Effect of Chronic Desipramine Treatment on Dihydroalprenolol, Imipramine, and Desipramine Binding Sites: A Quantitative Autoradiographic Study in the Rat Brain

Desmethylimipramine (DMI) administered once daily for 10 days caused a significant decrease in beta-adrenergic receptor binding, as measured by quantitative autoradiography in discrete brain regions. The decrease was observed 72 h after the last injection throughout the cortex and in hippocampus but not in other regions, much richer in beta-receptors, such as the caudate, olfactory tubercle, superior colliculus, dorsomedial thalamus, substantia nigra, or pineal. The same paradigm did not affect imipramine (IMI) binding in the cortex or in regions with high concentrations of IMI binding sites. DMI binding was not decreased, either. Significant increases in DMI binding were observed in frontal cortex and in the ventral aspect of the bed nucleus of the stria terminalis. We conclude that a reduction in tricyclic binding is not a general phenomenon following chronic treatment with tricyclic antidepressants, and changes in binding, when they do occur, are not correlated with areas of high binding site density.     

Autoradiographic Localization of the Putative D4 Dopamine Receptor in Rat Brain

The putative dopamine D4 receptor protein in rat brain was labelled and quantified autoradiographically using two selective benzamides: [³H]YM-09151-2 which labels D2, D3 and D4 dopamine receptors and [³H]Raclopride which labels D2 and D3. The difference in densities of both ligands at saturable concentrations, show a regional distribution for the putative D4 receptor in the following rank order: hippocampus > caudate putamen > olfactory tubercle = substancia nigra > nucleus accumbens core > cerebral cortex > cerebellum. A calculated value of 0.34 pmol/mg protein was attributable to D4 receptor maximum capacity in caudate putamen and was obtained after subtracting the Bmax of the ligands. Our results show that the distribution of D4 receptor only partially overlaps with the D4 mRNA localization reported earlier and is not only associated to limbic structures but to motor areas as well.     

Thyrotropin-Releasing Hormone Receptor Binding Sites: Autoradiographic Distribution in the Rat and Guinea Pig Brain

Thyrotropin-releasing hormone (TRH) binding sites were labeled in vitro in mounted brain tissue sections from rat and guinea pig brains with [3H]methyl TRH and localized autoradiographically using 3H-sensitive film. Regional densities of TRH binding sites were measured by computer-assisted microdensitometry. The distribution of sites in both species was highly heterogeneous. In both guinea pig and rat brains, the highest densities of binding sites were seen in the amygdaloid nuclei and the perirhinal cortex. In contrast, in other brain areas, a clear difference between the distribution of sites in rat and guinea pig was found. The temporal cortex, pontine nuclei, and interpeduncular nucleus, which contained high densities of binding in the guinea pig, were scarcely labeled in the rat. The accessory olfactory bulb and the septohippocampal area presented in the rat higher concentrations of binding sites than in the guinea pig. Other brain areas showing intermediate to low densities in both species were accumbens nucleus, bed nucleus of the stria terminalis, dentate gyrus, facial and hypoglossal nuclei, and gelatinosus subnucleus of the trigeminal nerve, among others. The anterior pituitary also presented low to intermediate concentrations of receptors. The distribution of TRH sites here described does not completely correlate with that of endogenous TRH, but is in good agreement with previous biochemical data. The results are discussed in correlation to the physiological effects that appear to be mediated by TRH.     

Pharmacological and Physicochemical Properties of Pre-Versus Postsynaptic 5-Hydroxytryptamine1A Receptor Binding Sites in the Rat Brain: A Quantitative Autoradiographic Study

