A neuropeptide Y (NPY)-related peptide is present in the river lamprey CNS

Pancreatic polypeptide (PP)-like material from brain + spinal cord, and retina extracts of Lampetra fluviatilis was studied by HPLC and RIA. The brain + spinal cord extract showed a complex elution profile with multiple peaks of immunoreactivity. The retina extract showed a much simpler pattern with a single significant peak along with a trace of a second peak corresponding to the latest and penultimate peaks in the brain extract. Twenty-one out of 36 residues could be sequenced from the latest eluting peak in the brain extract. This sequence showed 81% identity with porcine neuropeptide (NPY) suggesting that both the brain/spinal cord and retina of the river lamprey contain a peptide homologous to NPY.     

Different Regulation of Atrial ANP Release through Neuropeptide Y2 and Y4 Receptors

Neuropeptide Y (NPY) receptors are present in cardiac membranes. However, its physiological roles in the heart are not clear. The aim of this study was to define the direct effects of pancreatic polypeptide (PP) on atrial dynamics and atrial natriuretic peptide (ANP) release in perfused beating atria. Pancreatic polypeptides, a NPY Y₄ receptor agonist, decreased atrial contractility but was not dose-dependent. The ANP release was stimulated by PP in a dose-dependent manner. GR 23118, a NPY Y₄ receptor agonist, also increased the ANP release and the potency was greater than PP. In contrast, peptide YY (3-36) (PYY), an NPY Y₂ receptor agonist, suppressed the release of ANP with positive inotropy. NPY, an agonist for Y_{1, 2, 5} receptor, did not cause any significant changes. The pretreatment of NPY (18-36), an antagonist for NPY Y₃ receptor, markedly attenuated the stimulation of ANP release by PP but did not affect the suppression of ANP release by PYY. BIIE0246, an antagonist for NPY Y₂ receptor, attenuated the suppression of ANP release by PYY. The responsiveness of atrial contractility to PP or PYY was not affected by either of the antagonists. These results suggest that NPY Y₄ and Y₂ receptor differently regulate the release of atrial ANP.     

Differential effects on [35S]GTPgammaS binding using muscarinic agonists and antagonists in the gerbil brain

In this work, we studied the in vitro G-protein activation induced by muscarinic agonists using [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) autoradiographic methods to characterize the M(2) and M(4) muscarinic subtypes response. Thus, we describe a detailed characterization of the increases in [(35)S]GTPgammaS binding elicited by carbachol (Cch) and oxotremorine (OXO) (binding in the presence minus binding in the absence of agonist) throughout the gerbil brain (Meriones unguiculatus). For both agonists, the strongest stimulations were found in the superficial gray layer of the superior colliculus, the anteroventral and anteromedial thalamic nuclei, the anterior paraventricular thalamic nucleus, and the caudate-putamen. The comparative study using OXO and Cch suggested that OXO is able to detect differences in the response of structures enriched in M(4) muscarinic receptors, showing a lower potency to stimulate these brain areas. Furthermore, using increasing concentrations of selective M(2) (AF-DX 116) and M(1)/M(4) (pirenzepine) antagonists to inhibit specific Cch- or OXO-induced [(35)S]GTPgammaS binding, significant differences were observed in M(2)-enriched structures but not in M(4)-enriched ones such as the caudate-putamen. These data indicate that appropriate muscarinic agonist stimulation, together with selective inhibition of this effect using functional autoradiography, can be used as a tool to unravel the M(2)- and M(4)-muscarinic subtype-mediated response.     

Distribution of [H]kainic acid and binding sites in the rat brain: in vivo and in vitro receptor autoradiography

Brain sections incubated in vitro with a-[³H]kainic acid (KA; spec. act. 62.5 Ci/mmol), reveal a heterogenous distribution of low and high affinity KA binding sites in the brain. The highest density of KA binding sites was localised to the hippocampus CA₃ region and to superficial layers of the entorhinal cortex (3.8 6.0 μCi/g tissue). Intravenous injection of [³H]KA (1 μCi/g) reveals limited overall penetration of [³H]KA across the blood-brain barrier. However, a dense labelling of the hippocampus, entorhinal cortex and lateral septal regions (2.5–3.8 μCi/g tissue) was observed. Behaviourally, these rats exhibited mild limbic seizure activity possibly as a result of a direct action of KA in the hippocampus or entorhinal cortex.     

