Binding Sites In Brain Research Articles

Probes for the heterogeneity of muscimol binding sites in rat brain.

Introduction: The plant-based alkaloid muscimol is a potent agonist of inhibitory GABAA-neurotransmitter receptors. GABAA receptors are a heterogeneous family of pentameric complexes, with 5 out of 19 subunits assembling around the central anion pore. Muscimol is considered to bind to all receptor subtypes at the orthosteric drug binding site at the β+/α- interface. Recently, we observed that the antipsychotic drugs clozapine (CLZ), loxapine (LOX) and chlorpromazine (CPZ) although exerting functional inhibition on multiple GABAA receptor subtypes showed diverging results in displacing 3H-muscimol. While a complete displacement could be observed in hippocampal membranes by bicuculline (BIC), and no displacement with CPZ, the compounds CLZ and LOX competed partially. Non-sigmoidal, complex dose response curves were indicative of multiple sites. In the current study we now aimed to investigate more extensively this heterogeneity of bicuculline sensitive muscimol sites in rat brain. Methods: We tested membranes from four different brain regions (hippocampus, cerebellum, thalamus and striatum) and selected recombinantly expressed subunit combinations with displacement assays. 3H-muscimol displacement was tested with BIC, LOX, CLZ and CPZ. In silico ligand structural analysis and computational docking was performed. Results: We observed a unique pharmacology of each tested compound in the studied brain regions. Combining two of the tested ligands suggests that in striatum all CLZ sites are contained in the pool of LOX sites, while the CPZ sites may in part be non-overlapping with LOX sites. Experiments on recombinantly expressed receptors indicate, that BIC can displace 3H-muscimol from all tested receptors, while LOX and CLZ display different and variable competition indicative of multiple sites. Molecular docking produced structural correlates of the observed diversity of muscimol sites on the basis of bicuculline bound experimental structures. Discussion: These findings indicate that 3H-muscimol binding sites in rat brain are heterogeneous, with different populations of receptors, which are CPZ, LOX or CLZ sensitive or insensitive. These binding sites show a varying distribution in different rat brain regions. Molecular docking suggests that the so-called loop F region of α subunits drives the observed differences.

Understanding reactive astrogliosis in degenerative proteinopathies through the lens of postmortem brain imaging techniques

AbstractBackgroundThe discovery and validation of biomarkers beyond amyloid‐β and tau is now considered as a research priority in the Alzheimer’s disease (AD) field. In this context, astrocytes ‘key homeostatic cells’ in the central nervous system (CNS) are potential targets thanks to their swift response in AD pathology. Astrocytes tend to changes in brain physiology after CNS injury/insults via a specific defence response called reactive astrogliosis. The role of astrocytes in AD pathology is still under examination, however recent studies have openly suggested that reactive astrogliosis is an early event which may precede other hallmarks of the disease. In our recent work, we have speculated, based on clinical/translational in vivo and in vitro brain imaging studies, that there are two waves, ‘first (early) and second (late)’, of reactive astrogliosis in AD pathology with a distinct, close‐knit relationship with other pathological markers. Reactive astrogliosis also seems to play a critical role in other proteinopathies, such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). The PET imaging field is advancing very fast and different astrocytic‐tracers are now available, such as 11C‐Deprenyl (DED) and SMBT‐1 (both targeting MAO‐B) as well as 11C‐BU99008 (detect imidazoline2 binding sites), which we believe is of utmost relevance to shed more light on the role of reactive astrogliosis, including astrocytic heterogeneity and improve our understanding of the underlying mechanism in these proteinopathies.MethodWe used a mixed battery of specialized in vitro radioligand binding assays (saturation and competition studies in brain tissues) and large frozen human brain section autoradiography in AD, PSP, CBD and control brains.ResultIn our studies, we demonstrated that BU99008 could visualize reactive astrogliosis and target multiple binding sites in AD brain. We observed that BU99008 and DED varied in their binding behavior in AD and control brains, suggesting that they might be targeting a specific state/or a different sub‐population of astrocytes.ConclusionPostmortem brain imaging techniques are valuable tools for validation (pre‐clinical) of novel PET‐tracers. Our ongoing studies in PSP and CBD brains as well as with SMBT‐1 will provide deeper insight into the role of reactive astrogliosis in these deadly diseases.

Opioids and obstructive sleep apnea.

Freire C, Sennes LU, Polotsky VY. Opioids and obstructive sleep apnea. J Clin Sleep Med. 2022;18(2):647-652.

