Regional Variations in [3H]MK801 Binding to Rat Brain N-Methyl-D-Aspartate Receptors

This study examined (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate [( 3H]MK801) binding to the N-methyl-D-aspartate (NMDA) receptor in membranes prepared from six regions of rat brain. Highest levels of binding were found in hippocampus and cortex, whereas much lower densities were found in brainstem and cerebellum. NMDA receptors in cerebellum exhibited a significantly lower affinity for [3H]MK801 than cortical NMDA receptors. To determine whether forebrain and hindbrain NMDA receptors were distinct, the actions of glutamate, NMDA, ibotenate, quinolinate, glycine, and spermine were investigated. These agents increased [3H]MK801 binding in all brain regions examined. However, agonists were uniformly less efficacious in hindbrain compared to forebrain regions. NMDA mimetics and spermine were less potent in cerebellum compared to cortex whereas glycine was equipotent. Antagonists that act at the various modulatory sites on the NMDA receptor were also examined. DL-Amino-phosphonopentanoic acid and 7-chlorokynurenate were approximately equipotent in cortex and cerebellum. However, antagonists that are believed to act inside the NMDA-operated ion channel, including Mg2+ and phencyclidine, were approximately threefold less potent in cerebellum. The diminished regulation of [3H]MK801 binding by glutamate and glycine in the cerebellum was associated with a smaller effect of these agonists on the dissociation of [3H]MK801 from its binding site. The levels of glutamate, aspartate, glycine, serine, and glutamine in the membrane preparations were determined. However, variations in the levels of endogenous amino acids were not sufficient to account for the regional differences in [3H]MK801 binding. These results do not support the hypothesis that a distinct NMDA receptor exists in hindbrian regions of the rat CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of Adenosine Binding Sites in Rat Forebrain and Cerebellum

The metabolically stable adenosine analogue N6-cyclohexyl [3H]adenosine ([3H]CHA) was used to label adenosine receptors in rat forebrain and cerebellum during development. [3H]CHA binding develops rather slowly, with adult binding levels obtained at 24 days in cerebellum and later in the forebrain. Ontogenic profiles in both areas are consistent with the onset of neuronal differentiation. High and low affinity sites appear to develop in parallel, since Scatchard analysis in forebrain tissue obtained from 5-day-old animals revealed both binding sites.     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMDA Receptors in Rat Cerebellum and Forebrain: Subtle Differences in Pharmacology and Modulation

Abstract: Binding of [³H]glutamate, [³H]glycine, and the glutamate antagonist [³H]CGS-19755 to NMDA-type glutamate receptors was examined in homogenates of rat forebrain and cerebellum. Most glutamate agonists had a higher affinity at the [³H]glutamate binding site of cerebellar NMDA receptors as compared with forebrain, whereas all the glutamate antagonists examined showed the reverse relationship. The [³H]glycine binding site of forebrain and cerebellar NMDA receptors showed a similar pharmacology in both brain regions. In the cerebellum, however, [³H]glycine bound to a second site with a 10-fold lower affinity and with a pharmacology that resembled that of the glycine/strychnine chloride channel. [³H]Glutamate binding was not affected by glycine agonists or antagonists, nor was [³H]glycine binding affected by glutamate agonists in either forebrain or cerebellum. Both CGS-19755 and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, glutamate antagonists, reduced [³H]glycine binding in cerebellum, whereas only CGS-19755 was effective in forebrain. Glycine agonists and antagonists modulated [³H]CGS-19755 binding in forebrain and cerebellum to different extents in the two brain regions. From these studies we conclude that the cerebellar NMDA receptor has a different pattern of modulation at glutamate and glycine sites and that glycine may play a more important role in the control of NMDA function in the cerebellum as compared with forebrain.     

A photoactivatable naltrexone derivative labels glycoproteins of different molecular weight corresponding to the mu- and kappa-opioid receptors.

