Positron emission tomographic studies of cerebral benzodiazepine-receptor binding in chronic alcoholics

Positron emission tomography was used with [¹¹C] flumazenil (FMZ) and [¹⁸F] fluorodeoxyglucose to study GABA type A/benzodiazepine (GABA-A/BDZ) receptors and cerebral metabolic rates for glucose (1CMRglc) in 17 male patients with severe chronic alcoholism (ALC), 8 with (ACD) and 9 without alcoholic cerebellar degeneration (non-ACD). In comparison with male normal controls of similar ages, the ALC group had significantly reduced FMZ ligand influx (K₁), FMZ distribution volume (DV), and 1CMRglc bilaterally in the medial frontal lobes, including superior frontal gyrus and rostral cingulate gyrus; the ACD group had significant reductions of K₁, DV, and ICMRglc bilaterally in the same distribution, and also in the superior cerebellar vermis; and the non-ACD group had significant reductions of K₁, DV, and ICMRglc bilaterally in the same regions of the frontal lobes but not in the superior cerebellar vermis. When compared with the non-ACD group, the ACD group had significant reductions of K₁ and DV bilaterally in the superior cerebellar vermis. The results suggest that severe chronic alcoholism damages neurons containing GABA-A/BDZ receptors in the superior medial apsects of the frontal lobes, and in patients with clinical signs of ACD, neurons containing GABA-A/BDZ receptors in the superior cerebellar vermis.     

In vivo [11C] flumazenil-PET correlates with ex vivo [3H] flumazenil autoradiography in hippocampal sclerosis

By using [¹¹C]flumazenil-positron emission tomography ([¹¹C]FMZ-PET), we have previously shown that reductions of central benzodiazepine receptors (cBZRs) are restricted to the hippocampus in mesial temporal lobe epilepsy (mTLE) caused by unilateral hippocampal sclerosis (HS). Receptor autoradiographic studies on resected hippocampal specimens from the same patients demonstrated loss of cBZRs that was over and above loss of neurons in the CA1 subregion. Here, we report the first direct comparison of in vivo cBZR binding with [¹¹C]FMZ-PET and ex vivo binding using [³H]FMZ autoradiography. We applied a magnetic resonance imaging-based method for partial volume effect correction to the PET images of [¹¹C]FMZ volume of distribution ([¹¹C]FMZ V_d) obtained in 10 patients with refractory mTLE due to unilateral, hisologically verifed HS. Saturation autoradiography was performed on the hippocampal specimens obtained from the same patients, allowing calculation of receptor availability ([³H]FMZ B_max). After correction for partial volume effect, [¹¹C]FMZ V_d in the body of epileptogenic hippocampus was reduced by a mean of 42.1% compred with normal controls. [³H]FMZ b_max, determined autoradiographically from the same hippocampal tissue, was reduced by a mean of 42.7% compared with control hippocampi. Absolute in vivo and ex vivo measurements of cBZR binding for the body of the hippocampus were significantly correlated in each individual. Our study demonstrates that reduction of available cBZR on remaining neuron in HS can be reliably detected in vivo by using [¹¹C]FMZ-PET after correction for partial volume effect.     

GABA-A receptor impairment in cerebellar ataxia with anti-glutamic acid decarboxylase antibodies

Antibodies against glutamic acid decarboxylase (GAD-Abs) are associated with cerebellar ataxia, which is refractory to treatment with GABAergic drugs. To investigate the GABAergic neuronal system in vivo, we performed a combined positron emission tomography (PET) study with [¹¹C]-flumazenil and [¹⁸F]-fluorodeoxyglucose (FDG) in three patients with cerebellar ataxia with GAD-Abs. The GABA-A receptor function was investigated using flumazenil, which is a selective GABA-A receptor ligand, while FDG-PET using a three-dimensional stereotactic surface projection analysis was performed to estimate the metabolic rates of glucose (MRGlc) in the patients. GABAergic drugs showed no efficacy for the cerebellar ataxia in all three patients, and all three displayed a significant decrease in flumazenil binding in the cerebellum. No MRGlc decrease in the cerebellum was found in the two patients who presented with amelioration of cerebellar ataxia following intravenous immunoglobulin (IVIG) therapy, whereas a significant MRGlc decrease in the cerebellar hemisphere was observed in another patient who showed severe cerebellar atrophy on magnetic resonance images and no response to the IVIG therapy. The decreased flumazenil binding in the present patients indicated cerebellar GABA-A receptor impairment, which may be due to either neuronal cell loss, as demonstrated by the decreased MRGlc, or a dysfunction in GABAergic neuronal inhibition. Although GAD-Abs have been postulated to prevent the synthesis of GABA, resulting in decreased GABAergic transmission, the GABA-A receptor impairment may play another pathogenic role in cerebellar ataxia associated with GAD-Abs resulting in a condition refractory to GABAergic drug therapy.     

