake inhibitor. In behavioral studies, they displayed decreased anxiety-like behavior in the open field and antidepressant-like effects in the forced swim and sucrose preference tests. These results suggest that strategies aimed at blocking 5-HT1B autoreceptors may be useful for the treatment of anxiety and depression.

Antidiabetic drugs restore abnormal transport of amyloid-ß across the blood-brain barrier and memory impairment in db/db mice.

Previous studies have shown significant changes in amyloid-ß (Aß) transport across the blood-brain barrier (BBB) under diabetic conditions with hypoinsulinemia, which is involved in diabetes-associated cognitive impairment. Present study employed db/db mice with hyperinsulinemia to investigate changes in Aß transport across the BBB, hippocampal synaptic plasticity, and restorative effects of antidiabetic drugs. Our results showed that db/db mice exhibited similar changes in Aß transport across the BBB to that of insulin-deficient mice. Chronic treatment of db/db mice with antidiabetic drugs such as metformin, glibenclamide and insulin glargine significantly decreased Aß influx across the BBB determined by intra-arterial infusion of (125)I-Aß(1-40), and expression of the receptor for advanced glycation end products (RAGE) participating in Aß influx. Insulin glargine, but not, metformin or glibenclamide increased Aß efflux across the BBB determined by stereotaxic intra-cerebral infusion of (125)I-Aß(1-40), and expression of the low-density lipoprotein receptor related protein 1 (LRP1) participating in Aß efflux. Moreover, treatment with these drugs significantly decreased hippocampal Aß(1-40) or Aß(1-42) and inhibited neuronal apoptosis. The drugs also ameliorated memory impairment confirmed by improved performance on behavioral tasks. However, insulin glargine or glibenclamide, but not metformin, restored hippocampal synaptic plasticity characterized by enhancing in vivo long-term potentiation (LTP). Further study found that these three drugs significantly restrained NF-κB, but only insulin glargine enhanced peroxisome proliferator-activated receptor γ (PPARγ) activity at the BBB in db/db mice. Our data indicate that the antidiabetic drugs can partially restore abnormal Aß transport across the BBB and memory impairment under diabetic context.

Sex, social status, and CRF receptor densities in naked mole-rats.

Naked mole-rats (Heterocephalus glaber) live in groups that are notable for their large size and caste structure, with breeding monopolized by a single female and a small number of males. Recent studies have demonstrated substantial differences between the brains of breeders and subordinates induced by changes in social standing. Corticotropin-releasing factor (CRF) receptors-which bind the hormone CRF as well as related peptides-are important regulators of stress and anxiety, and are emerging as factors affecting social behavior. We conducted autoradiographic analyses of CRF1 and CRF2 receptor binding densities in female and male naked mole-rats varying in breeding status. Both globally and in specific brain regions, CRF1 receptor densities varied with breeding status. CRF1 receptor densities were higher in subordinates across brain regions, and particularly in the piriform cortex and cortical amygdala. Sex differences were present in CRF2 receptor binding densities, as is the case in multiple vole species. CRF2 receptor densities were higher in females, both globally and in the cortical amygdala and lateral amygdalar nucleus. These results provide novel insights into the neurobiology of social hierarchy in naked mole-rats, and add to a growing body of work that links changes in the CRF system with social behavior.

Clinical characteristics of idiopathic normal pressure hydrocephalus with Lewy body diseases.

BACKGROUND: Comorbidity of idiopathic normal pressure hydrocephalus and neurodegenerative diseases presents a diagnostic challenge. The aim of this study was to elucidate the clinical features of iNPH patients who are possibly comorbid with Lewy body diseases. METHODS: In this study, we retrospectively analyzed the records of consecutive 127 patients with definite idiopathic normal pressure hydrocephalus. (123)I-Metaiodobenzylguanidine myocardial scintigraphy was undertaken to 21 patients with idiopathic normal pressure hydrocephalus because of suspicion for comorbid Lewy body diseases. RESULTS: As a result, 7 of the 21 patients with idiopathic normal pressure hydrocephalus exhibited cardiac sympathetic abnormality. Idiopathic normal pressure hydrocephalus patients with cardiac sympathetic dysfunction had the constellation of clinical findings, such as younger onset, less severe urinary dysfunction, kinesie paradoxale, and cogwheel rigidity. CONCLUSIONS: Our results suggested that comorbidity of idiopathic normal pressure hydrocephalus and Lewy body diseases are not rare condition and careful screening for potentially-curative surgery is important especially in atypical cases.