The synthesis and characterization of a novel opioid receptor photoaffinity probe [3H]naltrexyl urea phenylazido derivative ([3H]NUPA) is described. In the absence of light, [3H]NUPA binds with high affinity in a reversible and saturable manner to rat brain and guinea pig cerebellum membranes. Dissociation constants and binding capacities (Scatchard plots) are 0.11 nM and 250 fmol/mg of protein for rat brain and 0.24 nM and 135 fmol/mg of protein for guinea pig cerebellum. Competition experiments indicate that this ligand interacts with high affinity at both mu- and kappa-opioid binding sites while exhibiting low affinity at delta sites (Ki = 21 nM). On irradiation, [3H]NUPA incorporates irreversibly into rat brain and guinea pig cerebellum membranes. SDS gel electrophoresis of rat brain membranes reveals specific photolabeling of a 67-kDa molecular mass band. Conversely, a major component of 58 kDa and a minor component of 36 kDa are obtained from [3H]NUPA-labeled guinea pig cerebellum membranes. Different photolabeling patterns are obtained in rat brain (mu/delta/kappa, 4/5/1) and guinea pig cerebellum (mu+delta/kappa, 1,5/8,5) membranes in the presence of selective opioid ligands indicating labeling of mu and kappa sites, respectively. Thus, [3H]NUPA behaves as an efficient photoaffinity probe of mu- and kappa-opioid receptors, which are probably represented by distinct glycoproteins of 67 and 58 kDa, respectively.     

Comparison of [3H] phencyclidine ([3H] PCP) and [3H] N-[1-(2-thienyl) cyclohexyl] piperidine ([3H] TCP) binding properties to rat and human brain membranes

The investigation of [3H] PCP and [3H] TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for [3H] PCP and [3H] TCP. However, low affinity binding sites were two times more numerous for [3H] PCP than for [3H] TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in the cerebrum. Taken together these results may indicate that in the cerebrum [3H] PCP labels other sites than NMDA/PCP receptor(s), maybe sigma receptors and/or the dopamine uptake complex. In human cerebral cortex samples [3H] TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity (5 times) than corresponding sites in the rat brain. In the human cerebellum [3H] TCP binding parameters were identical to those measured in the same region in the rat.     

Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirenzepine ([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.     

Ontogenetic changes in [3H]-spiroperidol binding sites in posthatch chick brain

The ontogenetic development of [3H]-spiroperidol binding sites was measured in the optic tectum, cerebellum, forebrain base, and forebrain roof of 1-, 4-, and 16-day-old chicks. In the chick optic tectum and cerebellum both the density and the total number of [3H]-spiroperidol binding sites increased from 4- to 16-days-posthatch, but no significant differences were found in either brain area across the initial four posthatch days. In the forebrain base, [3H]-spiroperidol receptor density and total binding increased significantly between 1- and 4-days-posthatch, but at 16-days-posthatch there was a slight decrease in receptor density. Binding sites in the forebrain roof were minimal at all ages. As expected, saturation experiments yielded curvilinear plots indicating the presence of high- and low-affinity binding sites. The high-affinity sites probably reflect dopamine D-2 receptors; whereas, the low-affinity sites may reflect other receptor types, possibly serotonin S-2. These results suggest that large doses of haloperidol, which are normally used in chick behavioral research, may produce behavioral effects by antagonizing multiple receptors.     

Changes of Ganglioside Pattern During Cerebellar Development of Normal and Staggerer Mice

Saturable and specific binding sites for 5-[3H]hydroxytryptamine (5-HT, serotonin) characterized by a KD of 3.5-4.5 nM were detected in the chick embryo brain and were shown to develop linearly as a function of age, weight, and protein content. Saturation and displacement studies using unlabeled 5-HT as the displacing ligand suggested a single population of binding sites. However, displacement studies using 5-methoxytryptamine, lysergic acid diethylamide (LSD), 2-bromo-lysergic acid diethylamide (BOL), methysergide, and spiperone as competing ligands suggested the existence of subclasses of [3H]5-HT binding sites because the Hill coefficients were less than unity. When compared with the reported [3H]5-HT binding sites (5-HT1) in the rat forebrain, the IC50 values of the competing ligands were similar. However, the Hill coefficients for LSD and methysergide were less than unity which suggested that the [3H]5-HT binding sites in the chick embryo brain may be more similar to those found in rat spinal cord than rat forebrain. To study [3H]5-HT binding site regulation and development, various serotonergic compounds were injected into the chorioallantoic fluid of the eggs at different times during embryonic development. Multiple pretreatments with d,l-5-hydroxytryptophan, 5-HT, or BOL were found to have no significant effects on either the affinity (KD) or number (Bmax) of specific [3H]5-HT binding sites. Multiple pretreatments with p-chlorophenylalanine were found to increase the Bmax of specific [3H]5-HT binding by 23% (p less than 0.01) whereas multiple pretreatments with LSD were found to decrease the Bmax of specific binding by 45% (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative autoradiography of Na+-dependent [3H]L-aspartate binding to L-glutamate transporters in rat brain: structure-activity studies using L-trans-pyrrolidine-2,4-dicarboxylate (L-t-PDC) and 2-(carboxycyclopropyl)-glycine (CCG)

