Common receptors for hallucinogens in rat brain: a comparative autoradiographic study using [125I]LSD and [125I]DOI, a new psychotomimetic radioligand

The S and R enantiomers of the psychotomimetic 5HT2 agonist DOI (2,5-dimethoxy-4-iodophenylisopropylamine) were labeled with 125I at high-specific activity. The regional distribution of binding sites for each of the enantiomers was investigated using in vitro quantitative autoradiography and compared to the regional distribution of [125I]LSD in the rat brain. Saturable, specific binding of the radioligands was determined in cortical membrane homogenates. All radioligands exhibited specific binding in localized regions throughout the rat brain. Binding of [125I]DOI enantiomers was completely displaced (greater than 90%) by 1 microM of the corresponding unlabeled enantiomer; [125I]LSD was completely displaced by 1 microM LSD. The choroid plexus showed the highest-density binding. Other regions showing high-density binding included the frontoparietal cortex (motor and somatosensory areas), anterior cingulate gyrus, lateral olfactory tubercle, nucleus accumbens, caudate nuclei, claustrum, nucleus of the lateral olfactory tract, dentate gyrus, mamillary nuclei, and motor trigeminal nuclei. In most regions, [125I]S-DOI, the less active enantiomer, exhibited 25-40% of the amount of total binding as [125I]R-DOI. In some regions, [125I]R-DOI and [125I]LSD had similar binding densities; in others, marked differences were apparent. The regional distribution of specific [125I]R-DOI binding sites correlated with the distribution of 5HT2 receptors reported in previous studies. DOI and its analogs may have potential clinical applications for in vivo localization of 5HT2-receptors using positron emission tomography (PET) and similar techniques.     

Dexamethasone downregulates chemokine receptor CXCR4 and exerts neuroprotection against hypoxia/ischemia-induced brain injury in neonatal rats

OBJECTIVE: Hypoxia/ischemia (H/I) induces rapid and massive brain damage in neonatal rat brain, resulting in long-term consequences on structural and functional maturation of the central nervous system. Inflammatory mediators contribute to these permanent pathological changes, which are sensitive to corticoid treatments. Since the chemokine receptor CXCR4, specific for the SDF-1 alpha/CXCL12 ligand, regulates both apoptotic and neuroregeneration processes, this receptor was quantified 2 days following H/I in neonatal rat brain in relation with dexamethasone (DEX) treatment. METHODS: Seven-day-old male rats were exposed to a 90-min hypoxia following unilateral carotid ligation (H/I) and were sacrificed 48 h later. Glucocorticoid-pretreated animals were injected subcutaneously 5 h prior to hypoxia with 0.5 microg/g DEX. Glial fibrillary acidic protein and cresyl violet staining were used for assessing the extent of brain lesion subdivided into necrotic and penumbra-like areas. The density of CXCR4 receptors was determined by quantitative autoradiography using [(125)I]SDF-1 alpha as a ligand. RESULTS: The H/I resulted in a massive lesion ipsilateral to the carotid ligation, which was extended to cortical, striatal, hippocampal and thalamic areas, while the contralateral hemisphere remained apparently unaffected. DEX decreased the lesion size by reducing mainly the necrotic area. H/I induced a marked increase in CXCR4 receptor binding in the penumbra-like areas. DEX pretreatment decreased CXCR4 receptor density in the penumbra and attenuated astrocytosis. Furthermore, DEX strongly lowered mortality rate and reduced functional recovery time right after hypoxia. CONCLUSION: The rapid enhancement in CXCR4 chemokine receptor binding in the affected brain areas suggests that SDF-1 alpha/CXCR4 may play a role in the hypoxia-induced inflammatory reaction in the neonatal brain. Attenuation of CXCR4 expression and astrogliosis could contribute to the neuroprotective effect of DEX pretreatment via influencing the inflammatory cascade induced by H/I in the neonatal brain.     

