Binding In Thalamus Research Articles

Detection and quantification of remote microglial activation in rodent models of focal ischaemia using the TSPO radioligand CLINDE

Neuroinflammation is involved in stroke pathophysiology and might be imaged using radioligands targeting the 18 kDa translocator protein (TSPO). We studied microglial reaction in brain areas remote from the primary lesion site in two rodent models of focal cerebral ischaemia (permanent or transient) using [125I]-CLINDE, a promising TSPO single photon emission computed tomography radioligand. In a mouse model of permanent middle cerebral artery occlusion (MCAO), ex vivo autoradiographic studies demonstrated, besides in the ischaemic territory, accumulation of [125I]-CLINDE in the ipsilateral thalamus with a binding that progressed up to 3 weeks after MCAO. [125I]-CLINDE binding markedly decreased in animals pre-injected with either unlabelled CLINDE or PK11195, while no change was observed with flumazenil pre-treatment, demonstrating TSPO specificity. In rats subjected to transient MCAO, [125I]-CLINDE binding in the ipsilateral thalamus and substantia nigra pars reticulata (SNr) was significantly higher than that in contralateral tissue. Moreover, [125I]-CLINDE binding in the thalamus and SNr was quantitatively correlated to the ischaemic volume assessed by MRI in the cortex and striatum, respectively. Clinical consequences of secondary neuronal degeneration in stroke might be better treated thanks to the discrimination of neuronal processes using in vivo molecular imaging and potent TSPO radioligands like CLINDE to guide therapeutic interventions.

High resolution micro‐SPECT scanning in rats using 125I β‐CIT: Effects of chronic treatment with carbamazepine

Carbamazepine (CBZ) is a first-line antiepileptic agent with mood-stabilizing effects in bipolar disorder. It has been reported to influence extracellular concentrations of serotonin and dopamine, suggesting an interaction with monoamine transporters. We have investigated this effect using in vivo single photon emission computed tomography (SPECT) in rats. Adult male rats received 3 mg/kg/h CBZ via mini-osmotic pump. After 14 days continuous treatment, animals underwent two consecutive SPECT scans, using 125I beta-CIT as a radiotracer to label serotonin transporter (SERT) and dopamine transporter (DAT) sites in the brain. Pharmacologic distinction was enabled by 125I beta-CIT SPECT imaging in rats acutely exposed to the serotonin and dopamine transporter inhibitors, fluoxetine and GBR12909. The interaction between CBZ and 125I beta-CIT binding to SERT and DAT was investigated using in vitro autoradiography. Carbamazepine (10 microm) did not affect binding of 125I beta-CIT to isolated rat brain slices, thereby excluding a direct effect on ligand binding to SERT and DAT. SPECT studies with fluoxetine and GBR12909 highlighted SERT binding in thalamus, hippocampus, centromedial nuclei, and occipital cortex, and DAT binding in the caudate. Prolonged treatment with CBZ failed to influence 125I beta-CIT binding to either SERT or DAT in any of the brain regions examined. This study employed the novel technique of small animal SPECT imaging to investigate the effects of CBZ on monoamine transporters in rat brain. Following prolonged treatment, the drug was without effect on SERT or DAT availability. The mechanism by which CBZ exerts its mood stabilizing effects remains elusive.

Measuring SSRI occupancy of SERT using the novel tracer [123I]ADAM: a SPECT validation study