Supporting evidence for negative modulation by protons of an ion channel associated with theN-methyl-D-aspartate receptor complex in rat brain using ligand binding techniques

The addition ofL-glutamic acid (Glu) alone, both Glu and glycine (Gly) or Glu/Gly/spermidine (SPD) was effective in potentiating[³H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding before equilibrium to an ion channel associated with theN-methyl-D-aspartate (NMDA) receptor complex in brain synaptic membranes extensively washed and treated with Triton X-100. The binding dependent on Glu almost linearly increased in proportion to decreasing proton concentrations at a pH range of 6.0 to 9.0 in external incubation medium, while a Gly-dependent portion of the binding increased with decreasing proton concentrations up to a pH of 7.5 with a plateau thereafter. In contrast, the SPD-dependent binding increased in proportion to decreasing proton concentrations up to a pH of 7.0 with a gradual decline thereafter. Similar profiles were also obtained with [³H]MK-801 binding at equilibrium, with an exception that significant binding of [³H]MK-801 was detected in the absence of any added agonists. The potency of SPD to potentiate [³H]MK-801 binding before equilibrium increased in proportion to decreasing proton concentrations, with those of both Glu and Gly being unchanged. In contrast, the ability of (+)MK-801 to displace [³H]MK-801 binding at equilibrium was not significantly affected by a decrement of external proton concentrations from pH 7.5 to pH 8.5 in the presence of Glu/Gly and Glu/Gly/SPD added. However, similar changes in external proton concentrations did not similarly affect binding of several radioligands for the NMDA and Gly domains on the receptor complex. Decreasing proton concentrations were effective in exponentially potentiating binding of [³H]SPD at a pH range of 6.0 to 9.0 without virtually altering [³H]D, L-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid binding. In addition, [³H]kainic acid binding markedly decreased with decreasing proton concentrations only in the presence of Ca²⁺ ions. These results suggest that protons negatively modulate neuronal responses mediated by the NMDA receptor ionophore complex through interference with opening mechanisms of the channel domain without disturbing association processes of the endogenous agonists with the respective recognition domains in rat brain. Moreover, possible modulation by protons of responses mediated by the kainate receptor in the presence of Ca²⁺ ions at concentrations that occur in vivo is also suggested.     

Differential coupling of GABA-A and GABA-B receptors to the noradrenergic system

Summary The GABA-A receptor agonist THIP, or the mixed GABA-A/GABA-B receptor agonist progabide dose dependency increased the release of norepinephrine (as measured by the production of MHPG) in the cerebral cortex and hippocampus. This effect was partially reversed by treatment with the GABA-A receptor antagonist bicuculline. In contrast, the GABA-B receptor agonist baclofen decreased the release of norepinephrine in the cerebral cortex and hippocampus. Pretreatment with the presynaptic noradrenergic neurotoxin DSP₄ increased the B_max for beta-adrenergic receptor binding in the cerebral cortex and hippocampus. This effect was partially prevented by chronic (14 day) treatment with either the beta-adrenergic agonist clenbuterol or the GABA-B receptor agonist baclofen. In contrast, chronic (14 day) administration with either the GABA-A receptor agonist THIP or the antidepressant imipramine failed to alter the increase in betaadrenergic receptor binding produced by DSP₄ pretreatment. These data suggest that the GABA-A receptor may be coupled to the presynaptic noradrenergic neuron and modulate the release of norepinephrine, while the GABA-B receptor is coupled to the postsynaptic noradrenergic neuron and likely functions through the cyclic AMP generating system.     