Type I interleukin-1 receptors in the mouse brain-endocrine-immune axis labelled with [125I]recombinant human interleukin-1 receptor antagonist.

Iodine-125-labelled recombinant human interleukin-1 (IL-1) receptor antagonist ([125I]IL-1ra) was utilized to further determine the characteristics of IL-1 receptors in the brain-endocrine-immune axis. The binding of [125I]IL-1ra in homogenates of mouse hippocampus, spleen and testis was linear over a broad range of membrane protein concentrations, saturable, reversible, and of high affinity (KD, 20-30 pM). In competition studies, IL-1ra, recombinant human IL-1 alpha, IL-1 beta and a weak IL-1 beta analog inhibited [125I]IL-1ra binding to mouse tissues in parallel with their biological activities. In autoradiographic studies, [125I]IL-1ra and [125I]IL-1 alpha binding showed comparable distribution patterns with highest densities of binding sites present in the dentate gyrus of the hippocampus, choroid plexus, anterior pituitary, marginal zones and red pulp regions of the spleen, epididymis and interstitial area of the testis. The binding characteristics and distribution of [125I]IL-1ra are comparable to those of previously characterized Type I IL-1 receptors. These data provide further support for a role for IL-1 in coordinating brain-endocrine-immune responses to physiological and pharmacological stimuli.     

125I]Iodomethyllycaconitine binds to alpha7 nicotinic acetylcholine receptors in monkey brain

We examined the binding of the novel nicotinic acetylcholine receptor (nAChR) ligand [125I]iodomethyllycaconitine (iodoMLA) in the brains of M. cynomologous (macaque) monkeys. [125I]iodoMLA bound throughout the brain with the greatest density in the thalamus and moderate intensity in the basal ganglia and cortical regions. The Kd and Bmax in whole brain tissue were similar whether 1 mM nicotine (Kd 33.25 +/- 15.17 nM, Bmax 5.80 +/- 1.06 fmol/mg) or 2 microM of the alpha7-selective antagonist alpha-bungarotoxin (Kd 46.12 +/- 18.45 nM, Bmax 6.30 +/- 1.06 fmol/mg) was used for nonspecific binding. The subtype-selectivity of this ligand was further studied with competition binding studies using nicotine, alpha-bungarotoxin and noniodinated MLA. Each ligand completely inhibited [125I]iodoMLA binding throughout the monkey brain, with Ki values of 2.23 +/- 0.85 microM for nicotine, 2.72 +/- 1.71 nM for alpha-bungarotoxin and 1.83 +/- 0.35 nM MLA in the caudate and 2.03 +/- 1.14 microM, 2.65 +/- 0.86 nM and 3.32 +/- 0.71 nM, respectively, in the putamen. The alpha3beta2/alpha6*-selective antagonist alpha-conotoxin MII failed to inhibit [125I]iodoMLA binding in any brain region. In monkeys with cognitive deficits resulting from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, [125I]iodoMLA binding was significantly increased in the striatum, similar to results previously observed for [125I]alpha-bungarotoxin. These results suggest that, under the present experimental conditions, [125I]iodoMLA was selective for alpha7-containing nAChRs and did not bind to alpha6-containing nAChRs. This radioligand may be a useful tool for selectively imaging alpha7-containing nAChRs in vivo.     

Characterization of the altered oligosaccharide composition of the insulin receptor on neural-derived cells