Serotonergic brain regions play a crucial role in the modulation of emotion, and serotonergic dysfunction may contribute to several neurological disorders. [123I]ADAM is a novel SPECT tracer which binds with high affinity to serotonin transporters (SERT). The objective of this study was to compare different methods for the quantification of tracer binding and to develop a simplified single-scan protocol for this tracer, as well as to investigate its potential for characterisation of the transporter occupancy versus plasma concentration curve of a selective serotonin re-uptake inhibitor (SSRI). Dynamic SPECT scans were performed on 16 healthy volunteers after administration of approximately 150 MBq [123I]ADAM. Data were acquired from the time of injection until approximately 5.5 h after injection in 30- or 45-min sessions. Each subject was scanned twice: with and without pre-treatment with the SSRI citalopram in various dosage regimens. The plasma concentration of citalopram (C(p)) was determined from venous samples. Images were reconstructed by filtered back-projection with scatter and attenuation correction. Tracer binding was quantified for midbrain, striatum and thalamus using cerebellum as a reference region. Quantification was done by kinetic modelling, graphical analysis and multi-linear regression, as well as by the ratio method, with binding potential (BP2) as the outcome measure. The SERT occupancy by citalopram was determined relative to the baseline scan for each subject, and the occupancy versus C(p) curve was fitted with the E(max) model. The highest binding of [123I]ADAM was in midbrain (mean baseline BP2+/-SD=1.31+/-0.29), with lower binding in thalamus (0.79+/-0.16) and striatum (0.66+/-0.13). There was good agreement between BP2 values obtained by different quantification methods. Using the ratio method, the best agreement with kinetic modelling was obtained with data from the time interval [200,260] min after injection. The fitting of the midbrain occupancy curve yielded a maximum occupancy of 84% and a plasma concentration required to reach 50% of the maximum of 2.5 ng/ml, with a goodness-of-fit variability of 13% (SD). Binding of [123I]ADAM to SERT in midbrain can be quantified with a single scan starting 200 min after injection. However, the variability of estimated occupancy values may be too high for critical assessment of occupancy of SERT by SSRI.

In vitro characterization of [ 3H]MethoxyPyEP, an mGluR5 selective radioligand

We have characterized the in vitro properties of 3-[ 3H]methoxy-5-(pyridin-2-ylethynyl)pyridine ([ 3H]MethoxyPyEP), an analogue of the mGluR 5 receptor subtype antagonist MPEP [2-methyl-6-(phenylethynyl)-pyridine], in rat tissue preparations using tissue homogenates and autoradiography. Binding of [ 3H]MethoxyPyEP to rat cortex, hippocampus, thalamus and cerebellum membrane preparations revealed saturable, high affinity binding (3.4 ± 0.4 nM, n = 4 in rat cortex) to a single population of receptors in all regions studied except for cerebellum. Binding was found to be relatively insensitive to pH and insensitive to DTT. High concentrations of NEM both reduce receptor concentration and binding affinity for the radioligand. In time-course studies at room temperature k on and k off were determined as 2.9 × 10 7 M −1 min −1 and 0.11 min −1 respectively. The rank order of affinities, as assessed by equilibrium competition studies, of a variety of ligands suggested binding of the radioligand selectively to mGluR5 (MPEP > trans-azetidine-2,4-dicarboxylic acid ≅ (S)-4-carboxyphenylglycine ≅ (+)MK801 ≅ CP-101,606 ≅ clozapine ≅ atropine ≅ ketanserin ≅ yohimbine ≅ benoxathian). Autoradiographic studies with [ 3H]MethoxyPyEP showed that binding was regioselective, with high density of binding in caudate and hippocampus, intermediate binding in thalamus and very low density in the cerebellum. These data show that [ 3H]MethoxyPyEP is a high affinity radioligand useful for the in vitro study of mGluR5 receptor distribution and pharmacologic properties in brain.

Pertussis toxin decreases absence seizures and GABA B receptor binding in thalamus of a genetically prone rat (GAERS)

Postsynaptic GABA B receptor-mediated events have previously been shown to be reduced by prior treatment with pertussis toxin in rat brain. In the present study genetic absence epilepsy rats from Strasbourg (GAERS) were given single bilateral injections of pertussis toxin (PTx 0.4 μg), denatured-PTx or vehicle saline into the relay nuclei of the thalamus under anaesthesia. After recovery the spike and wave discharge duration (SWD) was monitored for up to 6 days following which the brains were removed and GABA B or GABA A receptor autoradiography performed on 10 μm transverse sections. By 6 days the SWD of the rats treated with PTx was suppressed by 96% compared with vehicle-injected rats with a significant (62%) reduction even after 1 day. Denatured toxin had no effect at any time. After 6 days GABA B, but not GABA A, receptor binding was significantly reduced by 70–80% in the ventrolateral and ventral posteriolateral thalamic nuclei. No changes in other brain regions were detected and denatured toxin failed to alter GABA A or GABA B receptor binding in any brain region. These data implicate G-protein mechanisms in the generation of SWD in GAERS and support the role of GABA B receptors in their induction within the thalamus.