A possible mechanism of the nucleus accumbens and ventral pallidum 5-HT1B receptors underlying the antidepressant action of ketamine: a?PET study with macaques

Ketamine is a unique anesthetic reagent known to produce various psychotic symptoms. Ketamine has recently been reported to elicit a long-lasting antidepressant effect in patients with major depression. Although recent studies provide insight into the molecular mechanisms of the effects of ketamine, the antidepressant mechanism has not been fully elucidated. To understand the involvement of the brain serotonergic system in the actions of ketamine, we performed a positron emission tomography (PET) study on non-human primates. Four rhesus monkeys underwent PET studies with two serotonin (5-HT)-related PET radioligands, [¹¹C]AZ10419369 and [¹¹C]DASB, which are highly selective for the 5-HT1B receptor and serotonin transporter (SERT), respectively. Voxel-based analysis using standardized brain images revealed that ketamine administration significantly increased 5-HT1B receptor binding in the nucleus accumbens and ventral pallidum, whereas it significantly reduced SERT binding in these brain regions. Fenfluramine, a 5-HT releaser, significantly decreased 5-HT1B receptor binding, but no additional effect was observed when it was administered with ketamine. Furthermore, pretreatment with 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), a potent antagonist of the glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, blocked the action of ketamine on the 5-HT1B receptor but not SERT binding. This indicates the involvement of AMPA receptor activation in ketamine-induced alterations of 5-HT1B receptor binding. Because NBQX is known to block the antidepressant effect of ketamine in rodents, alterations in the serotonergic neurotransmission, particularly upregulation of postsynaptic 5-HT1B receptors in the nucleus accumbens and ventral pallidum may be critically involved in the antidepressant action of ketamine.     

Decrease in δ-opioid receptor density in rat brain after chronic [d-Ala,d-Leu]enkephalin treatment

Chronic treatment of Sprague-Dawley rats with [d-Ala²,d-Leu⁵]enkephalin (DADLE) resulted in the development of tolerance to the antinociceptive effect of this opioid peptide. When opioid receptor binding was measured, time-dependent decreases in [³H]diprenorphine binding to the P₂ membranes prepared from the cortex, midbrain and striatum were observed. Scatchard analysis of the saturation binding data revealed a decrease in B_max values and no change in the K_d values of [³H]diprenorphine binding to these brain regions, indicative of down-regulation of the receptor. This reduction in the opioid receptor binding activities could be demonstrated to be due to the DADLE effect on the δ-opioid receptors in these brain regions. When [³H]DADLE binding was carried out in the presence of morphiceptin, a significant reduction in the δ-opioid receptor binding was observed in all brain areas tested. μ-Opioid receptor binding decrease was observed only in the striatum after 5 days of DADLE treatment. Additionally, the onset of δ-opioid receptor decrease in the midbrain area was rapid, within 6 h of the initiation of the chronic DADLE treatment. Thus, analogous to previous observations in which chronic etorphine treatment preferentially reduced μ-opioid receptor binding, chronic DADLE treatment preferentially reduced δ-opioid receptor binding activity.     

Development of a micromethod to study the Na-independentl-[H]glutamic acid binding to rat striatal membranes. II. Effects of selective striatal lesions and deafferentations

An apparent single class of Na⁺-independentl-[³H]glutamate (l-[³H]Glu) binding sites was biochemically and pharmacologically identified on rat striatal tissue. The K_d value was 1.75 μM and the B_max3.89 nmol/g protein. In order to further elucidate the putative physiological role of these binding sites and to valid our binding assay, experiments were conducted to determine the anatomical location of thel-[³H]Glu binding sites in the striatum. Local injection of the neurotoxin, kainic acid into the striatum caused degeneration of target cells in the structure followed by an important decrease (-37%) in the number of these binding sites, with no significant change in the affinity constant. Lesions of the cortical frontal and parietal areas projecting to the striatum via putative glutamatergic fibers led, on the other hand, after 3 weeks to one month, to a significant increase (+23%) in the number ofl-[³H]Glu binding sites. The K_d value does not significantly change after decortication. Finally, suppression either of the nigrostriatal dopaminergic input or of the partially cholinergic thalamostriatal tract do not affect [³H]Glu binding to striatal tissue. These results suggest that about 40% of our [³H]Glu binding sites are located on striatal target cell sensitive to the neurotoxic effect of kainic acid. Therefore, they seem to be partly postsynaptic. The existence of a relation between these binding sites and the corticostriatal glutamatergic input was shown by the development of a supersensitivity response after suppression of this cortical input. These sites might therefore constitute one of the receptor subclass for Glu linked to the excitatory action of the corticostriatal afferent. Since degeneration of either the nigrostriatal dopaminergic or the thalamostriatal fibres did not affect the binding ofl-[³H]Glu characterized in our study, the binding sites will probably not occur at the presynaptic level on these nerve terminals in the striatum.     

