Binding of [3H]Imipramine to Mouse Cerebrocortical Membranes and to Glass Fiber Filters

It was suggested in a recent report by Phillips et al. [J. Neurochem. 43, 479-486 (1984)] that the low-affinity binding of [3H]imipramine in the mouse cerebral cortex could in fact represent binding of [3H]imipramine to the GF/B glass fiber filters used to terminate the assays. The present study demonstrates that this is not the case and advances two lines of evidence: (a) For saturation analysis, mouse cerebrocortical membranes were incubated with [3H]imipramine concentrations between 0.8 nM and 3.6 microM, and parallel incubations were carried out with buffer replacing the brain membranes. The same low-affinity component, in addition to the high-affinity component, was present in the binding of [3H]imipramine to brain membranes plus GF/B filters (uncorrected data), and in that to brain membranes alone (corrected data). (b) Dissociation experiments, in which filter binding is equal for all samples and dissociation time is the only variable, clearly indicated the nonhomogeneity of [3H]imipramine binding. Our results, however, do show that binding to recently purchased GF/B filters is not a negligible phenomenon in saturation experiments. Relatively lower binding was found to GF/C, GF/F, Gelman A/E, and Reeves Angel 934 AH filters; pretreatment of GF/B filters with polyethyleneimine (PEI) reduced binding to a greater extent in the single manifold than in the cell harvester.     

Developmental Changes of Cerebral Cortical [3H]Clonidine Binding in Rats: Influences of Guanine Nucleotide and Cations

Abstract: Cerebral cortical [³H]clonidine binding and influences of GTP and cations were investigated in developing rats. The results from Scatchard plots were compatible with the presence of two populations of binding sites [high-affinity binding ( K_D = 0.59 n M ) and low-affinity binding ( K_D = 7.12 n M )] in 70-day-old rats but only high-affinity binding ( K_D = 0.27 n M ) on day 1. Low-affinity binding was detectable on day 7. K_D values in high- and low-affinity binding were not significantly changed during development after 7 days. B_max of high-affinity binding reached a peak on day 15, and the value of low-affinity binding gradually increased with age. The addition of 10 μ M GTP caused a significant reduction in B_max of high-affinity binding after day 7. Neither K _D nor B_max of low-affinity binding was affected by 10 μ M GTP during development. NaCl (10 and 100 m M ) diminished the binding on days 7 and 70. MnCl₂ (0.1 and 1.0 m M ) markedly increased the binding on days 15 and 70 but not on day 7. It is suggested that: (1) single binding sites of α₂-adrenoceptors with higher affinity seem to be present on day 1; (2) low-affinity binding appears on day 7; (3) the number of high-affinity binding sites reaches a peak on day 15, followed by changes in populations of high-affinity as well as low-affinity sites without changing affinity; (4) the regulatory mechanism in α₂-receptors by guanine nucleotide reaches functional maturity between days 1 and 7; and (5) the involvement of Na⁺ and Mn²⁺ in α₂-receptor binding becomes functional by days 7 and 15, respectively.     

Taurine Interactions with Chick Retinal Membranes

Abstract: Binding of [³H]taurine to whole retinal membranes and to membranes obtained from retinal subcellular fractions was studied. [³H]Taurine bound to chick retinal membranes with high affinity and specificity. Two types of [³H]taurine binding associated to retinal membranes were observed, one with a K_D= 0.68 μM and the other one with a K_D,= 9.32 μM. Both types of binding were highly Na⁻-dependent. The Na⁺-dependent taurine binding was antagonized by strychnine. Bound [³H]taurine was effectively displaced by β-alanine but not by GABA or glycine. Taurine binding was preferentially localized in membranes obtained from the crude synaptosomal fraction, although it is also present in substantial amounts in all retinal membranes. A Na⁺-independent [³H]taurine binding exhibiting properties which might represent interaction with postsynaptic receptor sites could not be demonstrated in the chick retina.     