Typical insulin receptors are present on neuroblastoma cell lines. High affinity binding for insulin was present in membrane preparations from NG108 (a hybrid mouse neuroblastoma-rat glioma) as well as in membranes from SK-N-MC and SK-N-SH, two human neuroblastoma cell lines. Specific [125I]insulin binding was 24.4% for NG108, 16.9% for SK-N-MC and 5.2% for SK-N-SH at membrane protein concentrations of 0.4 mg/ml. IC50 for [125I]insulin binding was 3.4 nM in NG108 membrane preparations and 0.9 nM for SK-N-SH and 1.8 nM in SK-N-MC membranes. Apparent mol. wt. for the alpha subunits (identified by specific immunoprecipitation using the anti-insulin receptor antiserum B10) on SDS PAGE was 134 kDa for NG108; 124 kDa for SK-N-MC and 120 kDa for SK-N-SH. Neuraminidase digestion increased the mobility of the alpha subunit from both NG108 and SK-N-MC receptors to 120 kDa, whereas that from SK-N-SH were unaffected. Endoglycosidase H and endoglycosidase F digestions increased the mobility of the alpha subunits of all 3 cell lines to varying degrees, suggesting the presence of N-linked glycosylation. Insulin induced autophosphorylation of the insulin receptor beta subunit in WGA-purified membranes from all 3 cell lines. In addition, phosphorylation of a protein with an apparent mol. wt. 105 kDa was stimulated by insulin in WGA purified membranes from NG108. Tyrosine-specific kinase activity was present in the membranes from each cell line and was stimulated by insulin in a dose-dependent manner from 10(-9) to 10(-6) M. Proinsulin was about 100 times less potent in stimulating phosphorylation of the artificial substrate poly (Glu, Tyr)4:1 when compared to insulin in accordance with its lower binding affinity to the insulin receptor. Hexose transport was stimulated by insulin in all 3 cell lines. These results indicate that neuroblastoma cells contain specific insulin receptors and that they may be useful as models for studying the role of insulin in nervous tissue.     

Binding of atrial and brain natriuretic peptides in brains of hypertensive rats

Displacement of bound [125I]alpha-atrial natriuretic peptide (alpha-ANP) by brain natriuretic peptide (BNP) was used to map receptors common to both peptides in rat brain by in vitro autoradiography. Both spontaneously hypertensive rats (SHR) and the normotensive Wistar-Kyoto control strain (WKY) were studied. In both strains, [125I]alpha-ANP bound densely to subfornical organ, choroid plexus and arachnoid mater. Binding at these sites in either strain was displaced similarly by 1 microM unlabelled alpha-ANP or BNP. However, no [125I]alpha-ANP was displaced by peptides unrelated to alpha-ANP or BNP. In WKY, both alpha-ANP and BNP competed with similarly high affinities for binding sites occupied by [125I]alpha-ANP. This was also true for SHR. However, SHR showed a substantial reduction in the maximum number of binding sites in the subfornical organ and choroid plexus which were competed for by the peptides. Therefore, BNP may be a significant high affinity ligand for brain receptors previously thought specific for atrial natriuretic peptides, including receptors which vary between WKY and SHR.     

Autoradiographic localization of 5HT2 receptors in rat brain using [125I]-DOI, a selective psychotomimetic radioligand

1. Binding sites for the R and S enantiomers of the 5HT2 agonist DOI (2,5-dimethoxy-4-iodophenylisopropylamine) were identified in rat brain using quantitative in-vitro autoradiography and compared with [125I]-LSD binding. 2. In most regions of the brain, binding density of the less active isomer [125I]S-DOI was 15 to 85% of that exhibited by the active [125I]R-DOI isomer. 3. Cortical membrane preparations exhibited two binding sites, of the enantiomers with high (KdH) and low (KdL) affinity constants of 1.2 +/- 0.02 nM and 29 +/- 7 nM for the [125I]R-DOI and 2.1 +/- 0.2 nM and 18 +/- 4 nM for [125I]S-DOI respectively. The respective high (BmaxH) and low (BmaxL) binding densities were 92 +/- 10 and 536 +/- 164 fmol/mg protein for the [125I]R-DOI and 67 +/- 19 and 245 +/- 60 fmol/mg protein for [125I]S-DOI. 4. Our results correlate with regional distribution of 5HT2 receptors reported in previous studies and indicate that DOI and its congeners have potential clinical applications for the in-vivo localization of 5HT2 receptors.     