Numerous data suggested that the pharmacological and biochemical properties of 5-hydroxytryptamine1A (5-HT1A) receptors exhibit some regional differences in the CNS, notably within the raphe nuclei compared with various forebrain areas (such as the hippocampus). This possibility has been further investigated in the dorsal raphe nucleus and two areas within the hippocampus, the dentate gyrus and the CA1 area, using the quantitative autoradiographic technique. The potencies of 5'-guanylylimidodiphosphate to inhibit the specific binding of 125I-Bolton-Hunter-8-methoxy-2-(N-propyl-N-propylamino)tetralin (125I-BH-8-MeO-N-PAT) to 5-HT1A sites and of N-ethylmaleimide to block these sites irreversibly were identical in the dorsal raphe nucleus and the hippocampal areas in rat brain sections. In contrast, slight but significant differences were noted in the pH dependence and pharmacological properties of 5-HT1A sites labeled by 125I-BH-8-MeO-N-PAT in these three regions. Similarly, heat denaturation experiments and tissue exposure to either phospholipase A2 or the alkylating agent 8-methoxy-2-(N-2'-chloropropyl,N-propyl)aminotetraline revealed regional differences in the properties of 5-HT1A sites. However, in most cases, the observed variations were of greater amplitude between the CA1 area and the dentate gyrus, where 5-HT1A sites are located postsynaptically, than between any one of these areas and the dorsal raphe nucleus where they act as (presynaptic) somatodendritic autoreceptors. These data further support that subtypes of 5-HT1A receptors probably exist in the rat brain, but this heterogeneity seems unrelated to the pre- or post-synaptic location of these receptors.     

Quantitative Autoradiography of [3H]Indalpine Binding Sites in the Rat Brain: II. Regional Distribution

The localization of binding sites for [3H]indalpine to sections of rat brain was studied by a quantitative autoradiographic technique. Binding sites for this specific neuronal 5-hydroxytryptamine (5-HT) uptake inhibitor are concentrated in areas rich in 5-HT neuronal cell bodies, fibers, and synaptic terminals. One of the most interesting features of this regional distribution is the very high density of these sites found in the dorsal raphe, substantia nigra, ventral tegmental area, and locus ceruleus. Components of the visual system also show pronounced labelling with [3H]indalpine. The finding that limbic structures are strongly labelled by this drug may be related to the antidepressant activity of indalpine. The anatomical distribution of binding sites demonstrated is consistent with the specific labelling of 5-HT neurons by [3H]indalpine and confirms previous studies carried out with another 5-HT uptake inhibitor, [3H]imipramine.     

Serotonin Modulation of Low-Affinity Ouabain Binding in Rat Brain Determined by Quantitative Autoradiography

Previous results showed that Na⁺/K⁺-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na⁺/K⁺-ATPase activity and [³H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which isoform is involved in the Na⁺/K⁺-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [³H]ouabain binding to rat brain slices was employed at different [³H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [³H]ouabain binding was sensitive to serotonin 10^–3 M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [³H]ouabain concentrations, strongly suggests the participation of the 2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K⁺ handling by the glial 2 isoform of the pump.     

Effect of Unilateral Perinatal Hypoxic-Ischemic Brain Injury in the Rat on Striatal Muscarinic Cholinergic Receptors and High-Affinity Choline Uptake Sites: A Quantitative Autoradiographic Study

The binding characteristics and distribution of M1 and M2 muscarinic cholinergic receptors and high-affinity choline uptake sites were studied in the striatum of the rat at 3-4 and 9-12 weeks of age after exposure to unilateral perinatal hypoxic-ischemic brain injury. High-affinity choline uptake sites were labeled with [3H]hemicholinium-3, M1 receptors with [3H]pirenzepine, and M2 receptors with [3H]AF-DX 116. Saturation experiments revealed a significant decrease in the maximal binding capacity (Bmax) for [3H]pirenzepine-labeled M1 receptors in the lesioned caudate/putamen complex in immature rats with moderate brain injury, in comparison with controls. In contrast, the Bmax value for [3H]hemicholinium-3-labeled high-affinity choline uptake sites was significantly increased. No changes in dissociation constants (KD) were observed. These changes were most pronounced in the dorsolateral region of striatum. Striatal regional distribution of [3H]AF-DX 116 was not affected. In mature rats, binding of [3H]pirenzepine returned to control values, whereas [3H]hemicholinium binding showed a persistent increase (23%). The increase in [3H]hemicholinium-3 binding, as a specific marker of cholinergic nerve terminals, is consistent with our prior morphologic studies demonstrating relative preservation of cholinergic neurons and neuropil, and supports the concept that striatal cholinergic systems are resistant to hypoxic-ischemic injury.     