Quantitative autoradiography of nicotinic [H]acetylcholine binding sites in rat brain

Quantitative autoradiography was used to localize nicotinic [³H]acetylcholine (ACh) binding sites in rat brain. High concentrations of nicotinic [³H]ACh binding sites were observed in the anterior and medial nuclei of the thalamus, the medial habenula and the superficial layer of the superior colliculus. Moderate levels of binding sites were observed in a variety of brain regions such as the frontoparietal cortex and the hippocampus. Low levels of nicotinic ACh sites occurred throughout the hypothalamus and the primary olfactory cortex.     

Neuroanatomical mapping of juvenile rat brain regions with prominent basal signal in [(35)S]GTPgammaS autoradiography

[(35)S]GTPgammaS autoradiography represents a powerful functional approach to detect receptor-dependent G(i/o) protein activity in anatomically defined brain structures. Inherent to this technique, however, is the notable basal signal evident in several brain regions in the absence of receptor stimulation by exogenously added agonist. In the rat brain, much of this basal labelling derives from tonic activation of adenosine A(1) and lysophosphatidic acid LPA(1) receptors in the gray and white matter regions, respectively. Despite the elimination of the two receptor activities, prominent basal [(35)S]GTPgammaS labelling is still evident in discrete brain structures, possibly reflecting regional enrichment of G(i/o) and/or constitutive receptor activity or the presence of still unknown endogenous ligands activating their orphan receptors. Here, the anatomical distribution of the enhanced basal signal was systematically mapped in brain sections of 4-week-old male Wistar rats. Regions with prominent basal [(35)S]GTPgammaS labelling represented neuroanatomically distinct structures, in particular various thalamic and hypothalamic nuclei. For instance, the paraventricular thalamic nucleus, the bed nucleus of the stria terminalis and the subfornical organ were highly labelled, as were the periaqueductal gray and the nucleus of the solitary tract. Pre-treatment with N-ethylmaleimide (NEM), an alkylating agent preventing all known receptor-driven G protein activity in cryostat sections markedly decreased the basal binding in all examined regions. In preliminary screening, selective antagonists for various brain-enriched G(i/o)-coupled receptors failed to suppress the basal signal in any of the studied regions.     

Kinetics of [35S]dATPalphaS interaction with P2Y1 purinoceptor in rat brain membranes

Kinetics of [35S]dATPalphaS (2'deoxyadenosine-5'-[alpha-35S]-thiotriphosphate) interaction with rat brain membrane fragments was studied at 25 degrees C and at radioligand concentrations from 2 to 250 nM. At least two different ways of [35S]dATPalphaS interaction with the membranes were distinguished on the basis of radioligand on-rate. Firstly, the binding sites characterized by 'fast' on-rate can be observed. Secondly, the 'slow' binding sites were kinetically identified and quantified. As in both cases the bound radioligand could be displaced by excess of ATP, all these binding sites can be defined as 'specific sites'. In the 'slow' binding sites isomerization of the receptor-ligand complex was observed, as is typical for interaction of antagonists with G-protein coupled receptors, and the kinetic parameters for this interaction were similar with the appropriate data for the hP2Y1 receptors expressed in 1321N1 astrocytoma cells Therefore these sites could be assigned to the same receptor subtype in brain membranes while the 'fast' binding sites belong to other membrane-bound proteins, also interacting with ATP and its analogues. The kinetic properties of the latter sites were not analysed in detail.     

In vivo autoradiography of [H]SCH 39166 in rat brain: selective displacement by D1/D5 antagonists

The purpose of this study was to examine the receptor occupancy of D1/D5 antagonists for D1-like dopamine receptors in rat brain using [³H]SCH 39166, a highly selective D1/D5 antagonist with low affinity for 5HT2 receptors. A single concentration of triated SCH 39166 was administered to rats, with or without competing doses of the D1/D5 antagonist SCH 23390 and unlabeled SCH 39166, the D2-like antagonists haloperidol or the 5-HT₂ antagonist ketanserin. The bound radioactivity in the cortex, striatum, nucleus accumbens and olfactory tubercle was then quantified using an in vivo autoradiographic procedure. The results indicated that [³H]SCH 39166 was dose dependently displaced by the D1/D5 antagonists in regions associated with both the nigro-striatal pathway and the mesolimbic dopamine pathway, particularly the nucleus accumbens. Neither haloperidol nor ketanserin displaced [³H]SCH 39166 in any of the regions examined. The data were compared with previously published data examining the in vivo binding of [³H]SCH 39166 in rat brain homogenates. The relative values obtained were comparable to values detected in rat brain homogenates after in vivo binding of [³H]SCH 39166.     

