Ivermectin Interactions with Benzodiazepine Receptors in Rat Cortex and Cerebellum In Vitro

Abstract: The anthelmintic macrolide, ivermectin, enhances the binding of benzodiazepine agonist ([³H]-diazepam) and antagonist ([³H]β-carboline ethyl ester) ligands to rat cortical and cerebellar membrane preparations. Enhancement of benzodiazepine agonist binding is partially additive with that of γ-aminobutyric acid (GABA) and is inhibited by etazolate, bicuculline, and the steroid GABA antagonist R5135. Ivermectin-stimulated benzodiazepine antagonist binding is enhanced by bicuculline and inhibited by GABA and etazolate. The modulatory effects of bicuculline are chloride-dependent. The stimulatory effects of ivermectin, while quantitatively different in cortex and cerebellum, are qualitatively similar in both brain regions and are reduced in the presence of chloride. Ivermectin effects on benzodiazepine ligand binding to the benzodiazepine receptor complex and the differences in the effects of GABA, bicuculline, and R5135 on ivermectin-stimulated agonist and antagonist binding may provide evidence for distinct differences in the recognition sites for the two classes of benzodiazepine receptor ligand and their interactions with other components of the receptor complex.     

Characterization of the Solubilized GABA and Benzodiazepine Receptors from Various Regions of Bovine Brain

GABA and benzodiazepine receptors were solubilized from bovine cerebral cortex, cerebellum, and hippocampus and then partially purified by gel filtration and characterized. The apparent molecular weights of all these receptors were determined to be 600,000-650,000 by gel filtration, the sedimentation coefficients being 11.0-11.3 S by sucrose density gradient centrifugation. [3H]Muscimol was bound to two classes of sites in fractions from all three regions, and [3H]flunitrazepam bound to one class of sites. A comparison of the ratios of Bmax for flunitrazepam binding to Bmax for muscimol binding revealed that the fractions from the hippocampus exhibited a much higher ratio of benzodiazepine binding sites than were detected in fractions from the cortex and cerebellum. GABA agonist and antagonist inhibited [3H]muscimol binding to the fractions from these regions, at similar concentrations. Benzodiazepine agonists and antagonists also inhibited [3H]flunitrazepam binding in these three fractions, with similar potency. CL 218,872, however, inhibited [3H]flunitrazepam binding in the cerebellar fraction with the lowest IC50 value and that in th hippocampal fraction with the highest IC50 value. Hill coefficients for CL 218,872 inhibition were 0.98, 0.64, and 0.58 for cerebellum, cortex, and hippocampus, respectively.     

Benzodiazepine Receptor Binding in Cerebellar Cortex: Observations in Olivopontocerebellar Atrophy

Benzodiazepine receptor binding was measured in cerebellar cortex of 15 patients with dominantly inherited olivopontocerebellar atrophy (OPCA). The majority of these patients had a moderate to marked Purkinje cell loss, as judged by the lowered levels of dentate nucleus gamma-aminobutyric acid (GABA), a marker of Purkinje cells. Despite the reduction in Purkinje cell number cerebellar cortical benzodiazepine receptor density was either normal or slightly elevated in the OPCA patients. These results are in contrast to the findings in a mutant strain of mice deficient in Purkinje cells in which the concentration of benzodiazepine receptors in cerebellum is greatly reduced. Our data indicate that in the human, cerebellar cortical benzodiazepine receptors are either not significantly associated with Purkinje cells or that in OPCA Purkinje cell loss triggers a de novo synthesis of extra benzodiazepine binding sites. It is concluded that, in contrast with the rodent, in the human benzodiazepine receptor binding may not serve as a marker for cerebellar Purkinje cells.     

Type I and Type II γ-Aminobutyric Acid/Benzodiazepine Receptors: Purification and Analysis of Novel Receptor Complex from Neonatal Cortex

The gamma-aminobutyric acid (GABA) type A receptor was purified several thousandfold by affinity chromatography from rat cerebellum, adult cortex, and neonatal cortex. Competition for the benzodiazepine binding site by CL 218872 indicated that cerebellar receptors were predominantly type I, adult cortical receptors were a mixture of subtypes, and neonatal cortex was enriched in type II receptor. The receptor purified from neonatal cortex contained predominantly a 54-kilodalton (kDa), beta-subunit-like protein, whereas receptors from cerebellum and adult cortex contained nearly equal amounts of a 50-kDa, alpha-subunit-like protein and a 54-kDa polypeptide. Peptide maps of trypsin-digested 54-kDa subunits from cerebellum, adult cortex, and neonatal cortex exhibited very similar profiles, a result indicating considerable homology between these proteins in the receptor subtypes. A 59-kDa subunit protein was detected in the receptor complex purified from neonatal cortex. Like the 50-kDa, alpha-subunit of the type I receptor, this protein was photolabeled with [3H]flunitrazepam. The photolabeled peptide fragments, produced by trypsin digestion of these alpha 50- and alpha 59-subunits, exhibited the same retention times on reverse-phase HPLC. A less highly purified GABAA receptor preparation from adult rat spinal cord possessed characteristics that were very similar to those of the receptors purified from neonatal cortex.     