Phylogenetic distribution of [H]kainic acid receptor binding sites in neuronal tissue

The phylogenetic distribution of specific binding sites for kainic acid was determined in 14 species including invertebrates and vertebrates. The highest level of binding was observed in brains of the frog (Xenopus laevis), followed by the spiny dogfish (Heterodontus francisci), the goldfish (Carasius auratus) and the chick (Gallus domesticus). Although significant specific binding was noted in some of the lowest forms tested (e.g. Hydra littoralis), this was not a consistent observation in the invertebrates. In most cases, specific binding to both high and low affinity sites was detected; notable exceptions were the cockroach brain (Periplaneta americana), which had negligible high affinity binding, and the crayfish brain (Procambarus) which had negligible low affinity binding. In the spiny dogfish, the smooth dogfish and the chick, the highest level of binding occurred in cerebellum with less in the forebrain and the least in the medulla; in the mammalian species, the highest level of binding occurred in the forebrain structures with less in the cerebellum and least in the medulla.Eadie plots of the saturation isotherms for [³H]kainic acid revealed similar kinetics of binding for frog whole brain, rat forebrain and human parietal cortex with two apparent populations of binding sites: K_D1 = 25–50nM andK_D2 = 3–14nM. While binding in the spiny dogfish forebrain and human caudate nucleus occurred exclusively at a high affinity component, the cerebella of chick, rat and man exhibited only a low affinity binding site. In the 3 species studied most extensively, frog, rat and man, unlabeled kainic acid was the most potent inhibitor of the specific binding of [³H] kainic acid. l-Glutamic acid was 20–200-fold less potent than kainic acid, and d-glutamic acid was 4–2500-fold less potent than its l-isomer. Reduction of the isopropylene side chain of kainic acid to form dihydrokainic acid decreased the affinity of the derivative 115–30,000-fold. Hill coefficients derived from these displacement curves were 1.0 for unlabeled kainic acid but approximately 0.5 for l- and d-glutamic acids and dihydrokainic acid, which is compatible with negative cooperativity. In summary, these studies demonstrated a widespread distribution throughout the animal kingdom of specific binding sites for kainic acid in neural tissue; the characteristics of these receptor sites are remarkably similar from primitive vertebrates to man.     

Changes in GABAA receptor properties in amygdala kindled animals: in vivo studies using [11C]flumazenil and positron emission tomography

Purpose: The purpose of the present investigation was to quantify alterations in GABA_A receptor density in vivo in rats subjected to amygdala kindling. Methods: The GABA_A receptor density was quantified by conducting a [¹¹C]flumazenil (FMZ) positron emission tomography (PET) study according to the full saturation method, in which each animal received a single injection of FMZ to fully saturate the GABA_A receptors. Subsequently, the concentration‐time curves of FMZ in blood [using high‐pressure liquid chromatography with UV detector (HPLC‐UV) or high‐performance liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS)] and brain (with PET‐scanning) were analyzed by population modeling using a pharmacokinetic model, containing expressions to describe the time course of FMZ in blood and brain. Results: The GABA_A receptor density (B_max) in kindled rats was decreased by 36% compared with controls. This is consistent with a reduction of 28% in electroencephalography (EEG) effect of midazolam in the same animal model, suggesting that a reduced number of GABA_A receptors underlies the decreased efficacy of midazolam. Furthermore, receptor affinity (K_D) was not changed, but the total volume of distribution in the brain (V_Br), is increased to 178% of control after kindling, which might indicate an alteration in the transport of FMZ across the blood–brain barrier. Conclusions: Both the GABA_A receptor density (B_max), and possibly also the blood–brain barrier transport of FMZ (V_Br) are altered after kindling. Furthermore, this study indicates the feasibility of conducting PET studies for quantifying moderate changes in GABA_A receptor density in a rat model of epilepsy in vivo.     

