Alterations in Dihydromorphine Binding in Cerebral Hemispheres of Aged Male Rats

Equilibrium binding of [3H]dihydromorphine was assayed in brain regions of young and aged male F344 rats. Young rats had significantly higher receptor densities than old rats in the frontal poles, anterior cortex, and striatum. In the frontal poles, the decline in receptor concentration with age was accompanied by a significant increase in the apparent affinity of dihydromorphine for receptors, which may be compensatory for the decrease in Bmax. This pattern of receptor alterations is different than that previously observed in aged female rats. Therefore, processes which underlie synaptic alterations with age may be different in males and females.     

Aging and rat brain muscarinic receptors as measured by quinuclidinyl benzilate binding

Measurement of cholinergic muscarinic receptor binding in various rat brain areas using the ligand [³H]quinuclidinyl benzilate indicates that receptor binding is decreased in striatum and cerebellum of aged female rats (22 months old) as compared to younger rats (4 months old). Decreases were not observed in cortex, hippocampus, hypothalamus, or amygdala areas. Further examination of [³H]quinuclidinyl benzilate binding in subcellular fractions of aged and young rat cerebellum and striatum indicated a decrease in binding in the crude nuclear and crude synaptosomal fractions. Binding data indicate the observed decrease in specific ligand binding is due to a decrease in number of binding sites while receptor affinity does not appear to change.Supported by the Research Service of the Veterans Administration and by Research Grant NS 13227 from NINCDS.     

Regional analysis of brain opiate receptors in rats with hereditary hypothalamic diabetes insipidus.

The regional distribution of stereospecific ³H-dihydromorphine binding sites in Brattleboro rats with hereditary hypothalamic diabetes insipidus was studied. Control animals were homozygous normal Brattleboro rats and rats heterozygous for the vasopressin deficiency. Scatchard plots of ³H-dihydromorphine binding in a washed membrane preparation showed that rats with diabetes insipidus exhibited higher receptor concentrations in all assayed areas of the cerebral hemispheres. In the diencephalon, receptor concentrations were lower in diabetes insipidus rats. The results point to the existence of interactions between brain opioid systems and neurohypophyseal peptides.     

Intracerebral distribution pattern of radioactive morphine and morphine-like drugs after intraventricular and intrathecal injection

Summary The intracerebral distribution patterns of ¹⁴C-morphine, ³H-dihydromorphine, and ³H-fentanyl after intraventricular injection were studied autoradiographically in rats and rabbits. The extent of permeation into the ventricular wall was measured at different times after injection. The hydrophilic morphine and dihydromorphine could be demonstrated within the tissue up to 4 hours. They seemed to be retained within the gray matter and hindered in crossing fiber bundles. On the other hand, the lipophilic fentanyl was quickly removed from the brain but remained relatively longer demonstrable within the white matter. Also, after intrathecal injection of ¹⁴C-morphine a time dependent spread from the injection site was observed. The use of autoradiography in pharmacological experiments as described was found advantageous. Thus, it is possible to correlate directly, the time course of the pharmacological effect and the respective distribution pattern of the drug applied. This may lead to better information about the probable sites of drug action.     

Opioid binding properties in bovine retina

Saturable binding sites for tritiated dihydromorphine ([³H]DHM), D-Ala²-D-Leu⁵-enkephalin ([³H]DADL) and etorphine were found in a crude synaptosomal preparation of bovine retina. Scatchard analysis of saturation binding curves of each ligand was curvilinear and the presence of two independent binding sites inferred. The density of binding sites of [³H]etorphine was similar to that reported in brain crude synaptosomal preparations, and the affinity for the high affinity binding site to each ligand was similar to values determined in brain. Moreover, the regulation of the binding sites by GTP and sodium was also similar to that observed in brain. Selective binding sites for [³H]DADL (δ-sites) were not detectable, although binding sites similar in nature to μ-binding sites were detected.     