Regional Distribution of the GABAA/Benzodiazepine Receptor (α Subunit) mRNA in Rat Brain

A human cDNA clone containing the 5' coding region of the GABAA/benzodiazepine receptor alpha subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP-labelled antisense RNA probe (860 bases) prepared from the alpha subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus much greater than pons = striatum = medulla. In situ hybridization revealed high levels of alpha subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor alpha subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.     

Photoaffinity labeling of [3H]flunitrazepam- and [3H]Ro15-4513-bound pellets in rat cerebral cortex and cerebellum

Irreversible incorporation of [3H]flunitrazepam and [3H]Ro15-4513 into GABA/benzodiazepine receptor subunits was studied by UV irradiation using ligand-bound membrane pellets from rat cerebral cortical and cerebellar synaptic membranes. Specific incorporation for [3H]flunitrazepam was greater in the pellet than in the suspension. The incorporation was identical for [3H]Ro15-4513 in both pellet and suspension. With the ligand-bound pellets, 50% of the available binding sites were photolabeled by both ligands in cortex and cerebellum. SDS polyacrylamide gel electrophoresis and fluorography of [3H]flunitrazepam photo-labeled receptor revealed the same number of major sites in both brain regions. In contrast, [3H]Ro15-4513 appears to label fewer sites in cortex and cerebellum. Photoaffinity labeling with [3H]flunitrazepam in ligand-bound membrane pellet provides a more selective and reliable method for studying the subunit structure of GABA/benzodiazepine receptor complex.     

Benzodiazepine Treatment Causes Uncoupling of Recombinant GABAA Receptors Expressed in Stably Transfected Cells

Abstract: GABA_A and benzodiazepine receptors are allosterically coupled, and occupation of either receptor site increases the affinity of the other. Chronic exposure of primary neuronal cultures to benzodiazepine agonists reduces these allosteric interactions. Neurons express multiple GABA_A receptor subunits, and it has been suggested that uncoupling is due to changes in the subunit composition of the receptor. To determine if uncoupling could be observed with expression of defined subunits, mouse Ltk⁻ cells stably transfected with GABA_A receptors (bovine α1, β1, and γ2L subunits) were treated with flunitrazepam (Flu) or clonazepam. The increase in [³H]Flu binding affinity caused by GABA (GABA shift or coupling) was significantly reduced in cells treated chronically with the benzodiazepines, whereas the K _D and B _max of [³H]Flu binding were unaffected. The uncoupling caused by clonazepam treatment occurred rapidly with a t _1/2 of ∼30 min. The EC₅₀ for clonazepam treatment was ∼0.3 µ M , and cotreatment with the benzodiazepine antagonist Ro 15-1788 (5.6 µ M ) prevented the effect of clonazepam. The uncoupling observed in this system was not accompanied by receptor internalization, is unlikely to be due to changes in receptor subunit composition, and probably represents posttranslational changes. The rapid regulation of allosteric coupling by benzodiazepine treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABA_A and benzodiazepine receptors as well as benzodiazepine tolerance.     

Modification of γ-Aminobutyric AcidA Receptor Binding and Function by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline In Vitro and In Vivo: Effects of Aging

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased Binding of [3H]Muscimol and [3H]Flunitrazepam in the Rat Brain Under Hypoxia

We examined the effects of in vivo hypoxia (10% O2/90% N2) on the gamma-aminobutyric acid (GABA)/benzodiazepine receptors and on glutamic acid decarboxylase (GAD) activity in the rat brain. Male Wistar rats were exposed to a mixture of 10% O2 and 90% N2 in a chamber for various periods (3, 6, 12, and 24 h). The control rats were exposed to room air. The brain regions examined were the cerebral cortex, striatum, hippocampus, and cerebellum. GABA and benzodiazepine receptors were assessed using [3H]muscimol and [3H]flunitrazepam, respectively. Compared with control values, GAD activity was decreased significantly following a 6-h exposure to hypoxia in all four regions studied. On the other hand, the numbers of both [3H]muscimol and [3H]flunitrazepam binding sites were increased significantly. The increase in receptor number tended to return to control values after 24 h. Treatment of the membrane preparations with 0.05% Triton X-100 eliminated the increase in the binding capacity. These results may represent an up-regulation of postsynaptically located GABA/benzodiazepine receptors corresponding to the impaired presynaptic activity under hypoxia.     