Steroid-inhibition of [ [formula omitted]]γ-hydroxybutyric acid (GHB) binding in thalamic relay nuclei increases during absence seizures

γ-Hydroxybutyric acid (GHB), a naturally occurring analog of GABA, induces absence-like seizures in rats. We characterized the interaction of 3α-hydroxy steroids, alphaxalone and tetrahydrodeoxycorticosterone (which are potent modulators of GABA A receptors) with GHB binding sites in rat brain cortical membranes. The steroids inhibited [ 3 H ]GHB binding in a dose-dependent fashion (IC 50∼1 μM). Neither bicuculline nor GABA altered the dose–response of steroids in the [ 3 H ]GHB assay, suggesting that there was no GABA A component involved in the steroid-inhibition of [ 3 H ]GHB binding. Also, non 3α-hydroxy steroids were inactive in displacing [ 3 H ]GHB. Because GHB-induced absence seizures evolve most readily from layers I–IV of frontoparietal cortex and thalamic relay nuclei, we determined if the interaction of steroids with GHB binding sites in layers I–IV of frontoparietal cortex and thalamic relay nuclei was altered during GHB-induced absence seizures. We found that during GHB-seizures steroid-inhibition of [ 3 H ]GHB binding was increased selectively in thalamic relay nuclei but not in the layers I–IV of frontoparietal cortex or any other brain regions tested. This increase in steroid-inhibition of [ 3 H ]GHB binding in thalamus was apparent about 30 min after the onset of seizures, but not at the seizure-onset. As the seizures dissipated, the IC 50 values for steroids rose to the pre-seizure level. These data suggest that the enhancement in steroid-inhibition of [ 3 H ]GHB binding in thalamic relay nuclei observed during GHB-seizures was caused by absence seizures.

Long-term ethanol treatment elicits changes in nicotinic receptor binding in only a few brain regions

Chronic nicotine treatment will produce an upregulation of brain nicotinic receptors, and rats treated for 5 months with ethanol had increased [ 3H]nicotine binding in two of the three brain regions that were studied. However, several studies using short-term treatment did not detect an effect of ethanol on mouse brain nicotinic receptor numbers. Therefore, LS and SS mice were force-fed ethanol (15%, v/v) in the drinking water for 6 months. The LS mice developed tolerance to ethanol as measured by Y-maze crossing and rearing activity, body temperature, and sleep time. No evidence for tolerance to ethanol was seen in the SS mice. However, the SS mice showed increases in [ 3H]nicotine binding in thalamus and an increase in [ 125I]α-bungarotoxin binding in the cerebellum and superior colliculus. LS mice had reduced levels of hippocampal [ 125I]α-bungarotoxin binding. Thus, long-term ethanol treatment may affect brain nicotinic receptor binding but the effect is limited to only a few brain regions and may be influenced by genetic factors.

Opioid receptor imaging and displacement studies with [6- O-[ 11C]methyl]buprenorphine in baboon brain

Buprenorphine (BPN) is a mixed opiate agonist-antagonist used as an analgesic and in the treatment of opiate addiction. We have used [6- O-[ 11C]methyl]buprenorphine ([ 11C]BPN) to measure the regional distribution in baboon brain, the test-retest stability of repeated studies in the same animal, the displacement of the labeled drug by naloxone in vivo, and the tissue distribution in mice. The regional distribution of radioactivity in baboon brain determined with PET was striatum > thalamus > cingulate gyrus > frontal cortex > parietal cortex > occipital cortex > cerebellum. This distribution corresponded to opiate receptor density and to previously published data (37). The tracer uptake in adult female baboons showed no significant variation in serial scans in the same baboon with no intervention in the same scanning session. HPLC analysis of baboon plasma showed the presence of labeled metabolites with 92% ± 2.2% and 43% ± 14.4% of the intact tracer remaining at 5 and 30 min, respectively. Naloxone, an opiate receptor antagonist, administered 30–40 min after tracer injection at a dose of 1.0 mg/kg i.v., reduced [ 11C]BPN binding in thalamus, striatum, cingulate gyrus, and frontal cortex to values 0.25 to 0.60 of that with no intervention. There were minimal (<15%) effects on cerebellum. Naloxone treatment significantly reduced the slope of the Patlak plot in receptor-containing regions. These results demonstrate that [ 11C]BPN can be displaced by naloxone in vivo, and they affirm the feasibility of using this tracer and displacement methodology for short-term kinetics studies with PET. Mouse tissue distribution data were used to estimate the radiation dosimetry to humans. The critical organ was the small intestine, with a radiation dose estimate to humans of 117 nrad/mCi.