Calcitonin gene-related peptide (hCGRP alpha) binding sites in the nucleus accumbens. Atypical structural requirements and marked phylogenic differences.

The distribution of [125I]hCGRP alpha binding sites was studied in tissue sections from rat brain and, at the level of the nucleus accumbens in the brains of 6 other species. In the rat, very high levels of binding were found in the nucleus accumbens, the amygdaloid complex and mammillary body while high amounts were localized to the superficial layers of the superior colliculus, temporal cortex, cerebellum (molecular layer), frontal cortex and inferior olive. Moderate densities of [125I]hCGRP alpha binding were observed in the medial geniculate nucleus, inferior colliculus and substantia nigra. Regional competition studies in rat brain showed that salmon calcitonin was almost as effective as hCGRP alpha in competing for [125I]hCGRP alpha binding sites in the nucleus accumbens but was mostly inactive in other regions such as the mesolimbic cortex and the striatum. On the basis of their atypical sensitivity to salmon calcitonin, [125I]hCGRP alpha binding sites in the rat nucleus accumbens, which appear between postnatal days 4 and 7, do not seem to correspond to either the CGRP1 or CGRP2 receptor subtypes. Marked species differences were observed in the distribution of [125I]hCGRP alpha binding sites, especially in the nucleus accumbens. In the mouse, low densities of hCGRP alpha sites were observed in striatum and fronto-parietal cortex while low to moderate levels were found in the medial and posterior aspects of the nucleus accumbens. A similar distribution was seen in the guinea pig brain albeit of generally higher density. In the rat, very high amounts of [125I]hCGRP alpha binding were seen in the nucleus accumbens while lower levels were found in the striatum and certain cortical areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

An autoradiographic study of the distribution of binding sites for the novel alpha7-selective nicotinic radioligand [3H]-methyllycaconitine in the mouse brain.

[3H]-Methyllycaconitine ([3H]-MLA) is a new radioligand with selectivity for alpha7-type neuronal nicotinic acetylcholine receptors (nAChRs). In our previous study [Davies, A.R.L., Hardick, D.J., Blagbrough, I.S., Potter, B.V.L., Wolstenholme, A.J. & Wonnacott, S. (1999) Neuropharmacology, 38, 679-690], this radioligand labelled a single class of site in rat brain membranes; its pharmacology and distribution in crudely dissected brain regions closely paralleled that of the well-established alpha7-ligand [125I]-alpha-bungarotoxin. However, a small population of [3H]-MLA binding sites was apparently insensitive to alpha-bungarotoxin. Here we have extended the study to mouse brain, using autoradiography to examine the distribution of [3H]-MLA and [125I]-alpha-bungarotoxin binding sites. [3H]-MLA labelled a single class of site in mouse brain membranes with a KD of 2.2 nM and a Bmax of 45.6 fmol/mg protein. Specific binding, defined by unlabelled MLA (Ki = 0.69 nM), was completely inhibited by (-)-nicotine (Ki = 1.62 microM), whereas alpha-bungarotoxin inhibited only 85% of specific binding (Ki = 3.5 nM). The distributions of [125I]-alpha-bungarotoxin and [3H]-MLA binding sites were compared by autoradiography, and binding was quantitated in 72 brain regions. Binding of both radioligands was highly correlated, with highest densities in the dorsal tegmental nucleus of the pons, colliculi and hippocampus. Serial sections labelled with [3H]-MLA in the absence or presence of unlabelled MLA or alpha-bungarotoxin provided no evidence for any alpha-bungarotoxin-resistant binding. The results are discussed in terms of binding sites that are inaccessible to alpha-bungarotoxin in membrane preparations. This study demonstrates the utility of [3H]-MLA for characterization of alpha7-type nicotinic receptors in mammalian brain, and suggests that it labels a population identical to that defined by [125I]-alpha-bungarotoxin.     