In Vitro Modulation by Avermectin B1a of the GABA/Benzodiazepine Receptor Complex of Rat Cerebellum

Avermectin B1a, a macrocyclic lactone anthelmintic agent, causes a concentration-dependent increase of [3H]flunitrazepam binding to membranes from rat cerebellum by increasing the affinity and the number of binding sites. This effect appears to be independent of the concentration of chloride ions. The effects of avermectin B1a occur with high affinity (EC50 = 70 nM), and they persist after washing of the membranes with drug-free buffer. Pretreatment of the membranes with Triton X-100 completely abolishes the action of avermectin B1a. GABA and the GABA-mimetic compounds piperidine-4-sulfonic acid and THIP diminish the effects of avermectin B1a on benzodiazepine receptor binding in a bicuculline-methiodide-sensitive mode. In addition, the stimulation of [3H]flunitrazepam binding by avermectin B1a is decreased by the pyrazolopyridines etazolate and cartazolate. These observations suggest that avermectin B1a stimulates benzodiazepine receptor binding by acting on a modulatory site which is independent of the GABA recognition site and of the drug receptor for the pyrazolopyridines, but which is in functional interaction with these sites.     

The Benzodiazepine/GABA Receptor Complex: Molecular Size in Brain Synaptic Membranes and in Solution

Abstract: The molecular size of the benzodiazepine (BZ) receptor in the synaptic membrane of brain cortex (bovine or rat) was determined by an improved version of the radiation inactivation method to be 220,000. An identical size was found simultaneously for the associated γ-aminobutyric acid (GABA) receptor and for the component binding β-carboline esters. It is proposed that all three activities reside in a single protein or protein complex in the membrane. The size in solution, after extraction into Triton X-100 medium from exhaustively washed membranes, was estimated by sedimentation constant (9.4S) and by gel filtration (∼230,000 apparent MW), again with the BZ and GABA binding activities behaving identically. This size applies to the component that undergoes photoaffinity labelling by [³H]flunitrazepam in the membrane, and contains a 51,000 M_r polypeptide as the BZ-binding subunit. It is concluded that a protein complex or oligomer of 200,000–220,000 MW carries a class of BZ-binding sites and an associated class of GABA_A sites.     

Characterization of Na+-Independent GABA and Flunitrazepam Binding Sites in Preparations of Synaptic Membranes and Postsynaptic Densities Isolated from Canine Cerebral Cortex and Cerebellum

The binding of [3H]GABA and [3H]flunitrazepam was performed with synaptic membranes and post-synaptic densities (PSDs) isolated from canine cerebral cortex and cerebellum. Two GABA binding sites were found with cerebral cortex membranes but only one with cerebellar membranes. PSDs isolated from these showed only single binding sites, with cerebellar PSDs exhibiting lower KD values and a larger concentration of sites than did cerebral cortex PSDs. In the case of flunitrazepam, only one binding site was found for all four preparations, with cerebellar PSDs having twice the concentration of sites of cerebral PSDs. Photoaffinity labeling of the flunitrazepam receptor in PSDs resulted in the binding to a 51,000 Mr protein in both cases, with cerebellar PSDs again showing an increased concentration over that found in cerebral cortex PSDs. Based on this work, and on earlier work of ourselves and of others, we conclude that both populations of isolated PSDs contain inhibitory sites, but that the intact PSDs in both preparations are derived from Gray type I, probably excitatory, synapses, and that the inhibitory sites are found in the broken-up material in the PSD fractions which are derived from Gray type II, probably inhibitory, synapses.     