Presynaptic GABA B-and γ-hydroxybutyric acid-mediated mechanisms in generalized absence seizures

γ-Hydroxybutyric acid (GHB) is a naturally occurring compound which has the ability to induce generalized absence seizures when given to animals. This effect of GHB may be blocked by either GHB or GABA B receptor antagonists. We sought to test the hypothesis that pre-synaptic GHB- and GABA B-mediated mechanisms in thalamus and cortex are operative in the GHB model of generalized absence seizures. Presynaptic Ca 2+-dependent K + efflux was determined using Ca 2+-stimulated Rb 86 efflux in synaptosomes prepared from thalamus and cortex in the presence of GHB, a specific GHB receptor antagonist, the specific GABA B agonist (−)baclofen, or the specific GABA B antagonists, phaclofen and CGP 35348. The effect of these compounds was determined also on basal and K +-stimulated 45Ca 2+ uptake and basal and K +-stimulated synaptosomal cytosolic Ca 2+([Ca 2] i) in synaptosomes prepared from thalamus and cortex and on [ 125I]ω-conotoxin binding in thalamus and cortex using autoradiographic binding techniques. There was no demonstrable change in Ca 2+-stimulated Rb 86 efflux in any experimental condition studied; however GHB and (−)baclofen both suppressed K +-stimulated 45Ca 2+ uptake and [Ca 2] i in synaptosomes and were associated with a decrease in [ 125I]ω-conotoxin binding which achieved statistical significance only in frontal cortex, a brain region selectively involved in the genesis of GHB-induced absence seizures. The effects of GHB and (−)baclofen on K +-stimulated 45Ca 2+ uptake and [Ca 2], in synaptosomes were additive. The effects of GHB in this regard were attenuated by the GHB antagonist and phaclofen while that of (−)baclofen was attenuated by CGP 35348. These data do not support the hypothesis that the GHB and GABA B receptor are one and the same. Rather, they raise the possibility that a presynaptic GHB/GABA B receptor complex might be involved in the pathogenesis of GHB-induced generalized absence seizures.

The distribution of ω-conotoxin MVIIC nle-binding sites in rat brain measured by autoradiography

An analogue of ω-conotoxin MVIIC,[ 125I]ω-MVIIC nle, has been employed in an autoradiographic assay to define the distribution of binding sites in rat brain of this neuronal calcium channel antagonist. In comparison with N-type channels (labeled by [ 125I]ω-conotoxin GVIA), ω-MVIIC nle sites are much denser in cerebellum (molecular layer) than in forebrain. Binding in thalamus is also comparatively high for ω-MVIIC nle. Under these conditions, [ 125I]ω-MVIIC nle binding to rat brain sections is not displaceable by the N-channel antagonist, ω-conotoxin GVIA. The calcium channel blocker [ 125I]ω-conotoxin MVIIC nle labels a unique set of binding sites in mammalian brain.

Regional changes in [ 3H]inositol 1,4,5-triphosphate binding in the gerbil brain following repeated sublethal ischemia

We investigated the regional changes in [ 3H]inositol 1,4,5-triphosphate (IP 3) binding in the brain following ischemia using in vitro autoradiography. Three 2-min ischemic insults at 1-hr intervals and a 6-min period of ischemia were induced in gerbils and they were killed after 1, 4, and 28 days. Normal animals had high [ 3H]IP 3 binding in the CA1 subfield of the hippocampus and the striatum. The binding in the CA1 decreased strikingly after both 6-min ischemia and three 2-min ischemic insults. The [ 3H]IP 3 binding also decreased in the lateral striatum after three 2-min ischemic insults but not after 6 min of ischemia. Histological observations confirmed neuronal damage to these areas of reduced binding. By contrast, we found a marked increase in [ 3H]IP 3 binding in the ventral thalamus 28 days after three 2-min ischemic insults. Histological observations with Nissl staining revealed an accumulation of fine granular deposits there. Thus, repeated ischemic insults produced more extensive neuronal damage and changes in [ 3H]IP 3 binding than a single equivalent period of ischemia. The increased [ 3H]IP 3 binding in the thalamus coincidentally with an accumulation of Nissl-positive granules at the chronic stage after repeated ischemia is of considerable interest.