Ibotenic Acid Analogues as Inhibitors of [3H]Glutamic Acid Binding to Cerebellar Membranes

Abstract: The L-[³H]glutamic acid binding capability of rat cerebellar membranes prepared with or without preincubation at 37°C followed by washing was investigated. The two preparations ( K_D = 820 nM, B_max= 54.5 pmol/mg protein; K_D = 509 nM, B_max=13.0 pmol/mg protein) showed no difference in specificity of the binding of the ibotenic acid analogues, consistent with the removal of an endogenous inhibitor by the preincubation at 37°C followed by washing. The order of potency of the ibotenic acid analogues as inhibitors of L-[³H]glutamic acid binding is different from the order of potency in vivo , suggesting that the binding sites found are different from the physiological glutamic acid receptor.     

Binding Characteristics and Interactions of Depressant Drugs with [35S]t-Butylbicyclophosphorothionate, a Ligand that Binds to the Picrotoxinin Site

Abstract: [³⁵S]r-Butylbicyclophosphorothionate (TBPT), a cage convulsant with picrotoxinin-like activity, binds to rat brain membranes to a single site with an apparent K_D of 25.1 ± 5.6 n M and a B_max of 1.40 ± 0.22 pmol/mg protein. TBPT binding to rat brain membranes was inhibited by a variety of convulsant, depressant, anxiolytic, and anticonvulsant drugs that had previously been shown to inhibit [³H]a-dihydropicrotoxinin binding. Depressant drugs such as pentobarbital and the nonbarbiturate (+)etomidate inhibited TBPT binding in an uncompetitive manner. Thus, pentobarbital and (+)etomidate decreased both the affinity and the number of binding sites of TBPT to whole brain membranes. The IC₅₀ values of (+)etomidate (9 μ M ) and pentobarbital (90 μ M ) are similar to the EC₅₀ values at which they enhance both [³H]-γ-aminobutyric acid and [³H]diazepam binding in cerebral cortex membranes. RO5–4864, which has recently been shown to be a convulsant, also inhibited TBPT binding (IC₅₀= 10 μ M ). These results suggest that TBPT binds to the picrotoxinin site and further supports the notion that the picrotoxinin site is an important modulatory site at the benzodiazepine-GABA receptor-ionophore complex.     

Distribution of Specific High-Affinity Binding Sites for [3H]Imipramine in Human Brain

Abstract: [³H]Imipramine binds with high affinity to membranes from different regions of the human brain. The highest density of binding sites was observed in the hypothalamus and substantia nigra and the lowest density in the white matter and cerebellum. As found in rat brain, tricyclic antidepressant drugs are potent inhibitors of [³H]imipramine binding. Atypical antidepressants are, however, much weaker at inhibiting the specific binding. The [³H]imipramine binding site in human cortex is apparently identical to the site already described in the rat brain and in human platelets.     

[3H]Imipramine Binding of Protein Nature in Human Platelets: Inhibition by 5-Hydroxytryptamine and 5-Hydroxytryptamine Uptake Inhibitors

Abstract: The nature of [³H]imipramine binding to human platelets was investigated. Desipramine and 5-hydroxytryptamine (5-HT) displaced the same amount of binding and the binding was sensitive to protease treatment. The nature of pharmacological inhibition of [³H]imipramine binding was investigated in saturation experiments. Increases in K d without changes in B _max were noted with the addition of 5-HT, desipramine, norzimeldine, or 5-methoxytryptoline. Reductions in B _max without alterations in K _D were obtained when citalopram or clomipramine was added. It is concluded that the [³H]imipramine binding site in human platelets is of protein nature and that this binding site contains the substrate recognition site for 5-HT uptake. In addition, [³H]imipramine and other 5-HT uptake inhibitors have bonds to other parts of the 5-HT uptake carrier or to the surrounding lipid membrane. This additional binding outside the substrate recognition site is not one single site but most likely represents sites that are specific for the chemical structure of each uptake inhibitor, respectively.     