Characterization of NPY mRNA-expressing cells in the human brain: co-localization with Y2 but not Y1 mRNA in the cerebral cortex, hippocampus, amygdala, and striatum

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system. Its effects on, for example, cognition, memory and motor functions are thought to be mediated mainly via its interactions with the NPY Y1 and Y2 receptor subtypes. We had previously described the neuroanatomical organization of the Y1 and Y2 mRNA expression in humans. However, in view of the lack of information regarding the overall detailed distribution of NPY mRNA expression in the human brain, a complete picture of the anatomical organization of the NPY-related genes was still missing. Thus, in the present study, the regional distribution of NPY mRNA-expressing cells was analyzed in the post-mortem human brain. In addition, double labeling in situ hybridization was performed to characterize the NPY neuronal populations in relation to the Y1 and/or Y2 receptor mRNA localization in the human cerebral cortex, striatum, and amygdala. NPY mRNA was found to be abundant in layers II and VI of the neocortex, polymorphic layer of the dentate gyrus, basal ganglia, and amygdala. Double labeling in situ hybridization showed the co-expression of NPY mRNA with the Y2, but not with the Y1, mRNA in the human cerebral cortex, hippocampus, amygdala, striatum, and nucleus accumbens, and the existence of co-expression of the Y1 and Y2 mRNAs in the cerebral cortex and amygdala. Overall, these results suggest a role for the Y2, but not Y1, as an autoreceptor in the NPY neuronal populations of the human brain.     

Autoradiographic analysis of [H]ryanodine binding sites in rat brain: Regional distribution and the effects of lesions on sites in the hippocampus

Quantitative and qualitative autoradiographic methods together with lesion approaches were used to determine the distribution of [3H]ryanodine binding sites in rat brain and the neuronal localization of these sites in the hippocampus. In normal animals, levels of [3H]ryanodine binding sites ranged from a low of about 1 fmol/mg tissue in subcortical structures to a high of 12-18 fmol/mg tissue in subregions of the hippocampus and the olfactory bulb. Relatively high densities of sites (5-9 fmol/mg tissue) were also seen in the olfactory tubercle, most areas of the cerebral cortex, accumbens nucleus, striatum, lateral septal nuclei, pontine nucleus, superior colliculus and granule cell layer of the cerebellum. Specific binding was undetectable in white matter. In experimental animals, intracerebral injections of kainic acid caused neuronal degeneration and a near total depletion of [3H]ryanodine binding sites in the dentate gyrus and in fields CA1, CA2 and CA3 of the hippocampus. Injections of kainic acid that left dentate granule cells largely intact while destroying all neurons in field CA3 had no effect on binding sites in the dentate gyrus. However, these lesions substantially reduced the density of binding in field CA3, leaving a narrow band of sites outlining the position of the degenerated CA3 pyramidal cells. Mechanical knife-cut lesions that severed the granule cell mossy fiber input to field CA3 reduced the density of binding sites in the CA3 region. The results indicate that [3H]ryanodine binding sites in brain are heterogeneously distributed and suggest that a proportion of these sites in the hippocampus may be contained in mossy fiber terminals where a presumptive calcium channel/ryanodine receptor complex may be involved in the regulation of calcium mobilization and/or neurotransmitter release.     