The Influence of Chromosomal Environment on X-Linked Gene Expression in Drosophila melanogaster.

Sex chromosomes often differ from autosomes with respect to their gene expression and regulation. In Drosophila melanogaster, X-linked genes are dosage compensated by having their expression upregulated in the male soma, a process mediated by the X-chromosome-specific binding of the dosage compensation complex (DCC). Previous studies of X-linked gene expression found a negative correlation between a gene’s male-to-female expression ratio and its distance to the nearest DCC binding site in somatic tissues, including head and brain, which suggests that dosage compensation influences sex-biased gene expression. A limitation of the previous studies, however, was that they focused on endogenous X-linked genes and, thus, could not disentangle the effects of chromosomal position from those of gene-specific regulation. To overcome this limitation, we examined the expression of an exogenous reporter gene inserted at many locations spanning the X chromosome. We observed a negative correlation between the male-to-female expression ratio of the reporter gene and its distance to the nearest DCC binding site in somatic tissues, but not in gonads. A reporter gene’s location relative to a DCC binding site had greater influence on its expression than the local regulatory elements of neighboring endogenous genes, suggesting that intra-chromosomal variation in the strength of dosage compensation is a major determinant of sex-biased gene expression. Average levels of sex-biased expression did not differ between head and brain, but there was greater positional effect variation in the brain, which may explain the observed excess of endogenous sex-biased genes located on the X chromosome in this tissue.

Autoradiographic mapping of synaptic vesicle glycoprotein 2A in non-human primate and human brain.

Synaptic vesicle glycoprotein 2A (SV2A) has been previously characterized as an imaging biomarker for assessment of synaptic density in positron emission tomography (PET) studies of patients with neurological conditions. To provide detailed maps of the brain localization of SV2A autoradiography studies were carried out using the SV2A radioligand [11 C]UCB-J and whole hemisphere sections of non-human primate (NHP) and human brain. Binding of [11 C]UCB-J was observed in all evaluated grey matter structures of the primate brain, with highest density in the caudate nucleus and cortex and lowest density in pons and globus pallidus. The density of [11 C]UCB-J binding sites in human brain showed a good correlation with that in NHP brain. Binding of [11 C]UCB-J in the white matter was very low relative to that in grey matter containing structures and was only inhibited to a minor extent by co-incubation with a saturating concentration of unlabelled UCB-J. The high-resolution images obtained in the present study may aid the interpretation of data acquired in human subjects examined using [11 C]UCB-J in PET studies. In addition, observation of low binding for [11 C]UCB-J in white matter (centrum semiovale) supports that this structure can be used as a reference region for quantitative analysis of [11 C]UCB-J PET data.

In vitro binding affinity vs. in vivo site occupancy: A PET study of four diastereomers of dihydrotetrabenazine (DTBZ) in monkey brain

IntroductionIn vivo imaging methods such as Positron Emission Tomography (PET) can be used to examine the relationship between in vitro binding affinity and in vivo occupancy of binding sites in the brain for new drug candidates. In this study, PET imaging in monkey brain was used to evaluate that correlation for a set of four diastereomers of the compound dihydrotetrabenazine (DTBZ), the pharmacologically active metabolite of the drug tetrabenazine. MethodsPET studies of DTBZ diastereomers were completed in a single monkey brain. In vivo occupancies (ED50) were estimated using multiple drug doses and the vesicular monoamine transporter 2 specific radioligand (+)-α-[11C] DTBZ, employing a test-retest sequence of control PET scan, drug administration and a second PET scan completed on a single day. ResultsDTBZ has three chiral carbon centers and eight possible stereoisomers, and in vivo occupancy of the target site VMAT2 was observed only for the four diastereomers of DTBZ having the 11bR absolute configuration. The estimated in vivo occupancies (ED50 values from 0.023 to >3.15 mg/kg) correlated well (R2 = 0.95) with the in vitro binding affinities (Ki values of 4 to 600 nM for the VMAT2), and an even better correlation (R2 = 0.99) was found for the three isomers with in vitro binding affinities <100 nM. ConclusionsIf the physiochemical (MW, log P, pKa) or physiological (metabolism, transport, protein binding) properties of a set of drug stereoisomers are considered similar, the binding affinities determined from in vitro assays may predict the in vivo occupancies of the target binding site in the monkey brain.