Subcellular Localization of Rat Brain Insulin Binding Sites

Fractions and subcellular structures were prepared from rat brain homogenate and their purity was assessed using enzyme markers, gamma-aminobutyric acid binding, DNA content, and electron microscopy. Insulin binding was highest on the plasma membrane preparations and approximately 50% less so on brain homogenate crude mitochondrial (P2), myelinated axon, and synaptosome preparations. Very low levels of binding were found on mitochondria and nuclei. Differences in binding between fractions were due to numbers of binding sites, and not variable binding affinity. There was a close relationship between insulin binding and the activity of Na/K ATPase (E.C. 3.6.1.4) in all fractions (r = 0.98). Insulin binding to the P2 was compared with plasma membrane fractions in seven brain regions, and the results demonstrated the same close relationship between insulin binding and plasma membrane content in all regions except hypothalamus. Plasma membrane insulin binding was well represented by the binding on P2 membranes in all regions except hypothalamus and brainstem. It was concluded that insulin binding is distributed evenly over the surface of brain cells and is not increased on nerve endings.     

Autoradiographic Visualization of the Receptor Subclasses for Vasoactive Intestinal Polypeptide (VIP) in Rat Brain

Vertongen, P., S. N. Schiffmann, P. Gourlet and P. Robberecht. Autoradiographic visualization of the receptor subclasses for Vasoactive Intestinal Polypeptide (VIP) in rat brain. Peptides 18(10) 1547–1554, 1997.—Vasoactive Intestinal Polypeptide (VIP) exerts its biological effects through interaction with two high affinity receptors named the VIP₁- and the VIP₂ receptors. Their messenger RNAs have been mapped in rat brain by in situ hybridization. A cyclic peptide (RO 25-1553) and a secretin analogue ([R¹⁶]chicken secretin) were identified as selective agonist peptides for the VIP₂- and VIP₁ receptors, respectively. The iodinated peptides retained the high affinity and selectivity of the unlabelled peptides and were used for the mapping of each receptor subclass in rat brain. VIP₁ receptors were present in the cerebral cortex, the piriform cortex, the claustrum, the caudate-putamen, the dentate gyrus, the lateral amygdaloïd nucleus, the anteroventral thalamic nucleus, the rhomboïd nucleus, the supraoptic nucleus and the choroïd plexus. VIP₂ receptors were present in the cerebral cortex, the claustrum, the caudate-putamen, the nucleus accumbens, the lateral septal nuclei, the bed nucleus of the stria terminalis, the basolateral amygdaloïd nucleus, the Ammon’s horn, the thalamic nuclei except some centromedial nuclei, the medial habenula, the suprachiasmatic nucleus, the periventricular nucleus, the mammilary nucleus, the superior colliculus and the choroïd plexus.     

Effect of Pertussis Toxin on Radioligand Binding to Rat Brain Adenosine A1 Receptors

In a previous study we showed that in vivo treatment with pertussis toxin could inhibit some, but not all, effects of adenosine in the rat hippocampus. In this study we investigated the effect of pertussis toxin on the binding of adenosine analogues to A1 receptors in rat brain. Intraventricular injection of pertussis toxin (10 micrograms into the lateral ventricle) did not affect A1 receptor binding in any brain region studied, as evaluated by autoradiography. In vitro treatment of brain sections (10 microns) with pertussis toxin for 5 h, under conditions when greater than 80% of the G proteins were ADP ribosylated, did not alter radioligand binding to adenosine A1 receptors. GTP (10 microM) virtually abolished the high-affinity agonist binding to the A1 receptor. On the other hand, in solubilized cortical membrane preparations, pertussis toxin pretreatment induced a complete shift of the A1 receptors to the low-affinity state. This suggests that the ability of pertussis toxin to affect G proteins coupled to A1 receptors in brain depends not only on the distribution of the toxin but also on the configuration of receptors and G proteins.     

Quinuclidinyl Benzilate Binding in House Fly Heads and Rat Brain

Abstract: House fly heads contain a binding site for 3-quinuclidinyl benzilate (QNB) that is quite similar in pharmacology to the muscarinic acetylcholine receptor of vertebrate tissues. The house fly site binds [³H]QNB reversibly with a K _d of 260 PM and B_max of 1 pmol/g of heads from direct binding measurements. The K_d calculated from the ratio of the dissociation rate constant (2 × 10⁻⁴ sec⁻¹) to the association rate constant (2.5 × 10⁶ M⁻¹ Sec⁻¹) was 80 pM. The house fly site binds (-)quinuclidinyl benzilate preferentially, as do classic muscarinic receptors. The binding is also sensitive to other muscarinic antagonists and agonists. Nicotinic and other drugs are no more effective on the house fly site than they are on the rat brain muscarinic receptor itself. These binding studies suggest that the house fly QNB binding site is a muscarinic receptor.     