Sodium-dependent binding of [3H]L-aspartate was studied in thaw-mounted horizontal sections of fresh-frozen (i.e. not fixed) rat brain. After the incubation with [3H]L-aspartate, the sections were exposed against a 3H-sensitive film and the resulting autoradiograms were evaluated by quantitative densitometry. Effects of several inhibitors were examined and their potency expressed as IC50 and nH. Together with previously published data, the present study supports the view that [3H]L-aspartate binding to fresh-frozen sections of rat brain represents interaction of the radioligand with the substrate-binding sites on glutamate transporters. The most potent inhibitors were (2S,3S,4R)-2-(carboxycyclopropyl)-glycine (L-CCG III) and (2S,4R)-4-methylglutamate. In contrast, L-anti,endo-3,4-methanopyrrolidine dicarboxylate (L-a,e-MPDC) was about an order of magnitude less potent. Only subtle regional variations in the characteristics of inhibitors of [3H]L-aspartate binding were detected. It is not certain whether these differences reflect regional variations in the distribution of individual glutamate transporters or regional peculiarities in their pharmacological characteristics. In particular, (2S,4R)-4-methylglutamate, shown previously to differentiate between GLT-1 (principal glutamate transporter in the forebrain) and GLAST (expressed mainly in the cerebellum), did not strongly differentiate between the binding of [3H]L-aspartate in forebrain and cerebellum. Computer-assisted molecular modelling using selected glutamate analogues with restricted conformation (L-trans-pyrrolidine-2,4-dicarboxylate and four isomers of 2-(carboxycyclopropyl)-glycine: L- and D-CCG I, L-CCG III and L-CCG IV) identified at least one area of unfavourable steric interaction. We conclude that the quantitative autoradiographic studies using [3H]L-aspartate or other transporter-specific ligands, will be a useful tool to study the pharmacology of substrate binding sites on glutamate transporters in the mammalian brain in situ.     

Characterization of "High-Affinity"[3H]Ouabain Binding in the Rat Central Nervous System

The characteristics of [3H]ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of "high-affinity" binding sites with an apparent binding affinity (KD) of 10.4 +/- 0.9 nM and an estimated binding capacity (Bmax) of 7.6 +/- 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus, and frontal cerebral cortex. [3H]Ouabain binding to rat brain was sodium- and ATP-dependent and strongly inhibited by potassium. Proscillariden A was the most potent cardiac glycoside tested in inhibiting specific [3H]ouabain binding to brain membranes, and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that previously reported for inhibition of heart Na,K-ATPase. To assess whether the high-affinity binding sites for [3H]ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal [3H]ouabain binding was examined. Kainic acid lesions of the striatum reduced [3H]ouabain binding to striatal homogenates by 79.6 +/- 1.6%. This suggests that the "high-affinity" [3H]ouabain binding sites measured in our experiments are localized to neuronal elements. Thus, the high-affinity binding of [3H]ouabain to brain membranes may selectively label a neuronal form or conformation of Na,K-ATPase.     

Allosteric Modulation of Flunitrazepam Binding to Rat Brain Benzodiazepine Receptors by Methyl β-Carboline-3-Carboxylate

The inhibition of flunitrazepam (FNP) binding to rat brain benzodiazepine (BZ) receptors by methyl beta-carboline-3-carboxylate (MCC) was studied. Biphasic dissociation was observed for [3H]FNP and [3H]MCC in cerebral cortex, cerebellum, and hippocampus, although the dissociation of [3H]MCC was much faster. The dissociation rate of [3H]FNP was increased by MCC in the cerebellum, but was not altered in cerebral cortex or hippocampus. [3H]FNP binding stimulated by gamma-aminobutyric acid was enhanced in the presence of MCC in all three regions examined. These results indicate that MCC exerts these effects by interacting with allosteric sites that are different from the FNP recognition sites on the BZ receptors.     