Reduced Binding Potential of GABA-A/Benzodiazepine Receptors in Individuals at Ultra-high Risk for Psychosis: An [18F]-Fluoroflumazenil Positron Emission Tomography Study

Background:

Altered transmission of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter, may contribute to the development of schizophrenia. The purpose of the present study was to investigate the presence of GABA-A/benzodiazepine (BZ) receptor binding abnormalities in individuals at ultra-high risk (UHR) for psychosis in comparison with normal controls using [¹⁸F]-fluoroflumazenil (FFMZ) positron emission tomography (PET). In particular, we set regions of interest in the striatum (caudate, putamen, and nucleus accumbens) and medial temporal area (hippocampus and parahippocampal gyrus).

Methods:

Eleven BZ-naive people at UHR and 15 normal controls underwent PET scanning using [¹⁸F]-FFMZ to measure GABA-A/BZ receptor binding potential. The regional group differences between UHR individuals and normal controls were analyzed using Statistical Parametric Mapping 8 software. Participants were evaluated using the structured interview for prodromal syndromes and neurocognitive function tasks.

Results:

People at UHR demonstrated significantly reduced binding potential of GABA-A/BZ receptors in the right caudate.

Conclusions:

Altered GABAergic transmission and/or the imbalance of inhibitory and excitatory systems in the striatum may be present at the putative prodromal stage and play a pivotal role in the pathophysiology of psychosis.Key words: GABA, schizophrenia, ultra-high, risk for psychosis, caudate, PET, fluoroflumazenil     

The distribution and function of DL-[H]2-Amino-4-Phosphonobutyrate binding sites in the rat striatum

The binding of the glutate-like radioligand,DL-[³H]2-amino-4-phosphhonobutyrate (DL-[³H]APB), to L-glutamate-sensitive sites in the rat striatum was investigated. A single, saturable population of binding sites, indistinguishable from that characterized previously on rat whole brain synaptic membranes, was identified. The effects of specific lesions of the striatum: (a) decortication; (b) striatal injection of kainic acid; and (c) 6-hydroxydopamine injections into the substantia nigra, were also examined. SpecificDL-[³H]APB binding in the striatum was elevated significantly following decortication. An increase in the number of binding sites was found to be responsible for this enhancement in binding. Lesions of the postsynaptic tartets of corticostriatal fibres reduced the number of DL-[³H]APB binding sites in the striatum without affecting binding site affinity. This finding suggests thatL-APB sensitive excitatory amino acid receptors are located predominantly on membranes derived from structures postsynaptic with regard to the glutamatergic innervation. The possible physiological role of these receptors was examined using an in vitro release technique. BothL-glutamate and L-APB were found to facilitate potassium evoked [³H]dopamine release from striatal slices. This finding supports the proposed existence of functional acidic amino acid receptors on dopaminergic terminals in the striatum. These receptors may play an important role in the control of motor function.     

Age-related modifications on the GABAA receptor binding properties from Wistar rat prefrontal cortex

In the present communication we have investigated the pharmacological properties of the GABA_A receptor from adult (3 months old) and aged (24 months old) Wistar rat prefrontal cortex. The prefrontal cortex is implicated in cognitive functions and stress and both processes seem to be altered during aging. These changes could be mediated by modifications in the GABA_A receptor properties. Our results indicated the absence of generalized age-related modifications on the pharmacological properties of the GABA_A receptor from prefrontal cortical membranes. Saturation experiments using the non-selective benzodiazepine [³H]flunitrazepam revealed that neither the K_d values or the B_max were modified during aging. Moreover, Cl 218 872 displacement of [³H]flunitrazepam showed no age-related modifications on either the K_is or the relative proportion between the Type I and Type II benzodiazepine binding sites. Therefore, the benzodiazepine binding sites are well preserved in aged prefrontal cortex. On the other hand, saturation experiments using the GABA agonist [³H]muscimol demonstrated a decrease in the B_max of the low affinity [³H]muscimol binding sites in aged rats (4.3 ± 0.8 pmol/mg protein vs. 2.3 ± 0.2 pmol/mg protein in adult and aged rats, respectively). However, no age-dependent modifications were observed in the allosteric interaction between GABA and benzodiazepine binding sites. These results demonstrate that the benzodiazepine binding sites and the GABA binding sites of the GABA_A receptor complex from rat prefrontal cortical membranes are differentially affected by the aging process.     

