Preparation of Antibodies to β Subunits of γ-Aminobutyric AcidA Receptors

Antisera were produced in rabbits against synthetic peptides based on two regions of the cDNA sequence of the beta 1 subunit of bovine gamma-aminobutyric acidA (GABAA) receptors. The deduced amino acid sequences were similar in other beta subunits of bovine, rat, and chick receptors, predicting cross-reactability with all beta subunits. One antiserum (anti-beta e) was raised against an extracellular moiety near the invariant disulfide loop thought to be located near the neurotransmitter binding domain; the other (anti-beta c) was raised against an intracellular moiety containing a consensus sequence for cyclic AMP-dependent protein kinase phosphorylation of a serine residue. Predicted secondary structures suggested high potential immunogenicity for the chosen antigen peptides. Both antisera at high dilutions recognized the same polypeptide bands on western blots of GABAA receptors purified from three regions of bovine brain (four bands at 57, 54, 53, and 52 kDa in cerebral cortex) but fewer bands (57, 54, and 52 kDa) in hippocampus and cerebellum (one major band at 54 kDa, traces at 57 and 53 kDa). This is consistent with the presence of multiple beta subunits whose expression varies with brain region, as shown by molecular cloning. The anti-beta c antibody was able to immunoprecipitate purified GABAA receptor [3H]-muscimol binding, 87% in bovine cortex and 75% in total rat brain; the anti-beta e was unable to immunoprecipitate any antigen. These antibodies indicate a region-dependent heterogeneity of beta subunits and should be useful for analyzing structure, function, and localization of GABAA receptor subtypes in brain.     

Regulation of the γ-Aminobutyric AcidA Receptor by γ-Aminobutyric Acid Levels Within the Postsynaptic Cell

Synaptosomes and synaptoneurosomes were prepared from rat cerebral cortex. Comparison of the amino acid levels in the two types of organelles and of the effects of gabaculine thereon indicated that the neurosome portion of synaptoneurosomes constituted the major influencing component of the organelles. Administration to rats of inhibitors of gamma-aminobutyric acid (GABA) degradation, such as gabaculine and L-cycloserine, resulted in elevated GABA levels in synaptoneurosomes and a decrease in muscimol-stimulated Cl- up-take by the organelles. Addition of gabaculine directly to the incubation medium for the uptake assay had no effect on the Cl- transport. In contrast, administration to rats of isonicotinic acid hydrazide, an inhibitor of GABA synthesis, decreased the GABA level in synaptoneurosomes and increased the muscimol-stimulated Cl- uptake by the organelles. Although the evidence is not unequivocal, it does support the concept of GABA released from nerve endings being taken up by the postsynaptic cell, from where it exerts a regulatory influence on the functioning of the GABA receptor/ion channel complex.     

Adrenergic Receptors in Aging and Alzheimer''s Disease: Increased β2-Receptors in Prefrontal Cortex and Hippocampus

Loss of pigmented noradrenergic locus ceruleus neurons occurs in Alzheimer's disease (AD) and, to a lesser extent, in aging. We studied beta-adrenergic receptors and their subtypes, beta 1 and beta 2, by the specific binding of 125I-pindolol to particulate membrane preparations from prefrontal cortex, hippocampus, putamen, and cerebellum and to sections from frontal cortex by in vitro autoradiography. In prefrontal cortex from controls, numbers of total beta- and beta 2-adrenoceptors did not significantly correlate with age, but number of beta 1-adrenoceptors showed a weak but significant negative correlation. Binding in tissue particulate preparations to total beta-receptors did not reveal significant differences in samples from prefrontal cortex between AD subjects and age-matched controls. However, beta 1-adrenoceptors were decreased and beta 2-adrenoceptors were increased in number by approximately 30-50% in AD subjects. Thus, the relative ratio of beta 1-/beta 2-receptors was decreased in AD. Binding by in vitro receptor autoradiography performed in a subset of samples of frontal cortex also showed beta 2-adrenoceptors, and less consistently total beta- and beta 1-receptors, to be increased significantly in number in cortical laminae II, III, IV, and V of tissue sections from AD subjects. In these subjects, number of locus ceruleus cells and norepinephrine concentrations in putamen and frontal cortex were markedly reduced compared with values in controls. In the hippocampus, total beta- and both beta 2- and beta 1-adrenoceptors were increased in number in AD. In contrast, in the putamen, where beta 1-receptors predominate, total beta- and beta 1-receptors were significantly decreased in number with no consistent change in content of beta 2-receptors in AD. There were no significant changes in the cerebellum. Specific pindolol binding was not affected by interval between death and sampling of tissue at autopsy. Our results indicate selective changes in number of beta-receptors in AD. These changes in the cortex and hippocampus suggest receptor upregulation in response to noradrenergic deafferentation from the locus ceruleus or may simply reflect glial proliferation in AD.     