Effect of Unilateral Perinatal Hypoxic-Ischemic Brain Injury in the Rat on Dopamine D1 and D2 Receptors and Uptake Sites: A Quantitative Autoradiographic Study

The effect of a unilateral perinatal hypoxic-ischemic brain injury on dopamine D1 and D2 receptors and uptake sites was investigated in rats by using in vitro quantitative binding autoradiography, 2-3 weeks after the insult. We observed significant decreases in the Bmax and KD for [3H]SCH 23390-labeled D1 and in the Bmax for [3H]spiperone-labeled D2 receptors in the lesioned caudate-putamen in rats with moderate brain injury (visible loss in hemispheric volume ipsilateral to the injury) compared with the nonlesioned contralateral caudate-putamen or with control rats. Changes in [3H]SCH 23390 and [3H]spiperone binding predominated in the dorsolateral part of the lesioned caudate-putamen. Pronounced reduction in [3H]SCH 23390 binding was also observed in the substantia nigra pars reticulata on the side of the lesion. In contrast, we did not observe any significant change in Bmax or KD for [3H]mazindol-labeled dopamine uptake sites. Similarly, no significant changes in the levels of dopamine or its metabolites were found on the side of the lesion. The observed reductions in striatal dopamine D1 and D2 receptors are a reflection of striatal cell loss induced by the hypoxic-ischemic injury. The absence of changes in [3H]mazindol binding or dopamine levels in the lesioned caudate-putamen indicates that the dopaminergic presynaptic structures are preserved.     

Identification of Strychnine Binding Sites in the Rat Retina

Abstract: [³H]Strychnine specifically binds to membrane fractions isolated from rat retinae. The binding is saturable, with an apparent dissociation constant, K _D, of 14.3 × 10⁻⁹ M and 205 fmol bound/mg protein. Specific binding is time-dependent and proportional to protein concentration. Glycine and taurine are equally potent inhibitors of [³H]strychnine binding ( K _i= 4 × 10⁻⁵ M); no other amino acids endogenously present in the retina inhibited [³H]strychnine binding.     

Serotonin Synthesis Increased in Terminals Four Days after Reserpine Treatment: An Autoradiographic Study in Rat Brain

The rate of 5-HT synthesis was determined in discrete rat brain regions 4 days after a single dose of reserpine (10 mg/kg) or reserpine carrier (controls), using an autoradiographic method with labelled -methyl-L-tryptophan as a tracer. The results show that the rate of 5-HT synthesis was unchanged in the dorsal and median raphe, significantly decreased in the raphe magnus, and significantly increased in areas rich in serotonergic nerve terminals (i.e., hypothalamus, hippocampus, median geniculate body, parietal and visual cortices). An increase in tryptophan hydroxylase activity could account for the increase in the rate of serotonin synthesis seen in some regions. Since the 5-HT synthesis rate showed regional variability there seems to be a need for regional studies of the effect of drugs on the 5-HT synthesis. In addition, the 5-HT synthesis rate was not significantly different from that in controls in many of the brain regions.     

CNS Adenosine A1 Receptors Are Altered After the Administration of Convulsant 3-Mercaptopropionic Acid and Cyclopentyladenosine: An Autoradiographic Study

Rat CNS adenosine A₁ receptors were studied by quantitative autoradiography after the administration of convulsant 3-mercaptopropionic acid (MP) and an adenosine analogue cyclopentyladenosine (CPA), using 2-chloro-N⁶-[cyclopentyl-2,3,4,5-³H adenosine]-([³H]CCPA) as radioactive ligand. Specific binding was quantified in hippocampus, cerebellum, cerebral cortex, thalamic nuclei, superior colliculus and striatum, and the highest densities were found in CA1, CA2, and CA3 hippocampus subareas and the lowest levels in superior colliculus and striatum. MP administration (150 mg/kg, i.p.) produced significant increases in [³H]CCPA binding in CA1 subarea at seizure (15%) and postseizure (21%) and in CA2 at seizure (15%) but a tendency to decrease in dentate gyrus. There was an increase in cerebellum at seizure (18%) but no significant changes in the other studied regions. CPA injection (2 mg/kg, i.p.) enhanced [³H]CCPA binding in CA1 and CA2 areas (17–18%) but not in CA3 area of the hippocampus. When CPA was administered before MP, which delayed seizure onset, an increase in [³H]CCPA binding in CA1 hippocampus subarea (19%) and cerebellum (28%) was also observed. Results showed that the administration of convulsant MP and adenosine analogue CPA exerts differential effects on adenosine A₁ receptors in CNS areas; hippocampus is the most affected area with all treatments, specially CA1 subarea, supporting an essential role in convulsant activity as well as in seizure prevention.     