Regional changes in [H]d-aspartate and [H]TCP binding sites in Alzheimer's disease brains

The specific binding of [³H]d-aspartate, a marker for the presynaptic glutamate uptake site, and [³H]N-(1-[2-Thienyl]cyclohexyl)-piperidine ([³H]TCP), a high affinity ligand for theN-methyl-d-aspartate (NMDA)-associated phencyclidine binding site, was measured in homogenates of brain from normal subjects and from subjects with neuropathologically confirmed Alzheimer's disease. Alzheimer's disease was associated with a reduction in [³H]d-aspartate binding density in temporal cortex and caudate nucleus. By contrast, a reduction in the receptor density for [³H]TCP binding was only recorded in the frontal cortex. Thus, glutamate-containing nerve terminals are severely reduced in Alzheimer's disease, whilst the postsynaptic NMDA-phencyclidine receptor complex is much less affected. These findings have implications for theories of glutamate neurotoxicity in Alzheimer's disease.     

Differential labeling of dopamine and sigma sites by [H]nemonapride and [H]raclopride in postmortem human brains

The difference between the binding of [³H]nemonapride and [³H]raclopride has been used to quantify dopamine D₄ receptors in postmortem schizophrenic brain studies. Recent work, however, has suggested that at least part of the differential between [³H]nemonapride and [³H]raclopride binding may represent σ rather than D₄ receptor sites. We applied the nemonapride-raclopride subtraction method to postmortem, non-schizophrenic human striatum to examine the variation in dopaminergic receptor binding labeled by these ligands. Variation in σ receptor binding labeled by [³H]nemonapride was studied in frontal cortex, striatum and cerebellum. Specific binding was defined by sulpiride (dopamine receptor ligand), PPAP (σ receptor ligand) and haloperidol (mixed dopaminergic/σ agent), respectively. Haloperidol defined a combination of sites, which were approximately the sum of the dopaminergic and σ components defined by sulpiride and PPAP, respectively. Significant inter-individual variation in the amount of specific binding for dopaminergic and σ receptor sites was observed. However, no significant nor consistent observation of striatal dopamine D₄ receptors or D₄-like binding sites was observed in the striatum even though two independent sets of tissues, with different dissections were used. The inconsistencies in some previous postmortem studies appear to be at least partially explained by the inclusion of both σ and dopaminergic components in [³H]nemonapride binding and the inherent high inter-individual variability of the different components.     

Quantitative analysis of [H]spiroperidol binding to rat forebrain sections: Plasticity of neostriatal dopamine receptors after nigrostriatal injury