Binding of γ-Aminobutyric AcidA Receptors to Tubulin

Abstract: The rate of axonal transport of tubulin, actin, and the neurofilament proteins was measured in the peripheral and central projections of the rat L5 dorsal root ganglion (DRG). [³⁵S]Methionine was injected into the DRG, and the "front" of the radiolabeled protein was located 7, 14, and 20 days postinjection. Transport rates calculated for the neurofilament triplet proteins, tubulin, and actin in the peripheral nerve were ∼ 1.5-fold faster than those in the dorsal root. A progressive decrease in the rate of transport was observed from 7 to 20 days after radiolabeling in both the central and peripheral directions (neurofilaments, ∼ 1.7-fold; tubulin/actin, 2.1-fold). A surgical preparation, leaving the peripheral sciatic nerve with predominantly sensory fibers, was the basis for ELISAs for phosphorylation-dependent immunoreactivity of the high-molecular-weight neurofilament protein. In both dorsal roots and peripheral sensory axons the degree of phosphorylation was greater in nerve segments further away from the cell bodies. The degree of phosphorylation-related immunoreactivity correlates with the slowing of transport of radiolabeled cytoskeletal protein.     

Peptide Subunits of γ-Aminobutyric AcidA/Benzodiazepine Receptors from Bovine Cerebral Cortex

The gamma-aminobutyric acidA/benzodiazepine receptor complexes from bovine cerebral cortex were purified by immunoaffinity chromatography, and the main component peptide subunits were characterized. The peptide band originally thought to be a single beta subunit [57,000 Mr band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] is composed of at least four different peptides of 54,000-57,000 Mr. Two peptides of 55,000 and 57,000 Mr were recognized by the beta subunit-specific monoclonal antibody 62-3G1. Peptides in the range of 54,000-57,000 Mr were photoaffinity-labeled with [3H]muscimol. A different 57,000 Mr peptide was photoaffinity-labeled by [3H]flunitrazepam, but neither was recognized by the monoclonal antibody 62-3G1 nor photoaffinity-labeled with [3H]muscimol. Some peptides could be identified by their differential mobility shift in SDS-PAGE after treatment with endoglycosidase H. Two additional subunit peptides of 51,000 and 53,000 Mr were also photoaffinity-labeled by [3H]flunitrazepam and reacted with antiserum A. However, the 57,000 Mr peptide that also was photoaffinity-labeled by [3H]flunitrazepam did not react with antiserum A.     

Molecular Size of the γ-Aminobutyric AcidA Receptor Purified from Mammalian Cerebral Cortex

The hydrodynamic behaviour of both the soluble and purified gamma-aminobutyric acidA (GABAA) receptor of bovine or rat cerebral cortex has been investigated in solution in Triton X-100 or in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS). In all the hydrodynamic separations made, it was found that the binding activities for GABA, benzodiazepine, and (where detectable) t-butylbicyclophosphorothionate comigrated. Conditions were established for gel exclusion chromatography and for sucrose density gradient velocity sedimentation that maintain the GABAA receptor in a nonaggregated form. Using these conditions, the molecular weight of the bovine GABAA receptor in the above-mentioned detergents was calculated using the H2O/2H2O method. A value of Mr 230,000-240,000 was calculated for the bovine pure GABAA receptor purified in sodium deoxycholate/Triton X-100 media. A value of Mr 284,000-290,000 was calculated for the nonaggregated bovine or rat cortex receptor in CHAPS, but the Stokes radius is smaller in the latter than in the former medium and the detergent binding in CHAPS is underestimated. Thus the deduced Mr, 240,000, is the best estimate by this method.     