Regional vesicular acetylcholine transporter distribution in human brain: A [18 F]fluoroethoxybenzovesamicol positron emission tomography study.

Prior efforts to image cholinergic projections in human brain in vivo had significant technical limitations. We used the vesicular acetylcholine transporter (VAChT) ligand [18 F]fluoroethoxybenzovesamicol ([18 F]FEOBV) and positron emission tomography to determine the regional distribution of VAChT binding sites in normal human brain. We studied 29 subjects (mean age 47 [range 20-81] years; 18 men; 11 women). [18 F]FEOBV binding was highest in striatum, intermediate in the amygdala, hippocampal formation, thalamus, rostral brainstem, some cerebellar regions, and lower in other regions. Neocortical [18 F]FEOBV binding was inhomogeneous with relatively high binding in insula, BA24, BA25, BA27, BA28, BA34, BA35, pericentral cortex, and lowest in BA17-19. Thalamic [18 F]FEOBV binding was inhomogeneous with greatest binding in the lateral geniculate nuclei and relatively high binding in medial and posterior thalamus. Cerebellar cortical [18 F]FEOBV binding was high in vermis and flocculus, and lower in the lateral cortices. Brainstem [18 F]FEOBV binding was most prominent at the mesopontine junction, likely associated with the pedunculopontine-laterodorsal tegmental complex. Significant [18 F]FEOBV binding was present throughout the brainstem. Some regions, including the striatum, primary sensorimotor cortex, and anterior cingulate cortex exhibited age-related decreases in [18 F]FEOBV binding. These results are consistent with prior studies of cholinergic projections in other species and prior postmortem human studies. There is a distinctive pattern of human neocortical VChAT expression. The patterns of thalamic and cerebellar cortical cholinergic terminal distribution are likely unique to humans. Normal aging is associated with regionally specific reductions in [18 F]FEOBV binding in some cortical regions and the striatum.

中枢神経系におけるセクレチン受容体の発現に関する組織学的研究(平成12年度プロジェクト研究報告(保健学部・保健学研究科),研究助成金による成果紹介)

中枢神経系におけるセクレチン受容体の発現に関する組織学的研究(平成12年度プロジェクト研究報告(保健学部・保健学研究科),研究助成金による成果紹介)

Characterization of [3H] oxymorphone binding sites in mouse brain: Quantitative autoradiography in opioid receptor knockout mice

Oxymorphone, one of oxycodone’s metabolic products, is a potent opioid receptor agonist which is thought to contribute to the analgesic effect of its parent compound and may have high potential abuse liability. Nonetheless, the in vivo pharmacological binding profile of this drug is still unclear. This study uses mice lacking mu (MOP), kappa (KOP) or delta (DOP) opioid receptors as well as mice lacking all three opioid receptors to provide full characterisation of oxymorphone binding sites in the brain. Saturation binding studies using [3H]oxymorphone revealed high affinity binding sites in mouse brain displaying Kd of 1.7nM and Bmax of 147fmol/mg. Furthermore, we performed quantitative autoradiography binding studies using [3H]oxymorphone in mouse brain. The distribution of [3H]oxymorphone binding sites was found to be similar to the selective MOP agonist [3H]DAMGO in the mouse brain. [3H]Oxymorphone binding was completely abolished across the majority of the brain regions in mice lacking MOP as well as in mice lacking all three opioid receptors. DOP and KOP knockout mice retained [3H]oxymorphone binding sites suggesting oxymorphone may not target DOP or KOP. These results confirm that the MOP, and not the DOP or the KOP is the main high affinity binding target for oxymorphone.

Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using 3H-HOCPCA

GHB (γ-hydroxybutyric acid) is a compound endogenous to mammalian brain with high structural resemblance to GABA. GHB possesses nanomolar-micromolar affinity for a unique population of binding sites, but the exact nature of these remains elusive. In this study we utilized the highly selective GHB analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version (3H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations, 3H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that 3H-HOCPCA labels only the high-affinity specific GHB binding site, found in high density in cortical and hippocampal regions. The experiments revealed markedly stronger binding at pH 6.0 (Kd 73.8 nM) compared to pH 7.4 (Kd 2312 nM), as previously reported for other GHB radioligands but similar Bmax values. Using 3H-HOCPCA we analyzed the GHB binding protein profile during mouse brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that 3H-HOCPCA is a highly sensitive radioligand, offering advantages over the commonly used radioligand 3H-NCS-382, and thus a very suitable in vitro tool for qualitative and quantitative autoradiography of the GHB high-affinity site.

