Higher expression of alpha7 nicotinic acetylcholine receptors in human fetal compared to adult brain

Neuronal nicotinic acetylcholine receptors are thought to be involved in regulation of several processes during neurogenesis of the brain. In this study the expression of the alpha7 nicotinic acetylcholine receptor subtype was investigated in human fetal (9-11 weeks of gestation), middle-aged (28-51 years) and aged (68-94 years) medulla oblongata, pons, frontal cortex, and cerebellum. The specific binding of the alpha7 receptor antagonist [(125)I]alpha-bungarotoxin was significantly higher in fetal than in both middle-aged and aged medulla oblongata and aged pons. No significant decrease in [(125)I]alpha-bungarotoxin binding sites was observed from fetal to adult cortex and cerebellum. The alpha7 mRNA expression was significantly higher in all fetal brain regions investigated, except for aged cortex, than in corresponding middle-aged and aged tissue. The high expression of alpha7 nicotinic acetylcholine receptors in fetal compared to adult brain supports the view that these receptors play an important role during brain development.     

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.     

Brain nicotinic cholinoceptor binding in spontaneous hypertension

To study the role of central cholinergic mechanisms in hypertension, we have determined nicotinic and muscarinic agonist binding sites in the brain regions of stroke-prone spontaneously hypertensive rats (SHRSP), using [³H]nicotine and [³H]cismethyldioxolane (CD). There was a significant decrease in specific [³H]nicotine binding in the cerebral cortex, thalamus, midbrain, cerebellum and medulla oblongata of SHRSP at 16–24 weeks of age compared to that of age-matched Wistar Kyoto rats (WKY). Scatchard analysis revealed 35% decrease in theB_max value for [³H]nicotine binding in the SHRSP medulla oblongata without a change in theK_d value, suggesting a change in the receptor density. Similar reduction of nicotinic cholinoceptor binding sites was also observed in the discrete brain regions of young (5-week-old) SHRSP. In contrast, there was no alteration in specific [³H]CD binding in the SHRSP brain regions, except the hypothalamus which showed a significant increase. The SHRSP medulla oblongata showed no change in the ChAT activity. Thus, the present study suggests an important role for medullary nicotinic cholinoceptors in the pathogenesis of spontaneous hypertension.     

Autoradiographic localization of receptors for neuropeptide FF, FLFQPQRFamide, in human spinal sensory system

The regional distribution of FLFQPQRFamide binding sites on fresh unfixed cryostata sections from post mortem specimens of human spinal cord and lower medulla oblongata was studied by quantitative autoradiographic methods using [¹²⁵I]YLFQPQRFamide as ligand. Samples were taken from fives cases who had died with no history of neurological disease at ages ranging from 5 months to 66 years. The biochemical and pharmacological characteristics of [¹²⁵I]YLFQPQRFamide binding to mounted tissue sections were comparable to those reported for the rat in a previous study. [¹²⁵I]YLFQPQRFamide appeared to interact reversibly with high affinity binding sites (K_d = 0.06nM), distinct from opiate receptors. Sites labelled with [¹²⁵I]YLFQPQRFamide were distributed unevenly within the human spinal cord and lower mmedulla oblongata, with the highest density in the superficial layers of the dorsal horn and the spinal trigeminal nucleus. Although moderate labelling was observed in the ventral part of spinal grey matter, dense labelling appeared in the gracile and cuneate nuclei. No binding sites were detected in white matter. These results show that, as in the rat, FLFQPQRFamide receptors in the human spinal cord and lower medulla oblongata, are mainly concentrated within spinal areas implicated in the analgesic action of opiates. The possible role of these receptors in modulating spinal nociceptive information is discussed with respect to the pharmacological effects of substances acting on FLFQPQRFamide receptors in animals.     

Selective nicotinic receptor consequences in APP(SWE) transgenic mice

The nicotinic (nAChRs) and muscarinic (mAChRs) acetylcholine receptors and acetylcholinesterase (AChE) activity were studied in the brains of APP(SWE) transgenic mice (Tg+) and age-matched nontransgenic controls (Tg-) that were between 4 and 19 months of age. A significant increase in the binding of 125I-labeled alpha-bungarotoxin (alpha7 nAChRs) was observed in most brain regions analyzed in 4-month-old Tg+ mice, preceding learning and memory impairments and amyloid-beta (Abeta) pathology. The enhanced alpha7 receptor binding was still detectable at 17-19 months of age. Increase in [3H]cytisine binding (alpha4beta2 nAChRs) was measured at 17-19 months of age in Tg+ mice, at the same age when the animals showed heavy Abeta pathology. No significant changes in [3H]pirenzepine (M1 mAChRs) or [3H]AFDX 384 (M2 mAChRs) binding sites were found at any age studied. The upregulation of the nAChRs probably reflects compensatory mechanisms in response to Abeta burden in the brains of Tg+ mice.     