Characterization and regional distribution of strychnine-insensitive [3H]glycine binding sites in rat brain by quantitative receptor autoradiography

Recent evidence suggests that a strychnine-insensitive glycine modulatory site is associated with the N-methyl-D-aspartate receptor-channel complex. A quantitative autoradiographic method was used to characterize the pharmacological specificity and anatomical distribution of strychnine-insensitive [3H]glycine binding sites in rat brain. [3H]Glycine binding was specific, saturable, reversible, pH and temperature-sensitive and of high affinity. [3H]Glycine interacted with a single population of sites having a KD of approximately 200 nM and a maximum density of 6.2 pmol/mg protein (stratum radiatum, CA1). Binding exhibited a pharmacological profile similar to the physiologically defined strychnine-insensitive glycine modulatory site. Binding was stereoselective; the rank order of potency of simple amino acids as displacers of binding was: glycine greater than D-serine greater than D-alanine greater than L-serine greater than L-alanine greater than L-valine greater than D-valine. Binding was not altered by the inhibitory glycine receptor ligand, strychnine, by the glutamate agonists, quisqualate and kainate, or by GABA receptor selective ligands. Most competitive agonists or antagonists of the N-methyl-D-aspartate recognition site were ineffective displacers of glycine binding. The exceptions were the aminophosphono series of antagonists, D-alpha-aminoadipate, gamma-D-glutamyglycine and beta-D-aspartylaminomethylphosphonic acid. However, the inhibition of [3H]glycine binding produced by the aminophosphono compounds could be accounted for by the level of glycine contamination present in these compounds. The non-competitive NMDA receptor-channel blockers, phencyclidine, its thienyl derivative, and MK-801 did not alter glycine binding. Kynurenate, glycine methylester, L-serine-O-sulfate, L-homocysteic acid, and several glycine-containing dipeptides were effective displacers of glycine binding. Structure-activity relations of agonists and antagonists of the strychinine-insensitive glycine binding site are discussed. The distribution of strychnine-insensitive [3H]glycine binding was heterogeneous with the following rank order of binding densities: hippocampus greater than cerebral cortex greater than caudate-putamen greater than or equal to thalamus greater than cerebellum greater than brain stem. This distribution of binding was correlated with N-methyl-D-aspartate-sensitive [3H]glutamate binding (r2 = 0.77; P less than 0.001; Pearson product-moment) and [3H]thienylcyclohexylpiperidine binding (r2 = 0.72; P less than 0.001). These observations are consistent with the hypothesis that the strychnine-insensitive glycine binding site is closely associated with the N-methyl-D-aspartate receptor-channel complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acute withdrawal from chronic escalating-dose binge cocaine administration alters kappa opioid receptor stimulation of [S] guanosine 5′-O-[gamma-thio]triphosphate acid binding in the rat ventral tegmental area

There is evidence that the kappa opioid system plays an important role in cocaine addiction and that chronic cocaine administration and withdrawal from chronic cocaine alter kappa opioid receptor (KOPr) density. The present study employed in situ [³⁵S]guanosine 5′-O-[gamma-thio]triphosphate acid (GTPγS) binding autoradiography to measure KOPr-stimulated activation of G-protein in the caudate putamen, nucleus accumbens core and shell, lateral hypothalamus, basolateral amygdala, substantia nigra compacta, substantia nigra reticulata and ventral tegmental area (VTA), in response to chronic cocaine administration or acute and chronic withdrawal from chronic cocaine. Male Fischer rats were injected i.p. with saline or cocaine three times daily at 1 h intervals in an escalating-dose paradigm for 14 days (from 3×15 mg/kg/injection on days 1–3 up to 3×30 mg/kg/injection on days 10–14). Identically treated separate groups were withdrawn from cocaine or saline for 24 h or 14 days. No significant change in KOPr agonist U-69593-stimulated [³⁵S]GTPγS was found in the seven regions studied 30 min or 14 days after chronic 14 days escalating-dose binge cocaine administration. However there was an increase in KOPr -stimulated [³⁵S]GTPγS binding in the VTA (P<0.01) of rats withdrawn for 24 h from chronic cocaine. Our results show a cocaine withdrawal induced increase of KOPr signaling in the VTA, and suggest that the KOPr may play a role in acute withdrawal from cocaine.     