The ontogeny of specific prolactin binding sites in the rat choroid plexus

The development of prolactin receptors in the choroid plexus of the rat was examined using the in vivo autoradiographic approach employing the principle of competitive binding. Experimental animals were injected with [125I]prolactin alone (total binding) while control animals received [125I]prolactin and a 500-fold excess of unlabelled prolactin (non-specific binding). Newborns as well as animals 10, 14 and 18 days postnatal were studied. Three minutes following hormone injection animals received an intracardiac perfusion with fixative and tissues were prepared for quantitative light microscopic autoradiography. The choroid plexus first demonstrated specific binding of prolactin, i.e. a statistically significant difference in the autoradiographic reactions between experimental and control animals, at 14 days postnatal. The lactogen specificity of these binding sites was further defined by the ability of [125I]prolactin to be displaced by unlabelled human growth hormone, which is lactogenic in rats, and not by unlabelled insulin, which is structurally dissimilar to prolactin. Morphometric analyses were performed on electron micrographs of choroid plexus from 10- and 14-day postnatal rats. The volume densities of constituents known to be enriched in polypeptide hormone receptors were measured and compared. Small cytoplasmic vesicles and tubules were statistically significantly more abundant in 10-day-old rats than in 14-day-old animals. It is conjectured that these vesicles and tubules contain an intracellular pool of prolactin receptors whose decrease at 14 days parallels the expression of specific binding sites at the cell surface.     

Complex effects of stromal cell-derived factor-1 alpha on melanin-concentrating hormone neuron excitability

Stromal cell-derived factor 1alpha (SDF-1alpha), a chemoattractant for leucocytes and neurons, and its receptor, CXCR4 are expressed in subsets of neurons of specific brain areas. In rat lateral hypothalamic area (LHA) we show, using immunocytochemistry, that CXCR4 is localized within melanin-concentrating hormone (MCH)-expressing neurons, mainly involved in feeding behaviour regulation. We investigated whether SDF-1alpha may control MCH neuronal activity. Patch-clamp recordings in rat LHA slices revealed multiple effects of SDF-1alpha on the membrane potential of MCH neurons, indirect through glutamate/GABA release and direct through GIRK current activation. Moreover, SDF-1alpha at 0.1-1 nM decreased peak and discharge frequency of action potential evoked by current pulses. These effects were further confirmed in voltage-clamp experiments, SDF-1alpha depressing both potassium and sodium currents. At 10 nM, however, SDF-1alpha increased peak and discharge frequency of action potential evoked by current pulses. Using a specific CXCR4 antagonist, we demonstrated that only the depressing effect on AP discharge was mediated through CXCR4 while the opposite effect was indirect. Together, our studies reveal for the first time a direct effect of SDF-1alpha on voltage-dependent membrane currents of neurons in brain slices and suggest that this chemokine may regulate MCH neuron activity.     

In vitro and in vivo characterization of [125I]iodomethyllycaconitine in the rat

The in vitro and in vivo binding characteristics of [125I]iodomethyllycaconitine ([125I]iodoMLA) were determined in the rat. [125I]iodoMLA binding to rat cerebral cortex membranes was saturable and reversible and its specific binding represented approximately 70-80% of the total binding. [125I]iodoMLA labeled a single site with Kd = 1.8 +/- 0.4 nM and Bmax = 68 +/- 3 fmol/mg protein. Kinetic analysis revealed a t1/2 for association and dissociation of 10.5 +/- 3.1 and 10.3 +/- 1.6 min, respectively. Pharmacological characterization of [125I]iodoMLA binding indicated that it was specific for the alpha7 nAChR. In vitro brain region binding studies revealed greater binding in regions known to contain high numbers of alpha7 nAChRs. The analysis of the biodistribution of intravenously administered [125I]iodoMLA indicated that it was rapidly cleared and exhibited poor brain penetration; nevertheless, the levels of [125I]iodoMLA in alpha7 nAChR-rich target regions were significantly increased compared to the nontarget region (cerebellum) 60-120 min after administration. No metabolism of MLA by human liver S9 fraction was detected. Our results suggest that [125I]iodoMLA will be a useful radioligand to study the alpha7 nAChR in vitro and in vivo.     