Anatomical location of LPA1 activation and LPA phospholipid precursors in rodent and human brain.

Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors: LPA1 -LPA6 . LPA evokes several responses in the CNS, including cortical development and folding, growth of the axonal cone and its retraction process. Those cell processes involve survival, migration, adhesion proliferation, differentiation, and myelination. The anatomical localization of LPA1 is incompletely understood, particularly with regard to LPA binding. Therefore, we have used functional [(35) S]GTPγS autoradiography to verify the anatomical distribution of LPA1 binding sites in adult rodent and human brain. The greatest activity was observed in myelinated areas of the white matter such as corpus callosum, internal capsule and cerebellum. MaLPA1 -null mice (a variant of LPA1 -null) lack [(35) S]GTPγS basal binding in white matter areas, where the LPA1 receptor is expressed at high levels, suggesting a relevant role of the activity of this receptor in the most myelinated brain areas. In addition, phospholipid precursors of LPA were localized by MALDI-IMS in both rodent and human brain slices identifying numerous species of phosphatides and phosphatidylcholines. Both phosphatides and phosphatidylcholines species represent potential LPA precursors. The anatomical distribution of these precursors in rodent and human brain may indicate a metabolic relationship between LPA and LPA1 receptors. Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein-coupled receptors (GPCR), LPA1 to LPA6 . LPA evokes several responses in the central nervous system (CNS), including cortical development and folding, growth of the axonal cone and its retraction process. We used functional [(35) S]GTPγS autoradiography to verify the anatomical distribution of LPA1 -binding sites in adult rodent and human brain. The distribution of LPA1 receptors in rat, mouse and human brains show the highest activity in white matter myelinated areas. The basal and LPA-evoked activities are abolished in MaLPA1 -null mice. The phospholipid precursors of LPA are localized by MALDI-IMS. The anatomical distribution of LPA precursors in rodent and human brain suggests a relationship with functional LPA1 receptors.

Assessment of [125I]WYE-230949 as a novel histamine H3 receptor radiopharmaceutical.

Histamine H3 receptor therapeutics have been proposed for several diseases such as schizophrenia, attention deficit hyperactivity disorder, Alzheimer's disease and obesity. We set out to evaluate the novel compound, [125I]WYE-230949, as a potential radionuclide imaging agent for the histamine H3 receptor in brain. [125I]WYE-230949 had a high in vitro affinity for the rat histamine H3 receptor (Kd of 6.9 nM). The regional distribution of [125I]WYE-230949 binding sites in rat brain, demonstrated by in vitro autoradiography, was consistent with the known distribution of the histamine H3 receptor. Rat brain uptake of intravenously injected [125I]WYE-230949 was low (0.11 %ID/g) and the ratio of specific: non-specific binding was less than 1.4, as determined by ex vivo autoradiography. In plasma, metabolism of [125I]WYE-230949 into a less lipophilic species occurred, such that less than 38% of the parent compound remained 30 minutes after injection. Brain uptake and metabolism of [125I]WYE-230949 were increased and specific binding was reduced in anaesthetised compared to conscious rats. [125I]WYE230949 is not a potential radiotracer for imaging rat histamine H3 receptors in vivo due to low brain uptake, in vivo metabolism of the parent compound and low specific binding.

Dose-dependent, saturable occupancy of the metabotropic glutamate subtype 5 receptor by fenobam as measured with [11 C]ABP688 PET imaging.

Fenobam is a negative allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5) with inverse agonist activity and is expected to contribute to the treatment of neuropsychiatric disorders involving dysfunction of mGluR5 including Fragile X syndrome. This study examined whether [11 C]ABP688, an antagonist PET radioligand, competes with fenobam for the same binding site in the nonhuman primate brain and would allow examination of occupancy-plasma concentration relationships in the evaluation of the drug for target disorders in the human brain. Four paired PET studies with [11 C]ABP688 were performed in baboons at a baseline condition and after intravenous treatment with fenobam at different dose levels (0.3-1.33 mg/kg). Total distribution volume (VT ) and binding potential (BPND ) using the cerebellum as a reference region were obtained by the plasma reference graphical method. Then it was examined whether occupancy follows a dose-dependent, saturating pattern that was predicted by a modified first-order Hill equation in individual regions. Baseline regional VT and BPND values agreed with previously published data. Occupancy showed dose-dependent and saturating patterns in individual regions, reaching >90% occupancy at 1.33 mg/kg dose of fenobam in the majority of regions. To our knowledge, this is the first use of PET to characterize the mGluR5 therapeutic drug fenobam. This study demonstrates a proof of principle for determining the in vivo occupancy of fenobam in primates. The results indicate that [11 C]ABP688 and PET may be useful for examination of occupancy of mGluR5 by fenobam, which should prove to be useful for designing future studies and treatment of human disease states. Synapse 68:565-573, 2014. © 2014 Wiley Periodicals, Inc.