Amino Acid Levels and γ-Aminobutyric AcidA Receptors in Rat Neostriatum, Cortex, and Thalamus After Neonatal 6-Hydroxydopamine Lesion

Abstract: The amino acid γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in brain, and GABAergic neurons have been proposed to play a major role in basal ganglia physiology. In the neostriatum (caudate putamen), medium-sized aspiny interneurons, as well as neostriatal output neurons that project to several brain regions, use GABA as their neurotransmitter. Dopamine fibers arising from the substantia nigra represent a major input to the neostriatum where, besides their classic neurotransmitter role, they are seemingly involved in the regulation of amino acid neurotransmitter release. To further characterize the nature of some of the amino acid/dopamine interactions, selective dopaminergic deafferentations were produced in neonatal rats (3 days postnatal) by intraventricular administration of the neurotoxin 6-hydroxydopamine (6-OHDA); the noradrenergic neurons were protected by prior administration of desmethylimipramine. After a 3-month survival, levels of catecholamines, indoleamines, and amino acids were determined in cingulate cortex, thalamus, and neostriatum. In addition, GABA_A receptors were measured in membrane preparations from these three regions, using the specific agonist [³H]muscimol. In the 6-hydroxydopamine-lesioned rats, levels of dopamine and its metabolites homovanillic acid, 3,4-dihydroxyphenylacetic acid, and 3-methoxytyramine were decreased, as expected, in cortex and neostriatum, but remained unmodified in thalamus. In all three regions, serotonin content was increased; its metabolite, 5-hydroxyindole-3-acetic acid, was also elevated, but only in cortex and neostriatum. The levels of GABA were increased in neostriatum and thalamus, but remained unmodified in cortex. Glycine was increased in all three regions examined. There were also increases of phosphatylethanolamine and serine in thalamus, and of aspartic acid and alanine in neostriatum. The density of GABA_A binding sites was increased in neostriatum, but remained unchanged in cortex and thalamus. The changes in amino acid levels and [³H]muscimol binding sites induced by a neonatal 6-hydroxydopamine treatment differ from those found after similar lesions in adult animals, possibly because of the plastic and synaptic rearrangements that can still occur during early postnatal development. The present results also demonstrate that adaptations occur in response to a dopaminergic deafferentation at an early age and that these exhibit a regional specificity.     

Immunological Identification of Multiple α-Like Subunits of the γ-Aminobutyric AcidA Receptor Complex Purified from Neonatal Rat Cortex

Antibodies were prepared against a synthetic peptide corresponding to amino acid sequences 174-203 of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1-subunit. The antibodies recognized this synthetic alpha 1-peptide, but failed to react with the homologous peptide sequence, 170-199, of the bovine beta 1-subunit. On Western blots, anti-alpha 1-subunit antibody recognized a 50-kilodalton (kDa) protein in affinity-purified receptor preparations from adult rat cortex and cerebellum. In receptor purified from neonatal cortex, the anti-alpha 1-antibody reacted with 50-kDa, 53-54-kDa, and 59-kDa proteins. After digestion with endoglycosidase F, these three protein bands retained differing electrophoretic mobilities. The 50-kDa and 59-kDa subunits of affinity-purified neonatal receptor, which were photoaffinity-labeled with [3H]flunitrazepam, were immunoprecipitated to different extents by alpha-subunit antibody. These data suggest the existence in GABAA receptor from neonatal cortex of three proteins (50 kDa, 53 kDa, and 59 kDa) which have immunological homology to alpha 1-subunit of bovine GABAA receptor. The presence of an alpha- and a beta-like subunit with similar mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis may account for the relatively high concentration of protein in the 53-54-kDa band which has been observed in receptor purified from neonatal cortex. The presence of multiple alpha-like subunits may be related to the presence of a relatively high concentration of type II GABA receptor in this tissue.     