Effect of Age on Human Brain Serotonin (S-1) Binding Sites

The effect of age on the binding of [3H]5-hydroxytryptamine [( 3H]5-HT, serotonin) to postmortem human frontal cortex, hippocampus, and putamen from individuals between the ages of 19 and 100 years was studied. One high-affinity binding site was observed in adult brains, with a mean KD of 3.7 nM and 3.2 nM for frontal cortex and hippocampus, respectively, and 9.2 nM for putamen. Decreased binding capacities (Bmax) with age were detected in frontal cortex and hippocampus. In putamen a decrease in affinity was noted. Postmortem storage did not significantly contribute to the age-related changes. No significant sex differences were detected. [3H]5-HT binding was also studied in brains from human neonates. The specific binding was 1.5-3 times lower than in adult frontal cortex and putamen, and Scatchard analysis suggested more than one binding site. In infant hippocampus a single binding site was observed and except for a premature individual, the binding capacity approximated adult values.     

Modification of [3H]MK801 Binding to Rat Brain NMDA Receptors after the Administration of a Convulsant Drug and an Adenosine Analogue: A Quantitative Autoradiographic Study

Specific [³H]MK801 binding to rat brain NMDA receptors after the administration of the convulsant drug 3-mercaptopropionic acid (MP) and the adenosine analogue cyclopentyladenosine (CPA) was studied by means of a quantitative autoradiographic method. MP administration (150 mg/kg, i.p.) caused significant decreases in [³H]MK801 binding in several hippocampus subareas and layers, mainly in CA1 and CA3 at seizure (11–27%) and postseizure (8–16%) and in cerebral occipital cortex at seizure (18–22%). In nucleus accumbens, a rise was observed at postseizure (44%) and a tendency to increase at seizure (24%). CPA (2mg/kg, i.p.) decreased ligand binding in hippocampus (CA1, CA2, CA3) (17–22%) and in occipital cerebral cortex (18–24%). When CPA was administered 30 minutes before MP (which delayed seizure onset) and rats were sacrified at seizure, decreases in [³H]MK801 binding in several layers of CA1 and CA3 of hippocampus (11–27%) and in CA1, CA2, CA3 (24–35%) after CPA+MP postseizure, and an increase in CA2 after CPA and CPA+MP postseizure (20–34%), were observed. A drop was found in the occipital subarea (18–24%) after CPA and in the frontal and occipital subarea after CPA+MP postseizure (24–34%) while no changes were observed in any treatment involving the other cerebral cortex regions, thalamic nuclei, caudate putamen and olfactory tubercle. These results show that [³H]MK801 binding changes according to drug treatment and the area being studied, thus indicating a different role in seizure activity.     

Characterization of N-[3H]Methylcarbamylcholine Binding Sites and Effect of N-Methylcarbamylcholine on Acetylcholine Release in Rat Brain

The present experiments show that N-[3H]-methylcarbamylcholine ([3H]MCC) binds specifically and with high affinity to rat hippocampus, frontal cortex, and striatum. The highest maximal density of binding sites was apparent in frontal cortex and the lowest in hippocampus. [3H]MCC binding was potently inhibited by nicotinic, but not muscarinic, agonists and by the nicotinic antagonist dihydro-beta-erythroidine in all three brain regions studied. The effect of unlabeled MCC on acetylcholine (ACh) release from slices of rat brain was tested. The drug significantly enhanced spontaneous ACh release from slices of hippocampus and frontal cortex, but not from striatal slices. This effect of MCC to increase ACh release from rat hippocampus and frontal cortex was antagonized by the nicotinic antagonists dihydro-beta-erythroidine and d-tubocurarine, but not by alpha-bungarotoxin or by the muscarinic antagonist atropine. The MCC-induced increase in spontaneous ACh release from hippocampal and frontal cortical slices was not affected by tetrodotoxin. The results suggest that MCC might alter cholinergic transmission in rat brain by a direct activation of presynaptic nicotinic receptors on the cholinergic terminals. That this alteration of ACh release is apparent in hippocampus and frontal cortex, but not in striatum, suggests that there may be a regional specificity in the regulation of ACh by nicotinic receptors in rat brain.     