Buprenorphine: High-Affinity Binding to Dorsal Spinal Cord

The binding of the mixed opiate agonist-antagonist [3H]buprenorphine was compared with [3H]naloxone and [3H]dihydromorphine binding in membranes prepared from rat whole brain and dorsal spinal cord. Scatchard analysis of binding to whole brain yielded KD values close to 1.0 nM for all three 3H-ligands studied, although [3H]buprenorphine labelled five times as many binding sites. [3H]Naloxone and [3H]dihydromorphine bound to dorsal spinal cord with approximately the same affinity as to whole brain, although both 3H-ligands labelled fewer sites in the spinal cord. In contrast, Scatchard analysis of [3H]buprenorphine binding to spinal cord yielded curvilinear Scatchard plots, suggesting the presence of a very high-affinity (KD = 0.12 nM) binding site in addition to the high-affinity site (KD = 1.0 nM) present in the brain. Studies on the displacement of [3H]buprenorphine by opiates and D-Ala2,Met5-enkephalinamide supported the presence of two binding sites for this ligand in the spinal cord.     

Effects of Prenatal Phenobarbital on Benzodiazepine Receptor Development

Phenobarbital (PB) was administered to pregnant mice during days 9-21 of gestation. Forebrain and cerebellar [3H]flunitrazepam ([3H]FLU) binding was assayed in the offspring at birth and at 21 days of age. Prenatal treatment produced a decrease in the number (Bmax) of [3H]FLU receptors in both the forebrain and cerebellum at birth. A small decrease in the [3H]FLU dissociation constant (KD) values in the forebrain was also detected at birth, but no changes were seen in the [3H]FLU KD values in the cerebellum. No changes were observed in forebrain and cerebellar [3H]FLU Bmax or KD values at 21 days of age, indicating that the effects of prenatal exposure to PB on [3H]FLU binding are eliminated during the postnatal development of the forebrain and cerebellum. The receptor affinity for the triazolopyridazine CL 218,872, which distinguishes the type I and type II benzodiazepine (BDZ) receptors, was not altered by prenatal PB treatment. The coupling of the BDZ receptor to the gamma-aminobutyric acid and pentobarbital binding sites was unaffected by exposure to PB in utero.     

Comparison of the endogenous heptapeptide Met-enkephalin-Arg6-Phe7 binding in amphibian and mammalian brain

In previous communications [4, 38] we published that [3H]Met-enkephalin-Arg6-Phe7 (MERF) binds to opioid (kappa2 and delta) and sigma2 sites in frog and rat brain membrane preparations, however no binding to kappa1 sites could be established. In the present paper we compare the frog, rat and guinea pig brain membrane fractions with respect to their MERF binding data. No qualitative differences were found between the three species but specific binding of labelled MERF was maximal in frog brain and lowest in guinea pig brain, which corresponds to their kappa2 opioid receptor distribution. The naloxone resistant binding was also present in all investigated species and varied from 25% in frog and guinea pig cerebrum, to 50% in rat cerebrum and cerebellum, but no naloxone inhibition was found in guinea pig cerebellum where no kappa2 opioid receptors have been found. The presence of sigma2-like receptor was demonstrated in each investigated membrane fraction with displacement experiments using (-)N-allyl-normetazocine as competitor of tritiated MERF. It was shown that this site was responsible for 60-80% of [3H]MERF binding. The remaining part of the naloxone resistant labelled MERF binding could be displaced only with endogenous opioid peptides as met-enkephalin, dynorphin and beta-endorphin. The eventual physiological role of multiple MERF receptors is discussed.     

Muscarinic antagonist binding site heterogeneity as evidenced by autoradiography after direct labeling with [3H]-QNB and [3H]-pirenzepine

Saturable, high affinity binding of tritiated pirenzepine [( 3H]-PZ) was obtained in slide mounted tissue sections prior to performing autoradiographic localization of these binding sites. The binding in tissue sections of rostral rat forebrain gave a KD of 18nM and a Bmax of 51 fmoles/mg tissue. These binding characteristics are similar to those previously obtained in homogenate membrane preparations and indicate the binding is taking place in a similar manner. The distribution of the binding sites labeled with [3H]-PZ represented a subpopulation of those which could be labeled with tritiated quinuclidinyl benzilate [( 3H]-QNB). Thus, [3H]-PZ and [3H]-QNB both label regions of the cerebral cortex, hippocampus, striatum and dorsal horn of the spinal cord, while sites in the cerebellum, nucleus tractus solitarius, facial nucleus and ventral horn of the spinal cord are labeled with [3H]-QNB and not by [3H]-PZ. These observations indicate separate regions of the brain where antagonists bind to subtypes of muscarinic receptors.     