Bovine γ-Aminobutyric AcidA Receptor Sequence-Specific Antibodies: Identification of Two Epitopes Which Are Recognised in Both Native and Denatured γ-Aminobutyric AcidA Receptors

Polyclonal antibodies have been raised against synthetic peptides whose sequences correspond to the N-terminal 15 amino acids and the C-terminal 17 amino acids of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit. These antibodies were shown to react with the denatured GABAA receptor alpha subunit, Mr 53,000, in Western blots with both purified receptor and brain membranes as antigens. Also, both antibodies recognised both the purified and detergent-solubilised GABAA receptor as demonstrated by dose-dependent specific immunoprecipitation of the GABA and benzodiazepine binding sites from solution. Evidence is also presented to show brain-regional distribution of the expression of the alpha 1 subunit.     

γ-Aminobutyric AcidB Receptor Activation Modifies Agonist Binding to β-Adrenergic Receptors in Rat Brain Cerebral Cortex

The interaction of isoproterenol with beta-adrenergic receptor (beta AR) binding sites was measured in membranes prepared from rat brain cerebral cortical slices previously incubated in the presence or absence of gamma-aminobutyric acid (GABA) receptor agonists. Both GABA and baclofen, but not isoguvacine, altered beta AR agonist binding by increasing the affinity of both the low- and high-affinity binding sites and by increasing the proportion of low-affinity receptors. The response to baclofen was stereoselective, and the effect of GABA was not inhibited by bicuculline. The results suggest that GABAB, but not GABAA, receptor activation modifies the coupling between beta AR and stimulatory guanine nucleotide-binding protein, which may in part explain the ability of baclofen to augment isoproterenol-stimulated cyclic AMP accumulation in brain slices.     

γ-Aminobutyric AcidA Receptor Pharmacology in Rat Cerebral Cortical Synaptoneurosomes

Equilibrium binding interactions at the gamma-aminobutyric acid (GABA) and benzodiazepine recognition sites on the GABAA receptor-Cl- ionophore complex were studied using a vesicular synaptoneurosome (microsacs) preparation of rat brain in a physiological HEPES buffer similar to that applied successfully in recent GABAergic 36Cl- flux measurements. NO 328, a GABA reuptake inhibitor, was included in the binding assays to prevent the uptake of [3H]muscimol. Under these conditions, the equilibrium dissociation constant (KD) values for [3H]muscimol and [3H]diazepam bindings are 1.9 microM and 40 nM, respectively. Binding affinities for these and other GABA and benzodiazepine agonists and antagonists correlate well with the known physiological doses required to elicit functional activity. This new in vitro binding protocol coupled with 36Cl- flux studies should prove to be of value in reassessing the pharmacology of the GABAA receptor complex in a more physiological environment.     

Antibodies to the Human γ2 Subunit of the γ-Aminobutyric AcidA/Benzodiazepine Receptor

Abstract: A γ-aminobutyric acid_A (GABA_A) receptor (GABA_AR) γ₂ subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage λgt11. The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors λgt11 and pGEX-3X. Both γ-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the γ₂IL were purified, and a rabbit antibody to the LacZ–γ₂IL was made. The antibody reacted with the γ₂IL of both LacZ and GST fusion proteins and immunoprecipitated the GABA_AR/ benzodiazepine receptor (GABA_AR/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABA_AR/BZDR immunoblots with a wide peptide band of 44,000–49,000 M_r. Immunoprecipitation studies with the anti-γ₂IL antibody suggest that in the cerebral cortex, 87% of the GABA_ARs with high affinity for benzodiazepines and 70% of the GABA_ARs with high affinity for muscimol contain at least a γ subunit, probably a γ₂. These results indicate that there are [³H]muscimol binding GABA_ARs that do not bind [³H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABA_AR/BZDR antibodies indicate that the most abundant combination of GABA_AR subunits in the cerebral cortex involves α₁, γ₂ (or other γ), and β₂ and/or β₃ subunits. These subunits coexist in >60% of the GABA_AR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20–25%) of the cerebellar GABA_AR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain α₆, also contain γ₂ (or other γ) subunit(s). The α₁ and β₂ or β₃ subunits are also frequently associated with γ₂ (or other γ) and α₆ in these cerebellar receptors.     