In vivo receptor occupation by benzodiazepines and correlation with the pharmacological effect

The existence of specific receptor sites for benzodiazepines has been well documented by in vitro binding studies. In this study, using a highly radiolabelled [3H]-flunitrazepam, we investigated the binding of benzodiazepines to their receptor sites under in vivo conditions. Tracer doses of [3H]flunitrazepam (0.001 mg/kg) were injected i.v. into mice and the concentration of the drug in the brain was monitored. The accumulation of [3H]flunitrazepam 20 min after injection was found to be highest in the hippocampus, cortex, hypothalamus; to be intermediate in the striatum, medulla oblongata/pons and midbrain and to be lowest in the cerebellum. This corresponds well with the different densities of benzodiazepine receptors which we found in in vitro studies, with the exception of medulla oblongata/pons and cerebellum. When increasing doses (0.01--10 mg/kg) of non-labelled benzodiazepine derivatives (flunitrazepam, clonazepam, the 3S and 3R enantiomers of 5-(o-fluorophenyl)-1,3-dihyrdo-1,3-dimethyl-7-nitro-2H-1,4-benzodiazepine-2-one, and chlordiazepoxide) were injected simultaneously with [3H]flunitrazepam, a dose-dependant, saturable and and stereo-specific decrease of [3H]flunitrazepam concentration in the mouse hippocampus was observed. The dose range in which the unlabelled benzodiazepines decreases the levels of [3H]flunitrazepam in the hippocampus corresponds closely to that which inhibited pentylenetetrazol- or picrotoxin-induced seizures, indicating that this in vivo method determines the occupation of pharmacologically relevant receptors.     

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.     

Regional responses of rat brain muscarinic cholinergic receptors to immobilization stress

The effect of immobilization stress (IM-stress) on the muscarinic cholinergic (m-Ch) receptor binding was determined in 8 brain regions using [³H]quinuclidinyl benzilate (QNB). IM-stress produced an increase in specific QNB binding in the septum, striatum, hippocampus and pons + medulla oblongata. Scatchard panalyisis revealed that IM-stresss produced an increase in the affinity of m-Ch receptors in the septum, hippocampus andm pons + medulla oblangata, but did not alter the maximum number of binding sites (B_max). In the striatum, an increase in specific QNB binding was due to both the increase in B_max and reduction of the dissociation constant (K_d). The present study suggest that IM-stress induces supersensitivity of postsynaptic m-Ch receptors probably due to a deacrease in presynaptic cholinergic activities in the septum, striatum, hippocampus and pons + medulla oblongata. As the m-Ch receptors in the striatum and pons + medulla oblongata. Scatchard analysis revealed that IM-stress produced an increase in the affinity of m-Ch receptors stressful situations in this regions as well as in the septum, hippocampus and cerebral cortex.     

Postnatal development of two nicotinic cholinergic receptors in seven mouse brain regions

The developmental profiles for binding of alpha-[125I]bungarotoxin and L-[3H]nicotine to putative nicotinic cholinergic receptors were determined in seven mouse brain regions. The overall pattern of development of alpha-bungarotoxin binding was similar in all of the regions. Neonatal binding values tended to be greater than those observed in adult brain regions. Maximal binding occurred within 10 days of birth and adult binding values were reached by 20 days of age. The patterns of development of nicotine binding in each of the seven brain regions differed according to region. Gross similarities in developmental profiles for nicotine binding were found among the more caudal and among the more rostral regions. In hindbrain and cerebellum, maximal nicotine binding was found at birth (5 days of age in cerebellum); binding declined approximately 4-fold by 20 days and remained relatively constant thereafter. In midbrain and hypothalamus, a less extensive decrease in nicotine binding occurred from birth to adulthood (midbrain, 25%; hypothalamus, 50%). Nicotine binding in hippocampus and cortex remained unchanged between birth and adulthood. The developmental pattern for nicotine binding in striatum differed from that found in the other brain regions. At 5 days of age, binding was about 65% of adult binding, which was reached at 30 days of age. In most of the brain regions the developmental profile for alpha-bungarotoxin binding was different from that of nicotine. This difference was especially notable in striatum, where adult nicotine binding was higher than neonatal nicotine binding, whereas adult alpha-bungarotoxin binding was lower than neonatal alpha-bungarotoxin binding.     