The binding of [³H]spiroperidol to rat coronal sections in vitro was investigated using two procedures: swabbing studies, in which the tissue sections are wiped from the microscope slides after incubation in the presence of [³H]spiroperidol, and autoradiographic studies, in which the autoradiographic negatives are analyzed using computer-assisted densitometry. In the swabbing studies, the pharmacological and kinetic properties of butaclamol-displaceable binding were investigated, and the following results suggest that [^3H]spiroperidol binds specifically to only a single site within the basal forebrain of tissue sections and that the site is the dopamine D-2 receptor. (1) The pseudo-first order and first order plots for the rate of association to and dissociation from tissue sections appeared to be linear. (2) Dopamine antagonists, such asd haloperidol and butaclamol, were much more effective than dopamine agonists or the serotonin S-2 ligand, ketanserin, in inhibiting [³H]spiroperidol binding. (3) The ability of dopamine agonists to inhibit [³H]speroperidol binding was markedly reduced by the guanine nucleotide, Gpp(NH)p. (4) Saturation analysis of specific [³H]speroperidol binding revealed aK_d and B_max of 0.93 nM and 447 fmol/mg protein, and a Hill coefficient of 1.05. The findings are also compatible with the possibility that [³H]spiroperidol binds to several sites that have identical affinities for this ligand.Densitometric studies were used to assess the effect of lesions on [³H]spiroperidol binding in the neostriatum. Intrastriatal injection of kainic acid substantially reduced 1 μM (+)—butaclamol-displaceable binding, indicating that the receptors are in large part on intrinsic striatal neurons. Neostriatal [³H]spiroperidol binding was investigated 7 days after destruction of the mesotelencephalic dopamine system by the ventral tegmental injection of 6-hydroxydopamine. As determined by saturation analysis, the average values forK_d and B_max were 0.66 nM and 1212 fmol/mg protein in the intact striatum, and 0.82 nM and 1504 fmol/mg in the denervated striatum. The finding of a significant 23.8% increase in receptor density by the end of the first postoperative week, a period during which behavioral supersensitivity to apomorphine increases rapidly, supports the hypothesis that a proliferation of D-2 receptors underlies the behavioral manifestations of denervation supersensitivity.     

Comparative studies on binding of 3 different ligands to theN-methyl-d-aspartate recognition domain in brain synaptic membranes treated with Triton X-100

Treatment with a low concentration of Triton X-100 almost tripled the binding of [³H]d,l-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653), a novel competetive antagonist at anN-methyl-d-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, in synaptic membranes of the rat brain. The binding linearly increased with increasing protein concentrations of up to 0.4 mg/ml and also increased in proportion to incubation time with a plateau within 60 min after the initiation of incubation at 2°C in Triton-treated membranes. Elevation of incubation temperature from 2°C to 30°C resulted in a marked decrease in the binding at equilibrium by 80%, and a maximal level was obtained within 1 min after the initiation of incubation at 30°C with a gradual decline of up to 10 min. Bound [³H]CGP 39653 was rapidly dissociated by the addition of excess unlabeledl-glutamic acid (Glu), and the time required to attain complete dissociation was 60 min at 2°C and 1 min at 30°C, respectively. Among several agonists and antagonists tested, Glu was the most potent displacer of [³H]CGP 39653 binding with progressively less potent displacement byd-2-amino-5-phosphonovaleric,(±)-3-(2-carboxypiperain-4-yl)propyl-1-phosphonic (CPP),d-2-amino-7-phosphonoheptanoic,N-methyl-d-aspartic andN-methyl-l-aspartic acids. Agonists at the glycine (Gly) domain on the NMDA receptor ionophore complex invariably inhibited the binding of [³H]CGP 39653 without virtually affecting the binding of [³H]CPP or [³H]Glu in a manner that was sensitive to antagonism by 4 different antagonists at the Gly domain. In contrast, the Gly antagonist 1-hydroxy-3-aminopyrrolidone-2 markedly potentiated [³H]CPP binding with the binding of both [³H]CGP 39653 and [³H]Glu being unaltered. These results suggest that [³H]CGP 39653 may predominantly label an antagonist-preferring form of the NMDA domain in a state different from that favorable to labeling by [³H]CPP.     

Characterization of adenosine receptors in brain using N cyclohexyl [H]adenosine

The presence of distinct binding sites for adenosine in both the CNS and PNS has been proposed in numerous studies. The recent availability of stable adenosine analogues such as cyclohexyladenosine, 2-chloroadenosine and diethylphenylxanthine has made the characterization of such a receptor feasible. In the present report the binding of N⁶ cyclohexyl [³H]adenosine ([³H]CHA) to rat brain synaptosomal membranes is characterized. [³H]CHA binding is saturable and exhibits a biphasic kinetic saturation profile characteristic of 2 binding sites. The high affinity site has a K_d of 0.7 nM and the low affinity site 2.4 nM. The respective B_max values are 230 and 120 fmol/mg protein in rat forebrain. The highest density of binding sites is found in the hippocampus and subcellular distribution studies indicate that the [³H]CHA site is predominantly synaptosomal. [³H]CHA binding is highly dependent on the presence of adenosine deaminase since only 30% of the binding capacity is observed in synaptosomal membranes not treated with this enzyme. Of the many cations and anions tested only copper and zinc have effects on [³H]CHA binding. Both metals are potent inhibitors of binding with copper having an IC₅₀ of 30 μM and zinc 150 μM. Sulfhydryl reducing and alkylating agents also inhibit binding indicating that the binding site is a sulfhydryl-dependent protein.     