Divergent Regulation of Muscarinic Binding Sites and Acetylcholinesterase in Discrete Regions of the Developing Human Fetal Brain

Summary The expression of muscarinic acetylcholine binding sites and of cholinesterases was studied in extracts prepared from discrete regions of the human fetal brain, between the gestational ages of 14 and 24 weeks. The specific binding of [³H]N-methyl-4-piperidyl benzilate ([⁴H]-4NMPB) to muscarinic binding sites ranged between 0.05 and 1.30 pmol/mg protein in the different brain regions, withK _d values of 1.2 ± 0.2 nM. Binding of the cholinergic agonist oxotremorine fitted, in most of the brain regions examined, with a two-site model for the muscarinic binding sites. The density of muscarinic binding sites increased with development in most regions, with different rates and onset times. It was higher by about sixfold in some areas destined to become cholinergic, such as the cortex and midbrain, than in noncholinergic areas such as the cerebellum. In other areas destined to become cholinergic, such as the hippocampus and the caudate putamen, the receptor density remained low. Average density values increased from 0.1 ± 0.1 at 14 weeks up to 0.7 ± 0.4 pmol/mg protein at 24 weeks.The variability in the specific activities of cholinesterase was relatively low, and extracts from different brain regions hydrolyzed from 5 to 30 nmol of [³H]acetylcholine/min/mg protein. These were mostly true acetylcholinesterase (EC 3.1.1.7) activities, inhibited by 10^–5 M BW284C51, with minor pseudocholinesterase (EC 3.1.1.8) activities, inhibited by 10^–5 M iso-OMPA. The enzyme from different brain regions and developmental stages displayed similarK _m values toward [³H]acetylcholine (ca. 4 × 10^–4 M ^–1). The ontogenetic changes in cholinesterase specific activities had no unifying pattern and/or relationship to the cholinergic nature of the various brain areas. In most of the brain regions, the arbitrary ratio between the specific activity of cholinesterase and the density of muscarinic binding sites decreased with development, with average values and variability ranges of 83 ± 50 and 19 ± 19 at 14 and 24 weeks, respectively. Our findings suggest divergent regulation for cholinergic binding sites and cholinesterase in the fetal human brain and imply that the expression of muscarinic receptors is related to the development of cholinergic transmission, while acetylcholinesterase is also involved in other functions in the fetal human brain.I.B. took part in this work as partial fulfillment of the requirements of the Sackler Faculty of Medicine for an M.D. degree.     

α-Bungarotoxin Binds to Low-Affinity Nicotine Binding Sites in Rat Brain

Reported differences in the pharmacology and distribution of [3H]nicotine and [125I]alpha-bungarotoxin binding sites in mammalian brain suggest that these ligands label separate receptor sites. Affinity purification of an alpha-bungarotoxin binding protein from rat brain failed to copurify the high-affinity nicotine binding site, which remained in the nonbound soluble fraction after the affinity chromatography step. This confirms the independence of these putative receptor sites. Nevertheless, the binding of [125I]alpha-bungarotoxin to P2 membranes was inhibited by (-)-nicotine (Ki = 9 X 10(-6) M), and this sensitivity was preserved after affinity purification. It is proposed that alpha-bungarotoxin binds to a population of low-affinity nicotine binding sites. Comparison of the enantiomers of nicotine in competition studies at both radioligand binding sites revealed an 80-fold preference for the (-) form at the high-affinity [3H]nicotine binding site, whereas the site labelled by [125I]alpha-bungarotoxin displayed little stereoselectivity. In this respect, the brain alpha-bungarotoxin binding site resembles the nicotinic acetylcholine receptor from Torpedo electric organ.