Effect of Ethanol Treatment on Rate and Equilibrium Constants for [3H]Muscimol Binding to Rat Brain Membranes: Alteration of Two Affinity States of the GABAA Receptor

Abstract: Equilibrium binding curves were biphasic in control and ethanol-treated rats. [³H]Muscimol binds to sites of high ( K _DA of ∼10 n M ) and low ( K _DB of ∼0.3–0.4 µ M ) affinity. Chronic ethanol treatment produced a decrease in B _maxA value, and the hyperbolic binding profiles were progressively affected by the chronic and in vitro ethanol treatments, with most of this effect corresponding to the high-affinity site. IC₅₀ and K _i values were calculated for several competing ligands, using membranes from both control and ethanol-treated animals. The association and dissociation curves were also biphasic, using a radioligand concentration precluding a significant occupancy of the low-affinity sites, which suggests the existence of two forms or affinity states of the monoliganded receptor. Chronic ethanol treatment did not produce changes in the values of the dissociation rate constants (fast and slow phases). By contrast, we report for the first time a decrease in the values of the association rate constants, with this decrease being higher for the slow phase. Consequently, the dissociation equilibrium constants are two times higher in chronically ethanol-treated animals for both phases.     

Identification of GABAA/Benzodiazepine Receptors on Clathrin-Coated Vesicles from Rat Brain

Abstract: To investigate the subcellular compartments that are involved in the endocytosis and intracellular trafficking of GABA_A/benzodiazepine receptors, we have studied the distribution and properties of clonazepam-displaceable binding of [³H]flunitrazepam to membrane fractions from rat brain. The microsomal fraction was subjected to density centrifugation and gel filtration to isolate clathrin-coated vesicles. Homogeneity of the coated-vesicle fraction was demonstrated by using electron microscopy and by analysis of clathrin subunits and clathrin light-chain kinase. Vesicles exhibiting specific binding of [³H]flunitrazepam eluted from the sieving gel as a separate peak, which was coincident with that for coated vesicles. Scatchard analysis of equilibrium binding of [³H]flunitrazepam to coated vesicles yielded a K_D value of 21 ± 4.7 nM and a B_max value of 184 ± 28 fmol/mg. The K_D value for coated vesicles was 12-19-fold that found with microsomal or crude synaptic membranes. This low-affinity benzodiazepine receptor was not identified on any other subcellular fraction and thus appears to be a novel characteristic of coated vesicles. The B_maxvalue for coated vesicles, expressed per milligram of protein, corresponded to 16 and 115% of that found for crude synaptic and microsomal membrane fractions, respectively. Because the trafficking of neurotransmitter receptors via clathrin-coated vesicles is most likely to occur through endocytosis, the data suggest that an endocytotic pathway may be involved in the removal of GABA_A/benzodiazepine receptors from the neuronal surfaces of the rat brain. This mechanism could play a role in receptor sequestration and down-regulation that is produced by exposure to GABA and benzodiazepine agonists.     

Ascorbic Acid Inhibits 125I-SCH 23982 Binding but Increases the Affinity of Dopamine for D1 Dopamine Receptors

Abstract: Effects of ascorbic acid (AA) on ¹²⁵I-SCH 23982 binding to D₁ dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µ M –0.33 m M inhibited 75% of specific binding of ¹²⁵I-SCH 23982 in a dose-dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 m M AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 m M AA, but reduced the inhibition by 3.3 m M AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 m M EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high-affinity ( K _h = 224.9 ± 48.9 n M ) and low-affinity ( K _l = 21,100 ± 2,400 n M ) binding sites. Although the maximum binding of ¹²⁵I-SCH 23982 decreased to 40% without affecting the K _D value in the presence of 1.67 m M AA, the value of the high-affinity site for dopamine was increased ( K _h = 23.3 ± 9.4 n M ) and that of the low-affinity site was decreased ( K _l = 136,800 ± 40,900 n M ). These results suggest that AA may affect D₁ dopamine receptor function by lipid peroxidation, competition with dopamine for low-affinity sites, and reduced oxidation of dopamine.     

Human Y-79 Retinoblastoma Cells Exhibit Specific Corticotropin-Releasing Hormone Binding Sites

Abstract: In this study we have identified specific binding sites for corticotropin-releasing hormone (CRH) in human Y-79 retinoblastoma cell membranes by using ¹²⁵I-Tyrovine CRH (¹²⁵I-oCRH) as radioligand. Binding at 19°C was rapid with steady state being reached within 20 min, reversible and linear with membrane protein concentration. The ¹²⁵I-oCRH binding was enhanced by Mg²⁺ and inhibited by the GTP analogue guanosine 5'- O -(3'-thiotriphosphate). Y-79 cell membranes exhibited two populations of binding sites, a high-affinity site with an apparent dissociation constant ( K _D) of 1 n M and a low-affinity site with an apparent K _D of 500 n M . ¹²⁵I-oCRH binding was completely antagonized by human/rat CRH, [Met(O)²¹]oCRH, α-helical CRH_9–41, urotensin I, and sauvagine with a rank order of potency similar to that displayed by CRH receptors of other tissues. These data describe for the first time the presence of specific CRH-binding sites in retinal cells. The Y-79 cell line may therefore constitute a valuable model in which to study CRH action on retinal cells.     