Conformational changes at benzodiazepine binding sites of GABA(A) receptors detected with a novel technique

Benzodiazepines are widely used for their anxiolytic, sedative, myorelaxant and anticonvulsant properties. They allosterically modulate GABA(A) receptor function by increasing the apparent affinity of the agonist GABA. We studied conformational changes induced by channel agonists at the benzodiazepine binding site. We used the rate of covalent reaction between a benzodiazepine carrying a cysteine reactive moiety with mutated receptor having a cysteine residue in the benzodiazepine binding pocket, alpha1H101Cbeta2gamma2, as a sensor of its conformation. This reaction rate is sensitive to local conformational changes. Covalent reaction locks the receptor in the conformation stabilized by positive allosteric modulators. By using concatenated subunits we demonstrated that the covalent reaction occurs either exclusively at the alpha/gamma subunit interface, or if it occurs in both alpha1 subunits, exclusively reaction at the alpha/gamma subunit interface can modulate the receptor. We found evidence for an increased rate of reaction of activated receptors, whereas reaction rate with the desensitized state is slowed down. The benzodiazepine antagonist Ro15-1788 efficiently inhibited the covalent reaction in the presence of 100 microm GABA but only partially in its absence or in the presence of 10 microm GABA. It is concluded that Ro15-1788 efficiently protects activated and desensitized states, but not the resting state.     

Antibodies Recognising the GABAA/Benzodiazepine Receptor Including Its Regulatory Sites

Polyclonal antibodies have been raised against the GABA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X-100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I-labelled gamma-aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immunoprecipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl-beta-carboline-3-carboxylate binding, the [35S]t-butylbicyclophosphorothionate binding (TBPS), the barbiturate-enhanced [3H]flunitrazepam binding, and the GABA-enhanced [3H]flunitrazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the alpha-subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators.     

γ-Aminobutyric Acid and Pentobarbital Enhance 2-[3H]Oxoquazepam Binding to Type I Benzodiazepine Recognition Sites in Rat and Human Brain

2-Oxoquazepam (2oxoquaz) is a novel benzodiazepine which shows preferential affinity for type I benzodiazepine recognition sites. In the present study, we analyzed the effect of gamma-aminobutyric acid (GABA), pentobarbital, and chloride ions on [3H]2oxoquaz and [3H]flunitrazepam ( [3H]FNT) binding to membrane preparations from rat and human brain. GABA stimulated [3H]-2oxoquaz and [3H]FNT binding in a concentration-dependent manner. The maximal enhancement produced by GABA on [3H]2oxoquaz binding was higher than that produced on [3H]FNT binding in both rat and human tissues. In the rat brain, the effect of GABA on [3H]2oxoquaz was similar throughout different brain areas, whereas the effect on [3H]FNT binding was lower in the cerebral cortex and hippocampus than in the cerebellum. Moreover, both [3H]2oxoquaz and [3H]FNT binding were stimulated by chloride ions and pentobarbital. The results are consistent with the hypothesis that type I benzodiazepine recognition sites are linked functionally to the GABA recognition site and the chloride ionophore.     

Prevalence between different alpha subunits performing the benzodiazepine binding sites in native heterologous GABA(A) receptors containing the alpha2 subunit

The presence of two heterologous alpha subunits and a single benzodiazepine binding site in the GABA(A) receptor implicates the existence of pharmacologically active and inactive alpha subunits. This fact raises the question of whether a particular alpha subtype could predominate performing the benzodiazepine binding site. The hippocampal formation expresses high levels of alpha subunits with different benzodiazepine binding properties (alpha1, alpha2 and alpha5). Thus, we first demonstrated the existence of alpha2-alpha1 (36.3 +/- 5.2% of the alpha2 population) and alpha2-alpha5 (20.2 +/- 2.1%) heterologous receptors. A similar alpha2-alpha1 association was observed in cortex. This association allows the direct comparison of the pharmacological properties of heterologous native GABA(A) receptors containing a common (alpha2) and a different (alpha1 or alpha5) alpha subunit. The alpha2 subunit pharmacologically prevailed over the alpha1 subunit in both cortex and hippocampus (there was an absence of high-affinity binding sites for Cl218,872, zolpidem and [3H]zolpidem). This prevalence was directly probed by zolpidem displacement experiments in alpha2-alpha1 double immunopurified receptors (K(i) = 295 +/- 56 nM and 200 +/- 8 nM in hippocampus and cortex, respectively). On the contrary, the alpha5 subunit pharmacologically prevailed over the alpha2 subunit (low- and high-affinity binding sites for zolpidem and [3H]L-655,708, respectively). This prevalence was probed in alpha2-alpha5 double immunopurified receptors. Zolpidem displayed a single low-affinity binding site (K(i) = 1.73 +/- 0.54 microM). These results demonstrated the existence of a differential dominance between the different alpha subunits performing the benzodiazepine binding sites in the native GABA(A) receptors.     