Differential Responses of Expressed Recombinant Human γ-Aminobutyric AcidA Receptors to Neurosteroids

Neuroactive steroids, in particular 3 alpha-hydroxypregnanes, are allosteric modulators of the gamma-aminobutyric acidA (GABAA) receptor. Regionally selective expression of receptor subunit subtypes may account for differential responsiveness of tissues to GABAergic inhibition and neurosteroid modulatory effects. The effect of 5 alpha-pregnan-3 alpha-ol-20-one (epiallopregnanolone) on heterotropic cooperativity on the GABAA receptor complex has been studied in three subtypes of expressed recombinant human receptors and in rat brain and spinal cord. Steroid potentiation of [3H]flunitrazepam binding was greatest for the alpha 3 beta 1 gamma 2 receptor complex, whereas alpha 1 beta 1 gamma 2 and alpha 2 beta 1 gamma 2 complexes showed less than 100% enhancement in binding. Previous studies suggest that the spinal cord is devoid of alpha 1, whereas cerebellum is rich in alpha 1 subunits. Correspondingly, a differential enhancement of [3H]flunitrazepam binding in spinal cord (51%) versus cerebellum (28%) was also observed. The structure of neuroactive steroids is important in determinikng the extent of neuromodulatory activity. The 5 beta-pregnanes,5 beta-pregnan-3 alpha-ol-20-one (epipregnanolone) and 5 beta-pregnan-3 alpha,21-diol-20-one (5 beta-tetrahydrodeoxycorticosterone), were both less potent than their corresponding 5 alpha derivatives. A 3 alpha hydroxyl group is essential for neuromodulatory activity in the expressed receptors, as demonstrated by the observation that 5 alpha-pregnan-3 beta-ol-20-one (allopregnanolone) and 4-pregnen-3, 20-dione (progesterone) were both inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist-Dependent Internalization of γ-Aminobutyric AcidA/Benzodiazepine Receptors in Chick Cortical Neurons

An impermeant benzodiazepine receptor ligand was prepared by derivatization of the aminobenzodiazepine 1012-S with 4-sulfophenylisothiocyanate. The resulting N-(4-sulfophenyl)-thiocarbamoyl derivative of 1012-S (SPTC-1012S) was purified by reverse-phase HPLC, and the predicted structure was verified by mass spectrometry. The apparent affinity of SPTC-1012S (IC50 = 9.8 +/- 2.9 nM) for displacement of [3H]flunitrazepam from intact chick cortical neurons was similar to that of 1012-S (IC50 = 4.0 +/- 0.3 nM). However, at concentrations from 0.1 to 10 microM, 1012-S was consistently more efficacious than SPTC-1012S, a finding indicating that 6-8% of the benzodiazepine receptor pool was not accessible to the impermeant compound. This inaccessible pool was eliminated by permeabilization of the cells with saponin or Triton X-100, a result suggesting that approximately 7% of neuronal benzodiazepine receptors are intracellular. Acute treatment (1-4 h at 37 degrees C) of neurons with 100 microM gamma-aminobutyric acid (GABA) or 100 nM clonazepam had little effect on the level of [3H]flunitrazepam binding but increased the proportion of intracellular receptors by 61 and 74%, respectively, compared with untreated controls. Similar treatment with 1 mM GABA increased the level of intracellular sites by 154-176%. The effect of GABA on receptor internalization was blocked by cotreatment with the GABAA receptor antagonist R 5135. The results suggest that SPTC-1012S can be used as a probe to study the internalization of the GABAA/benzodiazepine receptor complex under normal conditions or following acute or chronic treatment with agonists.     