P>海洛因依赖对大鼠中脑腹侧被盖区、伏隔核神经元P物质和神经肽Y表达的影响 /P>

目的 探讨海洛因依赖对大鼠中脑腹侧被盖区（VTA）、伏隔核（NAc）神经元P物质（SP）、神经肽Y（NPY）表达的影响,为进一步揭示海洛因依赖的中枢机制提供重要的原位形态学资料。 方法 成年雄性SD大鼠55只,随机分为海洛因依赖组、生理盐水对照组及正常对照组。建立海洛因依赖模型,并分别于第10、17、24、31、38天取脑组织,免疫组织化学SABC法显示VTA、NAc区神经元SP、NPY免疫反应细胞并用图像分析法测定平均灰度值。结果 海洛因依赖组VTA、 NAc区的SP、NPY免疫反应细胞与生理盐水及正常对照组比较,免疫反应强度明显减弱、染色变浅。平均灰度值测定,海洛因依赖组各时间点大鼠VTA、NAc区 的SP、NPY免疫反应细胞平均灰度值均高于正常对照组及生理盐水对照组,差异有统计学意义（P ＜0.05）。结论 实验结果提示,海洛因依赖使VTA、NAc区SP、NPY的分泌受到抑制,可能导致内源性阿片肽的分泌受影响,SP、NPY可能是药物依赖形成机制中的关键信号分子。     

Localization of the dopamine uptake complex using [3H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine ([3H]BTCP) in rat brain

[³H]N-[1-(2-Benzo(b)thiophenyl)cyclohexyl]piperidine ([³H]BTCP) is a novel phencyclidine derivative with considerable selectivity for dopamine uptake sites. [³H]BTCP was used to label dopamine uptake sites in vitro, in rat brain, and the regions containing these sites were visualized with an autoradiographic technique. The binding was found to be highest in the striatum, where > 90% of binding was specific. Furthermore, 6-hydroxydopamine lesions of the nigrostriatal pathway obliterated striatal [³H]BTCP binding ipsilaterally, whereas ibotenic acid injection into the caudate-putamen failed to significantly reduce [³H]BTCP binding in that structure. These results indicate that [³H]BTCP labels dopamine uptake sites in mammalian brain and that it can be employed for autoradiographic studies of this transport complex.     

Localization of the dopamine uptake complex using [H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine ([H]BTCP) in rat brain

[³H]N-[1-(2-Benzo(b)thiophenyl)cyclohexyl]piperidine ([³H]BTCP) is a novel phencyclidine derivative with considerable selectivity for dopamine uptake sites. [³H]BTCP was used to label dopamine uptake sites in vitro, in rat brain, and the regions containing these sites were visualized with an autoradiographic technique. The binding was found to be highest in the striatum, where > 90% of binding was specific. Furthermore, 6-hydroxydopamine lesions of the nigrostriatal pathway obliterated striatal [³H]BTCP binding ipsilaterally, whereas ibotenic acid injection into the caudate-putamen failed to significantly reduce [³H]BTCP binding in that structure. These results indicate that [³H]BTCP labels dopamine uptake sites in mammalian brain and that it can be employed for autoradiographic studies of this transport complex.     

Quantitative autoradiographic distribution of NPFF1 neuropeptide FF receptor in the rat brain and comparison with NPFF2 receptor by using [125I]YVP and [(125I]EYF as selective radioligands

The selectivity of two new radioligands, [(125)I]YVP ([(125)I]YVPNLPQRF-NH(2)) and [(125)I]EYF ([(125)I]EYWSLAAPQRF-NH(2)), for neuropeptide FF (NPFF) receptor subtypes was determined using HEK293 cells expressing hNPFF(1) and CHO cells expressing hNPFF(2) receptors. Saturation binding and displacement experiments showed that [(125)I]YVP and [(125)I]EYF bound selectively with a very high affinity, K(D)=0.18 nM and 0.06 nM, to NPFF(1) and NPFF(2) receptors respectively.By using in vitro autoradiography with these radioligands and frog pancreatic polypeptide (PP) as selective unlabelled competitor of NPFF(2) binding sites, NPFF(1) and NPFF(2) receptor distribution was analyzed throughout the rat CNS.The highest densities of [(125)I]EYF binding sites were seen in the most external layers of the dorsal horn of the spinal cord, the parafascicular thalamic nucleus, laterodorsal thalamic nucleus and presubiculum of hippocampus. All specific binding of this radioligand was inhibited by 200 nM frog PP. The density of 0.1 nM [(125)I]YVP binding was much smaller in all brain areas and frog PP-insensitive binding sites (NPFF(1) receptor subtype) were detected in septal, thalamic and hypothalamic areas but were absent in the spinal cord.The restricted distribution of NPFF(1) receptors in the CNS supports its specific role in a limited number of neuronal functions. In contrast to the rat spinal cord where the NPFF(1) system is absent, there is no strict separation between NPFF(1) and NPFF(2) system at the supraspinal level.     