Benzodiazepine-sensitive GABA(A) receptors limit the activity of the NMDA/NO/cyclic GMP pathway: a microdialysis study in the cerebellum of freely moving rats

In the cerebellum, infusion of NMDA (200 microM) for 20 min evoked a marked (200%) increase of extracellular cyclic GMP (cGMP) levels. The selective GABA(A) receptor agonist muscimol (0.01-100 microM) was able to counteract the NMDA effect with an EC(50) of 0.65 microM; the inhibitory effect of muscimol (10 microM) was prevented by bicuculline (50 microM). Diazepam (10 microM) significantly potentiated the muscimol (1 microM) inhibition; furthermore, when coinfused with 0.1 microM muscimol (a concentration not affecting, on its own, the cGMP response to NMDA), diazepam (10 microM) reduced the NMDA effect. Similar results were obtained with zolpidem (0.1-1 microM). Finally, local infusion of the benzodiazepine site antagonist flumazenil (10 microM), together with muscimol and diazepam, almost completely restored the effect of NMDA on extracellular cGMP levels. It is concluded that GABA(A) receptors potently control the NMDA/nitric oxide/cGMP pathway in the cerebellum in vivo. In terms of the alpha subunit composition, we can deduce that the cerebellar GABA(A) receptor does not contain alpha(6) or beta(4) subunits because it is diazepam-sensitive. Moreover, the observation that zolpidem is active at a rather low concentration, in combination with localization studies present in the literature, tend to exclude the presence of alpha(5) subunits in the receptor composition and suggest the involvement of an alpha(1) subunit.     

Heterogeneity in the Allosteric Interaction Between the γ-Aminobutyric Acid (GABA) Binding Site and Three Different Benzodiazepine Binding Sites of the GABAA/Benzodiazepine Receptor Complex in the Rat Nervous System

In the present communication we have investigated the allosteric coupling between the gamma-aminobutyric acidA (GABAA) receptor and the pharmacologically different benzodiazepine (BZD) receptor subtypes in membranes from various rat nervous system regions. Two types of BZD receptors (type I and type II) have been classically defined using CL 218.872. However, using zolpidem, three different BZD receptors have been identified by binding displacement experiments in membranes. These BZD receptor subtypes displayed high, low, and very low affinity for zolpidem. The distribution of the high- and low-affinity binding sites for zolpidem was similar to that of type I and type II subtypes in cerebellum, prefrontal cortex, and adult cerebral cortex. On the other hand, the very-low-affinity binding site was localized in relative high proportion in spinal cord, hippocampus, and newborn cerebral cortex and, to a minor extent, in superior colliculus. The allosteric coupling between the GABAA receptor and the BZD receptor subtypes was different. The high- and low-affinity binding sites for zolpidem seemed to have a similar high degree of coupling, except in spinal cord. On the other hand, the very-low-affinity binding site for zolpidem displayed a low degree of coupling with the GABAA receptor. These results seem to indicate that the different efficacy of GABA in enhancing the [3H]flunitrazepam binding could be due to the different BZD receptor subtypes present in the GABAA/BZD receptor complex and, moreover, led us to speculate that the low GABA efficacy found in membranes from spinal cord, hippocampus, and newborn cerebral cortex might be due to the presence in relatively high proportion of the very-low-affinity binding site for zolpidem.     

A Gaba/Benzodiazepine Receptor Complex from Bovine Brain: Purification,Reconstitution and Immunological Characterization

Abstract

A GABA/benzodiazepine receptor complex was purified from bovine cerebral cortex. The receptor fraction displayed binding sites for benzodiazepines as well as high and low affinity binding sites for GABA which are characteristics of the membrane-bound receptor. Two monoclonal antibodies of which one was directed against the 50 kd and the other against the 55 kd subunit were used for immunoprecipitation studies. Both of them were shown to quantitatively precipitate the entire receptor population. These results indicate that the binding sites for benzodiazepines and GABA (high and low affinity sites) reside on the same receptor complex containing a mixture of 50 kd and 55 kd subunits. Reconstitution of the receptor in phospholipid vesicles was achieved.     

Effect of aging on neurotransmitter receptor binding in rat and human brain.

β-Adrenergic and GABA receptor binding were measured in brain areas of rats 3 to 24 months of age. While GABA receptor binding was not significantly different across age in any area, β-adrenergic receptor binding was significantly reduced in the cerebellum and brain stem, but not cerebral cortex, of 24-month-old animals. The loss in β-adrenergic receptor binding does not correlate in a temporal fashion with the reported decrease in norepinephrine-stimulated cyclic AMP accumulation in the cerebellum which occurs as early as 12 months of age. An age-related reduction in β-adrenergic binding was also noted in human cerebellar tissue obtained at autopsy, suggesting that the cerebellar dysfunction seen with aging may be related to a loss of cerebellar neurons which receive noradrenergic input.