Comparison of [3H]WIN 35,428 Binding, a Marker for Dopamine Transporter, in Embryonic Mesencephalic Neuronal Cultures with Striatal Membranes of Adult Rats

Abstract: In contrast to striatal membranes of adult rats, where high- ( K _D1= 34 n M ) and low- ( K _D2= 48,400 n M ) affinity binding sites for [³H]WIN 35,428 are present, in primary cultures of ventral mesencephalon neurons (CVMNs) only low-affinity binding sites were found ( K _D= 336,000 n M ). The binding of [³H]WIN 35,428 in CVMNs prepared from rat embryos was reversible, saturable, and located in cytosol. Although dopamine (DA) uptake blockers inhibited [³H]DA uptake at nanomolar concentrations in CVMNs, the displacement of [³H]WIN 35,428 binding in CVMNs by DA uptake inhibitors required 100-8,000 times higher concentrations than were needed to displace [³H]WIN 35,428 binding in striatal membranes. Piperazine derivatives, e.g., GBR-12909, GBR-12935, and rimcazole, inhibited [³H]WIN 35,428 binding in CVMNs more effectively than did cocaine, WIN 35,428, mazindol, nomifensine, or benztropin. A positive correlation ( r = 0.779; p < 0.001) was found between drug affinities for the striatal membrane sites labeled by [³H]WIN 35,428 and their abilities to inhibit DA uptake in CVMNs, whereas no correlation existed between the IC₅₀ values of drugs that inhibited [³H]WIN 35,428 binding and [³H]DA uptake in CVMNs. The cytosolic [³H]WIN 35,428 binding sites may be a piperazine acceptor and may not be involved in the regulation of the DA transporter.     

Two Affinity States for [3H]Imipramine Binding to the Human Platelet 5-Hydroxytryptamine Carrier: An Explanation for the Allosteric Interaction Between 5-Hydroxytryptamine and Imipramine

5-Hydroxytryptamine (5-HT) showed a biphasic effect on the dissociation rate of [3H]imipramine from human platelet membranes: At low concentrations (EC50, approximately 2.5 microM), 5-HT stimulated the rate, as expected for mutually exclusive binding of 5-HT and imipramine; at higher concentrations (EC50, approximately 40 microM), 5-HT reduced this stimulated rate, a result consistent with 5-HT binding at a site that is physically distinct from both the imipramine binding site and the 5-HT transport recognition site of the 5-HT carrier. This modulatory effect could be mimicked by tryptamine, was saturable and independent of Na+ concentration, and could also be demonstrated for detergent-solubilized carriers. Monophasic association kinetics for [3H]imipramine binding were found. Heat stability experiments showed biphasic thermal inactivation curves. These results are consistent with [3H]imipramine binding to two classes of binding sites at the 5-HT carrier on human platelet membranes, with affinities three- to fivefold different. 5-HT can convert the lower-affinity state into the higher-affinity state.     

[3H]Citalopram Binding to Brain and Platelet Membranes of Human and Rat

Citalopram, a selective serotonin (5-HT) uptake inhibitor with antidepressant properties, was found to bind with high affinity to the 5-HT transporter from human neuronal and platelet membranes. At 20 degrees C, KD was about 1.5 nM in both tissues. [3H]Citalopram bound to rat neuronal membranes with higher affinity than to human neuronal and platelet membranes; at 20 degrees C KD was about 0.7 nM. The Bmax value for the binding of [3H]citalopram to platelet membranes was close to that found using the 5-HT uptake inhibitors [3H]imipramine and [3H]paroxetine, suggesting that all three 5-HT uptake inhibitors bind to the 5-HT transporter. The dissociation rate of [3H]citalopram increased twofold with each 4-5 degree C increase in temperature in both human and rat membranes, although at any given temperature, the dissociation rate was about four times faster in the human neuronal and platelet membranes than in rat neuronal membranes.     