Evidence for heterogeneity of muscarinic receptors in the mollusc Pleurobranchaea

The properties of the specific binding of the muscarinic antagonist [125I]3-quinuclidinyl-4-iodobenzilate ([125I]4IQNB] to nervous tissue of Pleurobranchaea california were characterized. The specific binding of [125I]4IQNB to Pleurobranchaea nervous tissue was characterized by its high affinity (Kd = 0.61 +/- 0.11 nM) and saturability (Bmax = 602 +/- 46 fmol/mg protein). A comparison of the numbers of binding sites recognized by [125I]4IQNB and l-[3H]QNB in nervous tissue of three invertebrate species indicated that in Aplysia and Cancer magister (crab) ganglia membranes the two radioligands labeled comparable numbers of binding sites; however, in Pleurobranchaea membranes l-[3H]QNB recognized only a subpopulation (8-10%) of the total number of [125I]4IQNB binding sites. The disparity in the numbers of binding sites labeled by these radioligands was consistent with our finding of a heterogeneity of muscarinic antagonist binding sites in l-QNB competition experiments in Pleurobranchaea. Computer-assisted analysis of l-QNB competition of [125I]4IQNB specific binding demonstrated that these data were best described by a two-site model with high- and low-affinity sites for l-QNB. The high-affinity site recognized by l-QNB possessed an IC50 value of 0.2 nM and comprised 18% of the total specific binding, while the lower affinity site had an IC50 value of 55.6 nM and comprised the remaining 82% of the total population of [125I]4IQNB recognition sites. The IC50 value for l-QNB at the high-affinity site in Pleurobranchaea membranes is in excellent agreement with Kd values for l-[3H]QNB labeling of classical muscarinic receptors in a variety of invertebrate and vertebrate species.     

Regional localization of specific [I]leptin binding sites in rat forebrain

Specific [¹²⁵I]leptin receptor binding sites have been identified in choroid plexus (CP), but have eluded regional localization within the brain parenchyma. To optimize specific [¹²⁵I]leptin binding in brain loci, we ran experiments varying the pH of incubation buffers. We found that specific [¹²⁵I]leptin binding in CP was strikingly pH dependent with the most acidic buffer, pH 5.5, resulting in a greater than 100% increase over the amount of specific binding measured at pH 7.5. While low pH permitted detection of specific binding in parenchymal loci, clear pH dependency was only observed in the CP. In the caudate putamen (CauP), a locus with low specific binding, values for specific binding did not differ significantly across the range of pH conditions tested. Using incubation buffers at pH 6.0 in subsequent binding experiments, we localized specific [¹²⁵I]leptin binding in several brain loci including thalamus and hypothalamus. In CP and thalamus, where the range of OD permitted analysis of binding parameters, [¹²⁵I]leptin binding was saturable with increasing concentrations of unlabelled leptin. In all loci, specific [¹²⁵I]leptin binding was insensitive to competition by high concentrations of other unlabelled compounds. Our results varying pH conditions of the incubation buffer suggest leptin receptors may be divided into subclassifications based on pH sensitivity of the specific binding. Furthermore, our results suggest that although densities are low, high affinity leptin receptors are present in neural loci implicated in food intake and energy balance, and are more widespread in the forebrain than previously determined.     