Transcriptome-wide Discovery of microRNA Binding Sites in Human Brain

The orchestration of brain function requires complex gene regulatory networks that are modulated, in part, by microRNAs (miRNAs). These noncoding RNAs associate with argonaute (Ago) proteins in order to direct posttranscriptional gene suppression via base pairing with target transcripts. In order to better understand how miRNAs contribute to human-specialized brain processes and neurological phenotypes, identifying their targets is of paramount importance. Here, we address the latter by profiling Ago2:RNA interactions using HITS-CLIP to generate a transcriptome-wide map of miRNA binding sites in human brain. We uncovered ∼ 7,000 stringent Ago2 binding sites that are highly enriched for conserved sequences corresponding to abundant brain miRNAs. This interactome points to functional miRNA:target pairs across >3,000 genes and represents a valuable resource for accelerating our understanding of miRNA functions in brain. We demonstrate the utility of this map for exploring clinically relevant miRNA binding sites that may facilitate the translation of genetic studies of complex neuropsychiatric diseases into therapeutics.

Chemotherapy-Induced Pseudobulbar Affect: A Novel Case

Chemotherapy-Induced Pseudobulbar Affect: A Novel Case

The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin

A novel, non-AT1, non-AT2 brain binding site for angiotensin peptides that is unmasked by p-chloromercuribenzoate (PCMB) has been identified as a membrane associated variant of neurolysin. The ability of different organic and inorganic oxidative and sulfhydryl reactive agents to unmask or inhibit 125I-Sar1Ile8 angiotensin II (SI-Ang II) binding to this site was presently examined. In tissue membranes from homogenates of rat brain and testis incubated in assay buffer containing losartan (10μM) and PD123319 (10μM) plus 100μM PCMB, 5 of the 39 compounds tested inhibited 125I-SI Ang II binding in brain and testis. Mersalyl acid, mercuric chloride (HgCl2) and silver nitrate (AgNO3) most potently inhibited 125I-SI Ang II binding with IC50s ~1–20μM. This HgCl2 inhibition was independent of any interaction of HgCl2 with angiotensin II (Ang II) based on the lack of effect of HgCl2 on the dipsogenic effects of intracerebroventricularly administered Ang II and 125I-SI Ang II binding to AT1 receptors in the liver. Among sulfhydryl reagents, cysteamine and reduced glutathione (GSH), but not oxidized glutathione (GSSG) up to 1mM, inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis. Thimerosal and 4-hydroxymercuribenzoate moderately inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis at 100μM; however, they also unmasked non-AT1, non-AT2 binding independent of PCMB. 4-Hydroxybenzoic acid did not promote 125 I-SI Ang II binding to this binding site indicating that only specific organomercurial compounds can unmask the binding site. The common denominator for all of these interacting substances is the ability to bind to protein cysteine sulfur. Comparison of cysteines between neurolysin and the closely related enzyme thimet oligopeptidase revealed an unconserved cysteine (cys650, based on the full length variant) in the proposed ligand binding channel (Brown et al., 2001) [45] near the active site of neurolysin. It is proposed that the mercuric ion in PCMB and closely related organomercurial compounds binds to cys650, while the acidic anion forms an ionic bond with a nearby arginine or lysine along the channel to effect a conformational change in neurolysin that promotes Ang II binding.