Substantia Nigra γ-Aminobutyric Acid Receptors in Huntington''s Disease

The specific binding of [3H]gamma-aminobutyric acid (GABA) to nigral GABA receptors has been studied in postmortem brains from controls and patients with Huntington's disease (HD). A specific increase in the number of high-affinity binding sites for [3H]GABA was observed in HD patients, analogous to changes observed in rat substantia nigra [3H]GABA binding after striatal kainic acid (KA) lesion. The results provide further support for the striatal KA lesion in the rat as an animal model of HD. The implications of the results for the proposed therapeutic potential of GABA agonists in HD are discussed.     

Purification of the γ-Aminobutyric AcidA/Benzodiazepine Receptor Complex by Immunoaffinity Chromatography

The bovine gamma-aminobutyric acidA/benzodiazepine receptor complex has been purified by a novel immunoaffinity chromatography method on immobilized monoclonal antibody 62-3G1. Immunopurification of the complex was achieved in a single step with an improved yield over affinity chromatography on the benzodiazepine Ro 7-1986/1. High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the immunoaffinity-purified receptor revealed three major peptide bands of 51,000, 55,000, and 57,000 Mr which were also present in the Ro 7-1986/1 affinity-purified receptor. Peptide mapping, immunoblotting with subunit specific antibodies, and photoaffinity labeling with [3H]flunitrazepam and [3H]muscimol have been used for the identification of receptor subunits, including several which comigrated in a single band in SDS-PAGE.     

Age-Related Loss of t-[35S]Butylbicyclophosphorothionate Binding to the γ-Aminobutyric AcidA Receptor-Coupled Chloride Ionophore in Rat Cerebral Cortex

Muscimol and t-butylbicyclophosphorothionate (TBPS) are known to label two distinct sites within the gamma-aminobutyric acidA (GABAA) receptor complex, i.e., the GABA recognition site and the chloride ionophore, respectively. Age-dependent changes in the specific binding of [3H]muscimol and [35S]TBPS were compared in membranes prepared from the cerebral cortex of rats, 2-800 days old. Perinatal (day 2) binding of muscimol and TBPS represented 8 and 20% of the respective values for adults (day 180). After the first week, muscimol binding increased more rapidly than TBPS binding. Levels near those of adults were reached at day 20 and remained practically unchanged in adulthood (day 180). In aged (780-day-old) rats, the binding of TBPS was significantly reduced, whereas muscimol binding did not change compared with adult values. This decrease of TBPS binding derived from a reduced density of binding sites, rather than from affinity changes. The allosteric responsiveness of TBPS binding to exogenous GABA was also reduced in aged animals. These findings indicate an age-related change in the molecular (structural) organization of the GABAA receptor-chloride ionophore complex in rat cerebral cortex.     

Interconvertible Kinetic States of t-Butylbicycloorthobenzoate Binding Sites of the γ-Aminobutyric AcidA Ionophores

The kinetics of t-[³H]butylbicycloorthobenzoate (TBOB) binding to the convulsant sites of the γ-aminobutyric acid_A (GABA_A) receptor-ionophore complex were examined in synaptosomal membrane preparations of rat brain. On and off rates of TBOB binding were accelerated by 1 μM GABA and decelerated by 1 μM bicuculline methochloride, a GABA_A antagonist. The presence of GABA and bicuculline methochloride created rapid and slow phases of dissociation, respectively. The three groups of rate constants distinguished for the dissociation of 4 nM and 30 nM [³H]TBOB represent multiaffinity states of the convulsant sites depending on the presence of GABA or bicuculline methochloride. Apparent association rate constants do not obey the equation k_app=k_off±k_on [TBOB] without assuming interconvertibility of the kinetic states during binding. Avermectin B_1a (AVM B_1a), a chloride channel opening agent, accelerated the association and dissociation of TBOB and resulted in a biphasic effect on TBOB binding, i.e., enhancement at low concentrations (EC₅₀, 7.8 nM) followed by displacement at high concentrations (IC₅₀ 6.3 μM) of AVM B_1a. AVM B_1a resulted in similar biphasic effects on t- [³⁵S]butylbicyclophosphorothionate binding. DIDS, an isothiocyanatostilbene derivative with irreversible anion channel blocking effect, selectively inhibited basal [³H]TBOB binding (IC₅₀ 125 μM DIDS) leaving the enhancement by AVM B_1a unaffected.