Leptin transport at the blood–cerebrospinal fluid barrier using the perfused sheep choroid plexus model

Leptin is secreted by adipose tissue and thought to regulate appetite at the central level. Several studies have explored the central nervous system (CNS) entry of this peptide across the blood–brain and blood–cerebrospinal fluid (CSF) barriers in parallel, but this is the first to explore the transport kinetics of leptin across the choroid plexus (blood–CSF barrier) in isolation from the blood–brain barrier (BBB). This is important as the presence of both barriers can lead to ambiguous results from transport studies. The model used was the isolated Ringer perfused sheep choroid plexus. The steady-state extraction of [¹²⁵I]leptin (7.5 pmol l⁻¹) at the blood face of the choroid plexus was 21.1±5.7%, which was greater than extraction of the extracellular marker, giving a net cellular uptake for [¹²⁵I]leptin (14.0±3.7%). In addition, trichloroacetic acid precipitable [¹²⁵I] was detected in newly formed CSF, indicating intact protein transfer across the blood–CSF barrier. Human plasma concentrations of leptin are reported to be 0.5 nM. Experiments using 0.5 nM leptin in the Ringer produced a concentration of leptin in the CSF of 12 pM (similar to that measured in humans). [¹²⁵I]Leptin uptake at the blood–plexus interface using the single-circulation paired tracer dilution technique (uptake in <60 s) indicated the presence of a saturable transport system, which followed Michaelis–Menten-type kinetics (K_m=16.3±1.8 nM, V_max=41.2±1.4 pmol min⁻¹ g⁻¹), and a non-saturable component (K_d=0.065±0.002 ml min⁻¹ g⁻¹). In addition, secretion of new CSF by the choroid plexuses was significantly decreased with leptin present. This study indicates that leptin transport at the blood–CSF barrier is via saturable and non-saturable mechanisms and that the choroid plexus is involved in the regulation of leptin availability to the brain.     

Leptin transport at the blood--cerebrospinal fluid barrier using the perfused sheep choroid plexus model

Leptin is secreted by adipose tissue and thought to regulate appetite at the central level. Several studies have explored the central nervous system (CNS) entry of this peptide across the blood-brain and blood-cerebrospinal fluid (CSF) barriers in parallel, but this is the first to explore the transport kinetics of leptin across the choroid plexus (blood-CSF barrier) in isolation from the blood-brain barrier (BBB). This is important as the presence of both barriers can lead to ambiguous results from transport studies. The model used was the isolated Ringer perfused sheep choroid plexus. The steady-state extraction of [(125)I]leptin (7.5 pmol l(-1)) at the blood face of the choroid plexus was 21.1+/-5.7%, which was greater than extraction of the extracellular marker, giving a net cellular uptake for [(125)I]leptin (14.0+/-3.7%). In addition, trichloroacetic acid precipitable [(125)I] was detected in newly formed CSF, indicating intact protein transfer across the blood-CSF barrier. Human plasma concentrations of leptin are reported to be 0.5 nM. Experiments using 0.5 nM leptin in the Ringer produced a concentration of leptin in the CSF of 12 pM (similar to that measured in humans). [(125)I]Leptin uptake at the blood-plexus interface using the single-circulation paired tracer dilution technique (uptake in <60 s) indicated the presence of a saturable transport system, which followed Michaelis-Menten-type kinetics (K(m)=16.3+/-1.8 nM, V(max)=41.2+/-1.4 pmol min(-1) g(-1)), and a non-saturable component (K(d)=0.065+/-0.002 ml min(-1) g(-1)). In addition, secretion of new CSF by the choroid plexuses was significantly decreased with leptin present. This study indicates that leptin transport at the blood-CSF barrier is via saturable and non-saturable mechanisms and that the choroid plexus is involved in the regulation of leptin availability to the brain.     

Neurotensin receptor binding sites in monkey and human brain: autoradiographic distribution and effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment