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Imaging of VMAT2 binding sites in the brain by 18F-AV-133: The effect of a pseudo-carrier

Recently, 9-[(18)F]fluoropropyl-(+)-dihydrotetrabenazine ((18)F-AV-133) was reported as a new vesicular monoamine transporter (VMAT2) imaging agent for diagnosis of Parkinson's disease (PD). To shorten the preparation of (18)F-AV-133 and to make it more widely available, we evaluated a simple, rapid purification with a solid-phase extraction method (SPE) using an Oasis HLB cartridge instead of high pressure liquid chromatography (HPLC). The SPE method produced doses containing a pseudo-carrier, 9-hydroxypropyl-(+)-dihydrotetrabenazine (AV-149). To test the possible side effects of this pseudo-carrier, comparative dynamic PET scans of the brains of normal monkeys (2 each) and uni-laterally 6-OH-dopamine-lesioned PD monkeys (2 each) were performed using (18)F-AV-133 doses prepared by either SPE (containing pseudo-carrier) or HPLC (containing no pseudo-carrier). Autoradiographs of post mortem monkey brain sections were evaluated to confirm the relative (18)F-AV-133 uptake in the PD monkey brains and the effects of the pseudo-carrier on VMAT2 binding. The radiochemical purity of the (18)F-AV-133, whether prepared by SPE or by HPLC, was excellent (>99%). PET scans of normal and PD monkey brains showed an expected reduction of VMAT2 in the lesioned areas of the striatum. It was not affected by the presence of the pseudo-carrier, AV-149 (maximally 250 μg/dose). The reduced uptake in the striatum of the lesioned monkey brains was confirmed by autoradiography. Ex vivo inhibition studies of (18)F-AV-133 binding in rat brains, conducted with increasing amounts of AV-149, suggested that at the highest concentration (3.5mg/kg) the VMAT2 binding in the striatum was only moderately blocked (20% reduction). The pseudo-carrier, AV-149, did not affect the (18)F-AV-133/PET imaging of VMAT2 binding sites in normal or uni-laterally lesioned monkey brains. The new streamlined SPE purification method will enable (18)F-AV-133 to be widely available for routine clinical application in determining changes in monoamine neurons for patient with movement disorders or other psychiatric illnesses.

Autoradiography of non‐AT‐1, non‐AT‐2 125‐I‐SI angiotensin II binding in neurolysin knock‐out and wild‐type mouse brains

Recently a novel, non‐AT1, non‐AT2 binding site for angiotensin II (Ang II) was identified as the metalloendopeptidase neurolysin. To assess the localization of this binding site in brain, 3 mice deficient in neurolysin and 3 wild‐type mouse brains were evaluated for radioligand binding with 125I‐Sar1, Ile8 Ang II (250 pM) in the presence of AT1 and AT2 receptor‐saturating concentrations of losartan and PD123319, and 150 μM p‐chloromercuribenzoate using in vitro autoradiography. Specific (10 μM Ang II displaceable) 125I‐Sar1, Ile8 Ang II binding in wild‐type mouse brains was abundant, with highest levels in the molecular layer of the cerebellum, cerebral cortex, hippocampus, amygdala, caudate‐putamen, hypothalamus, lateral septum, and external plexiform layer of the olfactory bulb. Specific 125I‐Sar1, Ile8 Ang II binding in the neurolysin‐deficient mouse brains was profoundly reduced, however, the extent of reduction was region‐specific. Large decreases were seen in the cerebral cortex, substantia nigra, hippocampus, paraventricular thalamus, lateral septum, nucleus accumbens. Cerebellar cortex and hypothalamus showed moderate reduction in binding. These results confirm neurolysin as the novel non‐AT1, non‐AT2 receptor binding protein, but may reveal additional non‐AT1 non‐AT2 binding sites in the mouse brain. Supported by NHLBI HL‐096357

Nicotine-Induced Upregulation of Native Neuronal Nicotinic Receptors Is Caused by Multiple Mechanisms

Nicotine causes changes in brain nicotinic acetylcholine receptors (nAChRs) during smoking that initiate addiction. Nicotine-induced upregulation is the long-lasting increase in nAChR radioligand binding sites in brain resulting from exposure. The mechanisms causing upregulation are not established. Many different mechanisms have been reported with the assumption that there is a single underlying cause. Using live rat cortical neurons, we examined for the first time how exposure and withdrawal of nicotine shape the kinetics of native α4β2-containing nAChR upregulation in real time. Upregulation kinetics demonstrates that at least two different mechanisms underlie this phenomenon. First, a transient upregulation occurs that rapidly reverses, faster than nAChR degradation, and corresponds to nAChR conformational changes as assayed by conformational-dependent, subunit-specific antibodies. Second, a long-lasting process occurs correlating with increases in nAChR numbers caused by decreased proteasomal subunit degradation. Previous radioligand binding measurements to brain tissue have measured the second process and largely missed the first. We conclude that nicotine-induced upregulation is composed of multiple processes occurring at different rates with different underlying causes.