Region-specific associations between sex, social status, and oxytocin receptor density in the brains of eusocial rodents.

Naturally occurring variations in neuropeptide receptor distributions in the brain contribute to numerous mammalian social behaviors. In naked mole-rats, which live in large social groups and exhibit remarkable reproductive skew, colony-related social behaviors vary with reproductive status. Here we examined whether variation in social status is associated with variations in the location and/or density of oxytocin binding in this species. Autoradiography was performed to assess forebrain oxytocin receptor (OTR) densities in breeding and non-breeding naked mole-rats of both sexes. Overall, males exhibited higher OTR binding in the medial amygdala in comparison to females. While there were no main effects of reproductive status in any region, a sex difference in OTR binding in the nucleus accumbens was mediated by status. Specifically, breeding males tended to have more OTR binding than breeding females in the nucleus accumbens, while no sex difference was observed in subordinates. These effects suggest that oxytocin may act in a sex- and region-specific way that corresponds to reproductive status and associated social behaviors.

Intranasal administration of glial-derived neurotrophic factor (GDNF) rapidly and significantly increases whole-brain GDNF level in rats.

Previous studies have shown that glial cell line-derived neurotrophic factor (GDNF) exerts significant neuroprotective effects on substantia nigra (SN) neurons in the rat 6-hydroxydopamine (6-OHDA) model of Parkinson's disease (PD). In this study we used enzyme-linked immunosorbent assay (ELISA) to determine GDNF brain levels and distribution to target regions (i.e. striatum and SN) following intranasal administration of GDNF at different time points after administration. Brain levels increased significantly within 1h following a single 50-µg dose of GDNF in a liposomal formulation, returning to baseline by 24h. In a second study, different doses of GDNF (10-150 µg) in phosphate-buffered saline (PBS) were studied at the 1-h time point. Dose-dependent increases in brain GDNF levels were observed with apparent saturation of uptake at doses above 100 µg. Liposomes delivered 10-fold more GDNF to brain than PBS despite yielding similar neuroprotective efficacy in the 6-OHDA model, suggesting incomplete release of GDNF from liposomes in tissue. In a third study, autoradiography was performed on brain sections taken 1h after intranasal (125)I-labeled GDNF. Radioactivity was detected throughout the brain along the rostral-to-caudal axis, indicating that nasally administered GDNF can reach target areas. Collectively, these results demonstrate that intranasal administration of GDNF in liposomes or PBS achieves significant increases in GDNF in target brain areas, supporting use of intranasal administration as a non-invasive means of delivering GDNF to the brain to protect dopamine neurons and arrest disease progression in PD.

Thyroid hormone level is associated with motor symptoms in de novo Parkinson's disease.

Sympathetic denervation has been observed not only in the myocardium but also in the thyroid of patients with Parkinson's disease (PD). We investigated whether sympathetic denervation as indicated by decreased cardiac (123)I-meta-iodobenzylguanidine uptake is associated with the levels of thyroid hormones and whether the levels of thyroid hormones affect clinical manifestations in patients with PD. The subjects were 75 patients with de novo PD and 20 age-matched healthy controls. We examined the levels of thyroid-stimulating hormone, free triiodothyronine, and free thyroxine, and evaluated the associations of these levels with cardiac (123)I-meta-iodobenzylguanidine uptake and motor symptoms. The results showed that the free triiodothyronine level was below the normal range in 29 patients (approximately 40 %) and was significantly lower in the patients with PD than in the controls. The decreased free triiodothyronine level was associated with akinetic-rigid motor subtype and washout ratio of cardiac (123)I-meta-iodobenzylguanidine scintigraphy. The free triiodothyronine level negatively correlated with disease severity. Thyroid-stimulating hormone level was within normal range. However, its level was lower in patients with tremor-dominant type or mixed type than in those with akinetic-rigid type. All correlations of these variables with the levels of thyroid hormones remained statistically significant on multiple regression analysis. Our results suggest that the thyroid hormone level, especially the free triiodothyronine level, is closely related to motor symptoms in patients with de novo PD. Further studies are needed to clarify whether the decreased hormone levels have functional roles in motor and non-motor symptoms.

Distinct Circuits Underlie the Effects of 5-HT1B Receptors on Aggression and Impulsivity.