Regionally-selective down-regulation of NPY receptor subtypes in the obese Zucker rat. Relationship to the Y5 ‘feeding’ receptor

Experiments were performed to examine whether there are regionally and subtype selective changes in the density of neuropeptide Y (NPY) receptors in the obese Zucker rat which has an increased synthesis and release of NPY confined to the hypothalamus. Competition binding assays were employed to examine the feasibility of using [¹²⁵I]peptide YY ([¹²⁵I]PYY) to measure neuropeptide Y (NPY) Y5 ‘feeding’ receptors in the hypothalamus, hippocampal and cerebral cortex following masking of Y1 and Y2 receptors. Y5 receptors could not be discriminated from the binding to Y1 and Y2 receptors in hypothalamic, hippocampal or cerebral cortex homogenates, possibly owing to the small population of Y5 receptors expressed in the brain and the lack of selective ligands for this receptor. Quantitative receptor autoradiography was used to examine for regional changes in NPY receptor subtypes in obese versus lean Zucker rats. The non-selective Y1, Y2, Y4 and Y5 receptor ligand [¹²⁵I]PYY and the more selective Y1, Y4 and Y5 ligand [¹²⁵I][Leu³¹,Pro³⁴]PYY were employed, in conjunction with masking compounds in an attempt to measure any regional changes in the recently cloned NPY Y5 ‘feeding’ receptor. Specific [¹²⁵I]PYY and [¹²⁵I][Leu³¹,Pro³⁴]PYY binding was significantly reduced in the hypothalamic dorsomedial and arcuate nuclei as well as in the dorsal and lateral (perifornical) areas of obese Zucker rats, as compared to lean rats. In addition there were significant reductions in binding to the thalamic reuniens and centromedial nucleus, and hippocampal dentate gyrus of obese rats as compared to lean rats. Masking [¹²⁵I]PYY binding to Y1 receptors using 1 μM BIBP3226 demonstrated that the reduced NPY receptor density was due to reductions in Y2 or Y5 receptor density. The binding which was sensitive to BIBP3226, i.e. Y1 receptor density, was not different between obese and lean rats. Attempts using [¹²⁵I]PYY and the relatively selective Y2 agonist, [13–36]NPY to mask Y2 receptors and reveal Y5 receptors failed to leave any specific binding suggesting that [13–36]NPY was not selective enough to separate binding to Y2 and Y5 receptors. However, using [¹²⁵I][Leu³¹,Pro³⁴]PYY, masking binding to Y1 receptors using 1 μM BIBP3226 and masking any binding to Y4 using 1 nM rat pancreatic polypeptide left a small amount of binding remaining in the thalamus and hypothalamus, presumably to Y5 receptors which was significantly reduced in obese versus lean rat brain. These data suggest that there is a selective down-regulation in Y5 ‘feeding’ receptors in the obese Zucker rat which is known to possess a hyperactive arcuate-paraventricular NPY system.     

Direct radiolabeling by [H]quisqualic acid of group I metabotropic glutamate receptor in rat brain synaptic membranes

[³H]Quisqualic acid (QA) was synthesized and used to label metabotropic glutamate receptor (mGluR) in rat brain synaptic membranes in the presence of three different ionotropic glutamate receptor agonists at respective saturating concentrations. Of several mGluR agonists tested, group I agonists were more potent in displacing [³H]QA binding than group II and group III agonists in the presence of the three ionotropic agonists. [³H]QA binding was markedly inhibited by guanine nucleotide analogues in a concentration-dependent manner at a concentration range of 10 nM to 1 mM. Scatchard analysis revealed that [³H]QA binding consisted of a single component with a K_d of 50.9±5.3 nM and a B_max of 431.6±18.3 fmol/mg protein. These results suggest that [³H]QA indeed labels group I mGluR functionally coupled to GTP binding protein in rat brain synaptic membranes when determined under the experimental conditions employed.