Effects of cervical spinal hemisection on dihydromorphine binding in brainstem and spinal cord in cat

Cats were sacrificed 1-3 weeks after cervical (C1-C2) hemisection and receptor binding experiments were carried out with 4.0 and 0.6 nM concentrations of [3H]dihydromorphine [( 3H]DHM); these concentrations were shown by Scatchard analysis to represent the approximate Kd values of high and low affinity dihydromorphine binding sites in brain homogenates. Unilateral cervical hemisection produced significant (P less than 0.05), bilateral, reductions in the levels of [3H]DHM binding in the periaqueductal gray (PAG; 35-40%) and mesencephalic reticular formation (MRF; 47-51%), medial pons (40-56%) and medial medulla (30-37%). In paramedial pons and medulla, numerical reductions in [3H]DHM binding were observed (18 and 28%) which did not achieve statistical significance. In spinal cord, significant reductions were observed in the dorsal (45%) and ventral (29%) ipsilateral but not contralateral quadrants. We believe that these results in the brainstem and spinal cord reflect in part the loss of opiate binding on spinobulbar terminals and bulbospinal terminals, respectively, following orthograde degeneration. These observations support the hypothesis that the analgetic effects of opiates in the brainstem may in part be mediated by the direct inhibition of transmission through spinobulbar terminals.     

Age-dependent changes in brain glycine concentration and strychnine-induced seizures in the rat

Glycine levels and receptor binding were measured in the medulla and spinal cord of 2-month, 10-month, and 24-month-old Fischer 344 rats. The behavioral response to the administration of the glycine antagonist, strychnine, was also evaluated in 2- and 24-month-old animals to investigate the relevance of these parameters to the susceptibility to seizures. Significant reductions in glycine in both the spinal cord and medulla occurred from 2 to 24 months of age. The glycine precursors, serine and threonine, were decreased only in the spinal cord. [3H]Strychnine binding was also decreased by 38% and 34% in the medulla and spinal cord, respectively, of 24-month-old rats compared to 2-month-olds. [3H]GABA binding was similarly reduced while no age-related changes in [3H]diazepam binding in the spinal cord were detected. Comparison of 2- and 24-month-old animals after systemic injection of 1.75 mg/kg strychnine showed that senescent animals have a higher incidence of seizures and mortality compared to young animals. Decreases in glycinergic neurotransmission may lower strychnine seizure threshold in the aged animal.     

Selective changes in the levels of nicotinic acetylcholine receptor protein and of corresponding mRNA species in the brains of patients with Parkinson's disease

Reductions in the number of neuronal nicotinic acetylcholine receptors (nAChRs) have been shown to occur in connection with Parkinson's disease (PD), but it is still unclear which subtype of this receptor is affected. In the present study we examined various nAChR subtypes employing ligand binding, as well as levels of subunit protein and mRNA in the brains of PD patients and age-matched controls. Binding of [3H]epibatidine and levels of alpha3 mRNA in the caudate nucleus and temporal cortex, but not in the hippocampus were significantly decreased in the PD brain. The level of the alpha3 protein subunit was significantly reduced in all these brain regions but there was no change in the level of alpha4. The level of the beta2 protein subunit in the temporal cortex and hippocampus and the beta2 mRNA in the temporal cortex was lowered. Both the levels of the alpha7 subunit protein and [125I]alpha-bungarotoxin binding were significantly increased in the temporal cortex of PD patients whereas the alpha7 mRNA level was unchanged. These findings reveal selective losses of the alpha3- and beta2-containing nAChRs and an increase in the alpha7 nAChRs that might be related to the pathogenesis of PD.     