Impulsive and aggressive behaviors are both modulated by serotonergic signaling, specifically through the serotonin 1B receptor (5-HT1BR). 5-HT1BR knockout mice show increased aggression and impulsivity, and 5-HT1BR polymorphisms are associated with aggression and drug addiction in humans. To dissect the mechanisms by which the 5-HT1BR affects these phenotypes, we developed a mouse model to spatially and temporally regulate 5-HT1BR expression. Our results demonstrate that forebrain 5-HT1B heteroreceptors expressed during an early postnatal period contribute to the development of the neural systems underlying adult aggression. However, distinct heteroreceptors acting during adulthood are involved in mediating impulsivity. Correlating with the impulsivity, dopamine in the nucleus accumbens is elevated in the absence of 5-HT1BRs and normalized following adult rescue of the receptor. Overall, these data show that while adolescent expression of 5-HT1BRs influences aggressive behavior, a distinct set of 5-HT1B receptors modulates impulsive behavior during adulthood.

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

Experimental evaluation of radioiodinated sennoside B as a necrosis-avid tracer agent.

Necrosis-avid agents are a class of compounds that selectively accumulate in the necrotic tissues after systemic administration, which can be used for in vivo necrosis imaging and targeted therapies. In order to search for a necrosis-avid tracer agent with improved drugability, we labelled iodine-131 on sennoside B (SB) as a naturally occurring median dianthrone compound. The necrosis targetability and clearance properties of (131)I-SB were evaluated in model rats with liver and muscle necrosis. On SPECT/CT images, a "hot spot" in the infarcted liver lobe and necrotic muscle was persistently observed at 24 h and 72 h post-injection (p.i.). Gamma counting of the tissues of interest revealed a radioactivity ratio of necrotic to viable liver at 4.6 and 3.4 and of necrotic to viable muscle at 7.0 and 8.8 at 24 h and 72 h p.i., respectively. The good match of autoradiographs and fluoromicroscopic images with corresponding histochemical staining suggested preferential uptake of (131)I-SB in necrotic tissue. Pharmacokinetic study revealed that (131)I-SB has an elimination half-life of 8.6 h. This study indicates that (131)I-SB shows not only prominent necrosis avidity but also favourable pharmacokinetics, which may serve as a potential necrosis-avid diagnostic agent for assessment of tissue viability.

Distribution of non-AT1, non-AT2 binding of 125I-sarcosine1, isoleucine8 angiotensin II in neurolysin knockout mouse brains.

The recent identification of a novel binding site for angiotensin (Ang) II as the peptidase neurolysin (E.C. 3.4.24.16) has implications for the renin-angiotensin system (RAS). This report describes the distribution of specific binding of 125I-Sarcosine1, Isoleucine8 Ang II (125I-SI Ang II) in neurolysin knockout mouse brains compared to wild-type mouse brains using quantitative receptor autoradiography. In the presence of p-chloromercuribenzoic acid (PCMB), which unmasks the novel binding site, widespread distribution of specific (3 µM Ang II displaceable) 125I-SI Ang II binding in 32 mouse brain regions was observed. Highest levels of binding >700 fmol/g initial wet weight were seen in hypothalamic, thalamic and septal regions, while the lowest level of binding <300 fmol/g initial wet weight was in the mediolateral medulla. 125I-SI Ang II binding was substantially higher by an average of 85% in wild-type mouse brains compared to neurolysin knockout brains, suggesting the presence of an additional non-AT1, non-AT2, non-neurolysin Ang II binding site in the mouse brain. Binding of 125I-SI Ang II to neurolysin in the presence of PCMB was highest in hypothalamic and ventral cortical brain regions, but broadly distributed across all regions surveyed. Non-AT1, non-AT2, non-neurolysin binding was also highest in the hypothalamus but had a different distribution than neurolysin. There was a significant reduction in AT2 receptor binding in the neurolysin knockout brain and a trend towards decreased AT1 receptor binding. In the neurolysin knockout brains, the size of the lateral ventricles was increased by 56% and the size of the mid forebrain (-2.72 to +1.48 relative to Bregma) was increased by 12%. These results confirm the identity of neurolysin as a novel Ang II binding site, suggesting that neurolysin may play a significant role in opposing the pathophysiological actions of the brain RAS and influencing brain morphology.

The effects of benzofury (5-APB) on the dopamine transporter and 5-HT2-dependent vasoconstriction in the rat.