Neurotensin (NT) receptor binding sites were characterized and localized by using membrane binding assay and in vitro receptor autoradiography in monkey and human brain. Additionally, the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment on NT binding sites were investigated in monkey. [125I]Tyr3-NT ([125I]NT) apparently binds to a single class of high-affinity sites (Kd in nanomolar range) in both species. Ligand selectivity patterns strongly suggest that the structural requirements of both monkey and human brain NT receptors are very similar to those previously reported in other tissues, such as those of the rat brain and rat stomach. In monkey brain, [125I]NT binding sites are discretely distributed with high densities of sites found in the cingulate cortex, amygdala, hippocampus, ventral tegmental area, substantia nigra, and periaqueductal gray matter. A similar pattern is observed in the human brain. However, the laminar distribution of [125]NT binding sites in cortex varies between monkey and human brain. In monkey brain, [125I]NT binding sites are mostly concentrated in deep cortical layers while the laminar distribution of NT sites changes with cortical areas in human brain. The densities of [125I]NT binding sites are markedly decreased in the caudate, putamen, and substantia nigra in MPTP-treated monkeys. These results suggest strong interactions between NT and dopaminergic systems in both monkey and human brain tissues.     

5-HT6 receptor binding sites in schizophrenia and following antipsychotic drug administration: autoradiographic studies with [125I]SB-258585

The 5-hydroxytryptamine (5-HT; serotonin)-6 receptor (5-HT6R) is a putative target of atypical antipsychotic drugs and its mRNA expression is altered in schizophrenia. [125I]SB-258585 is a selective 5-HT6R antagonist which has been well characterized for use in the rat brain. The present study evaluated its suitability for receptor autoradiography in the human brain and its application to quantitative studies. The affinity (K(d) approximately 1.2 nM) and relative distribution of binding sites (striatum > cortex approximately hippocampus) were similar to the rat. The distribution of [125I]SB-258585 binding in these regions was also consistent with that of 5-HT6R mRNA, determined in parallel using in situ hybridization. [125I]SB-258585 binding site densities were measured in dorsolateral prefrontal cortex of 20 patients with chronic schizophrenia and compared with 17 normal subjects. No differences were seen between groups. Neither were [125I]SB-258585 binding site densities affected in the frontal cortex or striatum of rats following 2 weeks' administration of the antipsychotic drugs haloperidol, chlorpromazine, olanzapine, risperidone, or clozapine. In summary, [125I]SB-258585 is a suitable radioligand for studies of human brain 5-HT6R binding sites and shows that their distribution is broadly similar to that of the rodent. The lack of effect of schizophrenia or antipsychotic drug administration on [125I]SB-258585 binding suggests that an altered receptor density does not contribute to any involvement which the 5-HT6R may have in the disease or its treatment.     

Stromal cell-derived factor 1alpha mediates neural progenitor cell motility after focal cerebral ischemia.

In the adult rodent, stroke induces an increase in endogenous neural progenitor cell (NPC) proliferation in the subventricular zone (SVZ) and neuroblasts migrate towards the ischemic boundary. We investigated the role of stromal cell-derived factor 1alpha (SDF-1alpha) in mediating NPC migration after stroke. We found that cultured NPCs harvested from the normal adult SVZ, when they were overlaid onto stroke brain slices, exhibited significantly (P<0.01) increased migration (67.2+/-25.2 microm) compared with the migration on normal brain slices (29.5+/-29.5 microm). Immunohistochemistry showed that CXCR 4, a receptor of SDF-1alpha, is expressed in the NPCs and migrating neuroblasts in stroke brain. Blocking SDF-1alpha by a neutralizing antibody against CXCR 4 significantly attenuated stroke-enhanced NPC migration. ELISA analysis revealed that SDF-1alpha levels significantly increased (P<0.01) in the stroke hemisphere (43.6+/-6.5 pg/mg) when compared with the normal brain (25.2+/-1.9 pg/mg). Blind-well chamber assays showed that SDF-1alpha enhanced NPC migration in a dose-dependent manner with maximum migration at a dose of 500 ng/mL. In addition, SDF-1alpha induced directionally selective migration. These findings show that SDF-1alpha generated in the stroke hemisphere may guide NPC migration towards the ischemic boundary via binding to its receptor CXCR 4 in the NPC. Thus, our data indicate that SDF-1alpha/CXCR 4 is important for mediating specific migration of NPCs to the site of ischemic damaged neurons.