Effects of donepezil treatment on rat nicotinic acetylcholine receptor levels in vivo and in vitro

Research on acetylcholinesterase inhibitors (ChEIs) indicates that long term exposure increases the level of nicotinic acetylcholine receptors (nAChRs) but the effects of donepezil on nAChRs are not well studied. Therefore, we investigated the effects of sub-chronic donepezil administration on nAChRs in rats and rat pheochromocytoma PC-12 cells. Male Sprague Dawley rats were administered donepezil (0.7 and 2.4 micromoles/kg), nicotine (2.5 micromoles/kg) or saline subcutaneously twice daily for 14 days, PC-12 cells were incubated with 10(-6) to 10(-4) M donepezil for 72 hours and nAChR levels were determined by receptor binding assay using the nAChR ligands [3H]-epibatidine (EPI) for non-alpha 7 nAChRs and [3H]-methyllyconitine (MLA) for alpha 7 nAChRs. Chronic donepezil administration at 1.4 micromoles/kg/day and 4.8 micromoles/ kg/day significantly increased [3H]-epibatidine binding in the cortex to 126 +/- 1.3% and 127 +/- 3.2% of the saline control animals, respectively. [3H]-MLA binding in the cortex increased to 114 +/- 4.4% and 124 +/- 2.8% of the control group for the high and low dose groups, respectively. Hippocampal [3H]-EPI binding in the low dose and high dose groups significantly increased to 135 +/- 3.6% and 125 +/- 4.6% of the controls, respectively while there were no changes in the level of [3H]-MLA binding. In striatal homogenates, neither [3H]-EPI nor [3H]-MLA binding were significantly effected at either dose of donepezil. In PC-12 cells, [3H]-EPI binding was increased at the non-physiological 10(-4)M concentration only. There was no effect of donepezil on [3H]-MLA binding at any concentration examined. These results indicate that donepezil increases cortical alpha 7 and non-alpha 7 nAChRs, hippocampal non-alpha 7 nAChRs but does not influence striatal nAChR levels. Furthermore, the lack of an effect on the alpha 7-nAChRs in PC-12 cells suggests that the increase in cortical alpha 7 nAChRs may be an indirect effect of increased acetylcholine levels in vivo.     

Distribution of nicotinic binding sites with respect to CRF and neurophysin immunoreactive perikarya within the rat hypothalamus

These studies determined the differential autoradiographic distribution of [125I]alpha-bungarotoxin versus [3H]nicotine relative to the histochemically defined perikarya for neurophysin and corticotropin releasing factor (CRF). Specific [3H]nicotine binding sites occurred in relatively greater density within the neuropil surrounding PVN and SON compared to within the nuclei. In contrast, the highest density of [125I]alpha-BTX sites codistributed with neurophysin immunoreactive perikarya within these nuclei.     

Analysis of NMDA Receptors in the Human Spinal Cord

NMDA receptors in postmortem human spinal cord were analyzed using [³H]MK-801 ligand binding and immunoblotting with NMDA receptor subunit-specific antibodies. The averageK_Dfor [³H]MK-801 binding was 1.77 nM with aB_maxof 0.103 pmol/mg. The EC₅₀for stimulation of [³H]MK-801 binding withl-glutamate was 0.34 μM. None of these parameters were affected by postmortem intervals up to 72 h. Immunoblotting of native NMDA receptors showed that NR1, NR2A, NR2C, and NR2D subunits could all be found in the human spinal cord of which NR1 was preferentially located to the dorsal half. Immunoprecipitation of solubilized receptors revealed that NR1, NR2C, and NR2D subunits coprecipitated with the NR2A subunit, indicating that native human spinal cord NMDA receptors are heteroligimeric receptors assembled by at least three different receptor subunits. These results provide a basis for the development of drugs selectively aimed at spinal cord NMDA receptors for the future treatment of spinal cord disorders.     

Characterization of nicotinic receptors inducing noradrenaline release and absence of nicotinic autoreceptors in human neocortex

Presynaptic facilitatory nicotinic receptors (nAChRs) on noradrenergic axon terminals were studied in slices of human or rat neocortex and of rat hippocampus preincubated with [3H]noradrenaline ([3H]NA). During superfusion of the slices, stimulation by nicotinic agonists for 2 min only slightly increased [3H]NA outflow in the rat neocortex, but caused a tetrodotoxin-sensitive. Ca(2+)-dependent release of [3H]NA in rat hippocampus and human neocortex. In both tissues a similar rank order of potency of nicotinic agonists was found: epibatidine > DMPP > nicotine approximately cytisine > or = acetylcholine; choline was ineffective. In human neocortex, the effects of nicotine (100 microM) were reduced by mecamylamine, methyllycaconitine, di-hydro-beta-erythroidine (10 microM, each) and the alpha3beta2/alpha6betax-selective alpha-conotoxin MII (100/200 nM). The alpha3beta4 selective alpha-conotoxin AuIB (1 microM), and the alpha7 selective alpha-conotoxin ImI (200 nM) as well as alpha-bungarotoxin (125 nM) were ineffective. Glutamate receptor antagonists (300 microM AP-5, 100 microM DNQX) acted inhibitory, suggesting the participation of nAChRs on glutamatergic neurons. On the other hand, nAChR agonists were unable to evoke exocytotic release of [3H]acetylcholine from human and rat neocortical slices preincubated with [3H]choline. In conclusion: (1) alpha3beta2 and/or alpha6 containing nAChRs are at least partially responsible for presynaptic cholinergic facilitation of noradrenergic transmission in human neocortex; (2) nicotinic autoreceptors were not detectable in rat and human neocortex.     