5-APB, commonly marketed as 'benzofury' is a new psychoactive substance and erstwhile 'legal high' which has been implicated in 10 recent drug-related deaths in the UK. This drug was available on the internet and in 'head shops' and was one of the most commonly sold legal highs up until its recent UK temporary ban (UK Home Office). Despite its prominence, very little is known about its pharmacology. This study was undertaken to examine the pharmacology of 5-APB in vitro. We hypothesised that 5-APB would activate the dopamine and 5-HT systems which may underlie its putative stimulant and hallucinogenic effects. Autoradiographic studies showed that 5-APB displaced both [(125)I] RTI-121 and [(3)H] ketanserin from rat brain tissue suggesting affinity at the dopamine transporter and 5-HT2 receptor sites respectively. Voltammetric studies in rat accumbens brain slices revealed that 5-APB slowed dopamine reuptake, and at high concentrations caused reverse transport of dopamine. 5-APB also caused vasoconstriction of rat aorta, an effect antagonised by the 5-HT2A receptor antagonist ketanserin, and caused contraction of rat stomach fundus, which was reversed by the 5-HT2B receptor antagonist RS-127445. These data show that 5-APB interacts with the dopamine transporter and is an agonist at the 5-HT2A and 5-HT2B receptors in the rat. Thus 5-APB's pharmacology is consistent with it having both stimulant and hallucinogenic properties. In addition, 5-APB's activity at the 5-HT2B receptor may cause cardiotoxicity.

Salivary peptide tyrosine-tyrosine 3-36 modulates ingestive behavior without inducing taste aversion.

Hormone peptide tyrosine-tyrosine (PYY) is secreted into circulation from the gut L-endocrine cells in response to food intake, thus inducing satiation during interaction with its preferred receptor, Y2R. Clinical applications of systemically administered PYY for the purpose of reducing body weight were compromised as a result of the common side effect of visceral sickness. We describe here a novel approach of elevating PYY in saliva in mice, which, although reliably inducing strong anorexic responses, does not cause aversive reactions. The augmentation of salivary PYY activated forebrain areas known to mediate feeding, hunger, and satiation while minimally affecting brainstem chemoreceptor zones triggering nausea. By comparing neuronal pathways activated by systemic versus salivary PYY, we identified a metabolic circuit associated with Y2R-positive cells in the oral cavity and extending through brainstem nuclei into hypothalamic satiety centers. The discovery of this alternative circuit that regulates ingestive behavior without inducing taste aversion may open the possibility of a therapeutic application of PYY for the treatment of obesity via direct oral application.

Extracellular pH and neuronal depolarization serve as dynamic switches to rapidly mobilize trkA to the membrane of adult sensory neurons.

Activation of the nerve growth factor (NGF) receptor trkA and tissue acidosis are critically linked to inflammation-associated nociceptor sensitization. This study explored how increased acidity is linked to sensory neuron sensitization to NGF. Adult Wistar rat primary sensory neurons grown at physiological pH 7.4, then either kept at pH 7.4 or challenged for 30 min in pH 6.5 medium, provided a model of acidosis. Nonpermeabilizing trkA immunofluorescence revealed a significant increase in trkA mobilization to the plasma membrane from intracellular stores in response to proton challenge. This was confirmed using a surface protein biotinylation assay and Brefeldin A disruption of the rough endoplasmic reticulum-Golgi-trans-Golgi network. Mobilization of trkA to the membrane at pH 6.5 was abolished in neurons treated with the acid-sensitive ion channel blocker, amiloride. While elevated levels of NGF-independent trkA phosphorylation occurred at pH 6.5 alone, the level of activation was significantly increased in response to NGF challenge. Exposure of sensory neurons to pH 6.5 medium also resulted in strong calcium (Ca(2+)) transients that were reversible upon reintroduction to physiological pH. The pH 6.5-induced mobilization of trkA to the membrane was Ca(2+) dependent, as BAPTA-AM Ca(2+) chelation abrogated the response. Interestingly, KCl-induced depolarization was sufficient to induce mobilization of trkA to the cell surface at pH 7.4, but did not augment the response to pH 6.5. In conclusion, increased mobilization of trkA to neuronal membranes in response to either acidosis or neuronal depolarization provides two novel mechanisms by which sensory neurons can rapidly sensitize to NGF and has important implications for inflammatory pain states.