High-Affinity Binding of [3H]Desipramine to Rat Brain: A Presynaptic Marker for Noradrenergic Uptake Sites

Abstract: High-affinity binding sites (apparent K _D= 1.5 nM) for [³H]desipramine have been demonstrated and characterized in membranes prepared from rat brain. The binding of [3H]desipramine was found to be saturable, reversible, heat-sensitive, sodium-dependent, and regionally distributed among various regions of the brain. High concentrations of [³H]desipramine binding sites were found in the septum, cerebral cortex, and hypothalamus, whereas lower concentrations were found in the medulla, cerebellum, and corpus striatum. A very good correlation ( r = 0.81, P < 0.001) was observed between the potencies of a series of drugs in inhibiting high-affinity [³H]desipramine binding and their capacity to block norepinephrine uptake into synaptosomes. In 6-hydroxydopamine-lesioned rats there was a marked decrease in [³H]norepinephrine uptake and [3H]desipramine binding with no significant alterations in either [³H]serotonin uptake or [³H]imipramine binding. These results suggest that the high-affinity binding of [³HJdesipramine to rat brain membranes is pharmacologically and biochemically distinct from the high-affinity binding of [3H]imipramine, and that there is a close relationship between the high-affinity binding site for [³H]desipramine and the uptake site for norepinephrine.     

Specific High-Affinity Binding of L-[3H]Aspartate to Rat Brain Membranes

Abstract: The binding of L-[³H]aspartate was investigated in washed membranes prepared from whole rat brain. We were able to differentiate two separate binding sites differing in their Na dependence. The Na-independent binding was saturable, reversible, and optimal at 20°C and at pHs in the neutral range. The dissociation constant (K_d) at 20°C was about 200 n M . This binding site seemed to be modulated by magnesium and calcium at physiological concentrations. None of the amino acids tested was a potent competitor for Na-independent L-[³H]aspartate binding. This binding site was unevenly distributed in the rat central nervous system: cerebellum = cerebral cortex > ponsmedulla > spinal cord. Destruction of the intrinsic neurons of the cerebellum by injecting kainic acid 30 days before sacrifice resulted in a 53% reduction in Na-independent binding in this region. The Na-dependent binding of L-[³H]-aspartate (K_d= 484 n M ) was strongly inhibited by D-aspartate, L-glutamate, D,L-aspartate β-hydroxamate; was unaffected by calcium and magnesium; and showed a different pattern of distribution: cerebral cortex > cerebellum = pons-medulla = spinal cord. This binding in cerebellum was unaffected by injections of kainic acid.     

Characterization of Membrane-Bound and Solubilized High-Affinity Binding Sites for 5'-N-Ethylcarboxamido[3H]adenosine from Bovine Cerebral Cortex

Abstract: A high-affinity binding site for 5'- N -ethylcarboxamido[³H]adenosine ([³H]NECA) from bovine cerebral cortex has been characterized in its membrane-bound and solubilized state after gel filtration on Sepharose CL-6B. For detection of this site in membranes, it was necessary to remove metabolites with high affinities for this site enzymatically, e.g., adenosine by addition of adenosine deaminase and inosine by addition of nucleoside phosphorylase. The pore-forming peptide antibiotic alamethicin further enhanced binding of [³H]NECA to this site in membranes. In contrast to adenosine receptors and the adenotin-like low-affinity binding protein, this novel site was extremely sensitive against treatment with the sulfhydryl alkylating agent N -ethylmaleimide. In competition experiments, this site could be differentiated from adenosine receptors by its high affinity for adenine nucleotides and its lack of affinity for adenosine receptor antagonists. Inosine and its derivative S -(4-nitrobenzyl)-6-thioinosine were relatively potent ligands with K _i values in the high nano- and low micromolar range, respectively. We conclude that the high-affinity NECA binding site described previously in bovine striatum is not exclusively located in the striatum, but can also be detected in membrane preparations and soluble extracts of bovine brain cortex.     