Quantitative autoradiographic mapping of 5-HT3 receptors in the rat CNS using [125I]iodo-zacopride and [3H]zacopride as radioligands.

Substitution of the chlorine atom by a radio-iodine in position 5 in the zacopride molecule yielded [125I]iodo-zacopride that bound with high affinity (Kd = 4.3 nM) to 5-HT3 receptors in the rat central nervous system. Assays with membranes from the posterior (mainly entorhinal) cortex confirmed that the pharmacological properties and regional distribution of [125I]iodo-zacopride-specific binding sites were identical with those of 5-HT3 sites labelled by the reference radioligand [3H]zacopride. Autoradiographic investigations for the visualization and quantification of 5-HT3 receptors yielded similar results with both radioligands, but autoradiograms could be obtained after only 1-3 days of exposure of sections labelled with [125I]iodo-zacopride, instead of 4-6 months using [3H]zacopride. The highest density of 5-HT3 sites was found in the nucleus tractus solitarius followed by, in decreasing order, the dorsal motor nucleus of the vagus nerve, the superficial layers of the dorsal horn in the spinal cord, the nucleus of the spinal tract of the trigeminal nerve, and the area postrema. Significant labelling of 5-HT3 receptors was also observed in limbic areas (amygdala, hippocampus, frontal and entorhinal cortex), and to a much lower extent in the dorsal raphe nucleus, striatum, and substantia nigra. These multiple locations further support the idea that 5-HT3 receptors are probably involved in several 5-HT-mediated functions in the central nervous system.     

Regional brain concentrations of vasoactive intestinal polypeptide in normotensive Wistar-Kyoto and spontaneously hypertensive rats

The regional brain and spinal cord concentrations of vasoactive intestinal polypeptide immunoreactivity (VIP) were measured in age-matched normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats. The relative order of distribution of VIP in the WKY strain was cortex (44 pmol/g) greater than hippocampus = striatum greater than midbrain = hypothalamus greater than medulla oblongata/pons = lumbar spinal cord (SC) greater than cervical SC greater than thoracic SC (2.5 pmol/g) whereas in the SH strain this order was cortex (35 pmol/g) greater than striatum = midbrain greater than hippocampus = hypothalamus greater than medulla oblongata/pons = lumbar SC greater than cervical SC greater than thoracic SC (1 pmol/g). The VIP concentrations of the thalamus, cerebellum and pituitary were at the level of assay sensitivity (0.5 pmol/g) in both strains. In comparison to the WKY, the SH rats had significantly lower VIP levels in the hippocampus (-42%) and cervical (-46%) and thoracic (-56%) spinal cord but significantly higher levels in the midbrain (+64%).     

Modification of the binding of [H]MK-801 to brain regions and spinal cord of rats treated chronically with U-50,488H, a κ-opioid receptor agonist

Male Sprague-Dawley rats were rendered tolerant to U-50,488H by twice-daily injections of the drug (25 mg/kg, i.p.) for 4 days. In tolerant rats, the binding of [³H]MK-801 was increased in pons and medulla and corpus striatum but decreased in midbrain and hippocampus and was due to changes in B_max values. In U-50,488H-abstinent rats, the binding of [³H]MK-801 was increased in pons and medulla and hippocampus, and decreased in midbrain and amygdala. In hippocampus, the B_max of [³H]MK-801 was increased but the K_d was decreased whereas in amygdala and pons and medulla, the changes were due to alterations in the B_max values. Previous studies have shown that NMDA receptor antagonists block the tolerance to the analgesic action of U-50,488H in rodents. The present studies demonstrate differential changes in the NMDA receptors of brain regions of U-50,488H-tolerant and -abstinent rats.