Determination of radioactivity in infant,juvenile and adult rat brains after injection of anti-influenza drug [C]oseltamivir using PET and autoradiography

Oseltamivir phosphate (Tamiflu^®) is an orally active anti-influenza drug, which is hydrolyzed to its metabolite Ro 64-0802 inhibiting the influenza virus with potent activity. The abnormal behavior of young influenza patients associated with the use of oseltamivir has developed to a social problem in countries where Tamiflu is often prescribed. It is important to determine the amount of oseltamivir in the brain and to elucidate the relationship between its presence and neuropsychiatric side effects. The aim of this study was to determine the radioactivity in the infant, juvenile and adult rat brains after injection of [¹¹C]oseltamivir into the rats using PET and autoradiography. After injection of this radioligand, the highest radioactivity was found in the infant brain and the radioactivity level decreased with age. Ex vivo autoradiography on the infant brain displayed a relatively higher radioactivity in the cerebellum than that in the cerebrum. Pretreatment with cyclosporin A (an inhibitor for P-glycoprotein) increased the brain radioactivity. These results give helpful insights into elucidating why the neuropsychiatric side effects of oseltamivir occur in young patients.     

Opposite alterations of NPFF1 and NPFF2 neuropeptide FF receptor density in the triple MOR/DOR/KOR-opioid receptor knockout mouse brains

Mice lacking the mu-delta-kappa-opioid receptor (MOR/DOR/KOR) genes and their corresponding wild-type littermates have been used to quantify NPFF(1) and NPFF(2) (neuropeptide FF) receptors by in vitro autoradiography in the central nervous tissues. Adjacent coronal sections were labelled with [125I]YVP ([125I]YVPNLPQRF-NH(2)) and [125I]EYF ([125I]EYWSLAAPQRF-NH(2)) as specific radioligands for NPFF(1) and NPFF(2) receptors, respectively. NPFF(2) receptors are predominantly expressed in both genotypes, but their density increases significantly in non cortical regions of mutant mice: 64% in the amygdaloid area, 89, 308, 1214 and 49% in the nucleus of the vertical limb of the diagonal band, substantia nigra, the vestibular nucleus and the spinal cord, respectively. In contrast, the density of the NPFF(1) subtype is lower than NPFF(2) in both genotypes and significantly decreased in some brain areas of mutant mice: -99, -90 and -90% in the nucleus of the vertical limb of the diagonal band, substantia nigra and the spinal cord, respectively. This study shows that mice lacking opioid receptors have brain region-dependent increases (NPFF(2)) and decreases (NPFF(1)) in NPFF receptors densities and suggests a different functional participation of each NPFF receptor subtype in the actions of opioids.     

Autoradiography of angiotensin-converting enzyme in fixed and unfixed rat brain using the specific enzyme inhibitor [I]351A or a polyclonal antibody and [I]staphylococcal protein A

Angiotensin-converting enzyme (ACE, kininase II, EC 3.4.15.1) was visualized in unfixed rat brain sections by autoradiography after incubation with a polyclonal goat anti-rabbit lung converting-enzyme antibody and [¹²⁵I]protein A. Paraformaldehyde fixation interfered with the recognition of ACE by its antibody in brain nuclei but not in the choroid plexus. Conversely, incubation of brain sections with the specific ACE inhibitor [¹²⁵I]351A allowed ACE visualization in either unfixed and paraformaldehyde-fixed brain sections, since [¹²⁵I]351A binding was not affected by our fixation conditions. Our results indicate that ACE could be visualized in unfixed brain sections directly by incubation with the specific inhibitor [¹²⁵I]351A or indirectly by radioimmunohistochemistry and autoradiography. Different autoradiographic methods could be used to visualize and quantify ACE in unfixed or fixed tissues.