Characterization of [3H]CP-96,501 as a Selective Radioligand for the Serotonin 5-HT1B Receptor: Binding Studies in Rat Brain Membranes

Abstract: 3-(1,2,5,6-Tetrahydro-4-pyridyl)-5- n -propoxyindole (CP-96,501) was found to be a more selective ligand at the serotonin 5-HT_1B receptor than the commonly used 5-HT_1B agonist, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [³H]CP-96,501, was found to bind with a high affinity ( K _D, 0.21 n M ) to a single binding site ( n _H, 1.0). The receptor density of this site ( B _max, 72 fmol/mg of protein) matched that of the 5-HT_1B receptor determined with [³H]5-HT. Competition curves of 16 serotonergic compounds in [³H]CP-96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5-HT_1B receptor determined with [³H]5-HT or [¹²⁵I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [³H]CP-96,501 binding. In the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p], [³H]CP-96,501 binding was reduced, while the potency of CP-96,501 to displace [¹²⁵I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP-96,501. The results of this study suggest that [³H]CP-96,501 is a useful agonist radioligand for the 5-HT_1B receptor.     

Modulation of Neuropeptide FF Receptors by Guanine Nucleotides and Cations in Membranes of Rat Brain and Spinal Cord

Abstract: Using a radioligand binding assay, we examined ionic modulation and G protein coupling of neuropeptide FF(NPFF) receptors in membranes of rat brain and spinal cord. We found that NaCl (but not KCl or LiCl) and MgCl₂ increased specific ¹²⁵I-YLFQPQRFamide (¹²⁵I-Y⁸Fa) binding to NPFF receptors in both tissues in a dose-dependent manner, with optimal conditions being 60 m M NaCl and 1 m M MgCl₂. Guanine nucleotides dose-dependently inhibited specific ¹²⁵I-Y⁸Fa binding to rat brain and spinal cord membranes with maximal effects of 64 ± 6 and 71 ± 2%, respectively. The order of potency was nonhydrolyzable GTP analogues > GTP GDP > GMP, ATP. The guanine nucleotide inhibition was observed in the absence and presence of NaCl and MgCl₂. The mechanism of inhibition in spinal cord membranes appeared to be a reduction in the number of NPFF receptors; in one experiment, control K_D and B_max values were 0.068 n M and 7.2 fmol/mg of protein, respectively, and with 0.1 μ M guanylylimidodiphosphate the respective values were 0.081 n M and 4.9 fmol/mg, a 32% reduction in receptor number. Similar results were obtained with guanosine 5'-0-(3-thiotriphosphate). Our data suggest that ¹²⁵I-Y8Fa binding sites in rat CNS are G protein-coupled NPFF receptors regulated by GTP and cations.     

AMPA Receptor Development in Rat Telencephalon: [3H]AMPA Binding and Western Blot Studies

Abstract: Telencephalic membranes from rats of different embryonic (E16, E19) and postnatal (P2, P7, P14, adult) ages were assessed for α-[³H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([³H]AMPA) binding and for immunoreactivity levels of AMPA receptor subunits (GluR1, GluR2/3, and GluR4). In addition, the synaptic markers synaptophysin and NCAM₁₄₀ (a neural cell adhesion molecule isoform) were examined by immunoblot. The density of [³H]AMPA binding sites increased steadily with advancing age. This increase was due mainly to the development of the large low-affinity component ( K _D = 400 n M ) that dominates the [³H]AMPA binding profile of adult rat brain membranes. As resolved by two-site regression analysis, the high-affinity component ( K _D = 15 n M ) of the [³H]AMPA binding increased by approximately twofold from E16 to adult, whereas the low-affinity component increased by 25-fold. Staining for GluR1 and GluR2/3 increased steadily with increasing age at all time points examined; synaptophysin and NCAM₁₄₀ exhibited similar ontogenic immunostaining profiles. GluR4 immunoreactivity was first evident at P14 and increased by adulthood. These results indicate that AMPA receptor density increases steadily during development and that this increase is coincident with the ontogenic expression of other synaptic components. Furthermore, there is a shift toward a preponderance of low-affinity [³H]AMPA binding, which occurs during the period when AMPA receptors are being sorted into postsynaptic regions, suggesting that some element of the postsynaptic membrane environment modulates AMPA receptor properties.