Identification of a Novel N-Methyl-D-Aspartate Receptor Population in the Rat Medial Thalamus

To evaluate the possibility of pharmacologically distinct N-methyl-D-aspartate (NMDA) receptor subtypes, quantitative autoradiography was used to determine the potency of several compounds as inhibitors of L-[3H]glutamate or [3H]MK-801 binding to rat brain NMDA receptors in 10 brain regions. Competitive NMDA receptor antagonists displayed differing pharmacological profiles in the forebrain, cerebellum, and medial regions of the thalamus (midline nuclei). For example, compared with other competitive antagonists, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonate (CPP) and LY-233536 were especially weak displacers of L-[3H]glutamate binding in the cerebellum. In the the medial thalamus, CPP and D-2-amino-5-phosphonopentanoate displayed relatively low affinities, whereas LY-233536 was relatively potent. The noncompetitive NMDA receptor antagonists also displayed regional variations in their pharmacological profiles. Relative to other regions, [3H]MK-801 binding in the cerebellum was weakly displaced by MK-801 and potently displaced by dextromethorphan and SKF-10047. In the medial thalamus, 1-[1-(2-thienyl)-cyclohexyl]piperidine was relatively potent and SKF-10047 was relatively weak. These results confirm previous suggestions that the cerebellum contains a distinct NMDA receptor subtype and indicate that nuclei of the medial thalamus contain a novel NMDA receptor subtype that is distinct from both those found in the cerebellum and in the forebrain.     

[3H]Diprenorphine Binding to k-Sites in Guinea-Pig and Rat Brain: Evidence for Apparent Heterogeneity

The binding of the unselective opioid antagonist [3H]diprenorphine to homogenates prepared from rat brain and from guinea-pig brain and cerebellum has been studied in HEPES buffer containing 10 mM Mg2+ ions. Sequential displacement of bound [3H]diprenorphine by ligands with selectivity for mu-, delta-, and kappa-opioid receptors uncovers the multiple components of binding. In the presence of cold ligands that occupy all mu-, delta-, and kappa-sites, opioid binding still remains. This binding represents 20% of total specific sites and is displaced by naloxone. The nature of these undefined opioid binding sites is discussed.     

Autoradiographic Distribution and Characteristics of High- and Low-Affinity Polyamine-Sensitive [3H]Ifenprodil Sites in the Rat Brain: Possible Relationship to NMDAR2B Receptors and Calmodulin

Abstract: We have studied the regional distribution and characteristics of polyamine-sensitive [³H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [³H]ifenprodil (40 nM) in a biphasic manner with IC₅₀ values ranging from 42 to 352 nM and 401 to 974 µM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC₅₀ values in the high micromolar range. Wiping studies using forebrain slices (containing both high- and low-affinity sites) or cerebellar slices (containing only the low-affinity site) showed that high- and low-affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [³H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [³H]ifenprodil from its low-affinity site in hindbrain and forebrain regions. High-affinity [³H]ifenprodil sites, defined either by ifenprodil displacement curves or by [³H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low-affinity [³H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [³H]Ifenprodil was found to bind to calmodulin-agarose with very low affinity (IC₅₀ of ifenprodil = 516 µM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [³H]ifenprodil from its low-affinity site in brain sections. However, the localization of the low-affinity [³H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high-affinity [³H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist.     

Postnatal Evolution of the γ-Aminobutyric Acid/Benzodiazepine Receptor Complex in a Model of Inherited Epilepsy: The Quaking Mouse

Binding assays of [3H]muscimol and [3H]-flunitrazepam have been performed on brain homogenates of brainstem, cerebellum, and forebrain of genetically epileptic quaking (qk) mutant mice 20, 40, 70, and 90 days old and their corresponding controls of the same strain (C57BL/6J). The endogenous gamma-aminobutyric acid (GABA) content has been determined in various brain regions of 70-day-old qk and control mice. Finally, the behavioral effects of diazepam, of the mixed GABAA/GABAB receptor agonist progabide, and of the selective GABAB receptor agonist baclofen have been assessed in adult qk mutants. Our results strongly suggest a lack of involvement of GABAergic neurotransmission in the inherited epilepsy of the qk mutant mouse.