Stereospecific interaction of opiate narcotics in binding of 3H-dihydromorphine to membranes of rat brain

Membranes from homogenates of corpus striatum bound ³H-dihydromorphine in a saturable fashion with a Km value of 1 × 10^?9M. The binding of ³H-dihydromorphine to the membranes was reduced to about 10% by 10^?7M levorphanol but not by 10^?7M dextrorphan. The binding of ³H-dihydromorphine became less sensitive to 10^?7M levorphanol when the concentration of ³H-dihydromorphine was greater than 2 × 10^?9M. Other opiate narcotics, e.g. morphine and $\text{l}$-methadone, were as effective as levorphanol in competition for the binding ³H-dihydromorphine with ED₅₀ values of 2–4 × 10^?9M. $\text{d}$-Methadone and dextrorphan were about 1/50 and 1/2000 as effective as their respective levo-isomers. The opiate antagonist, naloxone, also competed effectively for the binding sites with an ED₅₀ value of 3.3 × 10^?9M. Substances like acetylcholine, choline, serotonin, norepinephrine and dopamine were ineffective. Only ionophores specific for divalent cations stimulated the binding of ³H-dihydromorphine suggesting that some endogenous divalent cations may be inhibitory to the binding of the opiate narcotic. The receptors of ³H-dihydromorphine probably exist in the membranes of nerve endings and have a density of 6 × 10¹² sites per g in corpus striatum. We conclude that the described technique can successfully detect the opiate narcotic receptors in the central nervous system without the usual method of displacement.     

Distribution of kappa opioid receptors in the brain of young and old male rats

The experiments to be described have been designed in order to: (a) provide new information on the concentrations of opioid kappa receptors in different regions of the brain of the male rats; and (b) to analyze whether the density of brain kappa receptors might be modified by the process of aging. The concentration of kappa receptors was investigated in the hypothalamus, amygdala, mesencephalon, corpus striatum, hippocampus, thalamus, frontal poles, anterior and posterior cortex collected from male rats of 2 and 19 months of age. 3H-bremazocine (BRZ) was used as the ligand of kappa receptors, after protection of mu and delta receptors respectively with dihydromorphine and d-ala-d-leu-enkephalin. The results obtained show that: (1) in young male rats, the number of kappa opioid receptors is different in the various brain areas examined: the hypothalamus and the striatum have a concentration of kappa binding sites which is significantly higher than that found in the mesencephalon and in the amygdala; much lower concentrations of kappa binding sites have been found in the thalamus, the frontal poles, the hippocampus, the anterior and posterior cerebral cortex. (2) Aging exerts little influence on the number of kappa receptors in the majority of the brain structures considered. However in the amygdala and in the thalamus the number of kappa receptors was increased in old animals. To the authors' knowledge, the data here presented are the first ones which suggest that age may increase rather than decrease the number of neurotransmitter receptors in the brain.     

Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats

The subcutaneous implantation of an estradiol pellet (10 mg) into female rats induced a hypophyseal hyperplasia with hyperprolactinaemia. Examination of neurotransmitter receptors in the hippocampus, striatum and cerebral cortex one month after the implantation revealed that estrogenization was associated with: an increased density of ³H-domperidone binding sites (D₂ receptors) in the striatum and reduced numbers of ³H-serotonin high affinity sites (5-HT₁ receptors) in the hippocampus and of ³H-muscimol binding sites (GABA receptors) in the hippocampus, striatum and cerebral cortex. In contrast, the characteristics of ³H-spiperone binding to 5-HT₂ receptors (in the cerebral cortex) and those of ³H-flunitrazepam binding to benzodiazepine sites (in the three brain regions examined) were not significantly different in estrogenized and in control female rats. However, the enhancing effect of GABA on ³H-flunitrazepam binding was markedly reduced in brain membranes from estrogenized animals. The respective roles of estradiol and prolactin in mediating these changes in neurotransmitter receptors are discussed notably with regard to the regional heterogeneity of estradiol binding capacity in the rat brain.     

Aging-Associated Changes in the Pharmacological Properties of the Benzodiazepine (ω) Receptor Isotypes in the Rat Hippocampus

Abstract: The aging-associated changes in hippocampal benzodiazepine (ω) receptor isotypes have been investigated in rats of the Wistar and Fischer 344 strains. Displacement experiments of [³H]flunitrazepam binding by zolpidem demonstrated that in hippocampal membranes from adult (3-month-old) Wistar strain rats, high (type I; ω₁)-, intermediate (type II_M; ω₂)-, and low (type II_L; ω₅)- affinity sites for this imidazopyridine account for 27.1 ± 7.5, 44.2 ± 7.5, and 28.8 ± 5.1%, respectively. In hippocampal membranes from aged (24-month-old) rats of the same strain, the relative abundance of these sites was 42.8 ± 9.3, 26.3 ± 4, and 36.0 ± 5.9%, respectively. In contrast, no significant difference was observed in the whole benzodiazepine (ω) binding site density between adult and aged rats. The increase in type I (ω₁) binding site density in the hippocampus of aged rats was also demonstrated in saturation experiments with [³H]zolpidem. This aging-induced increase in [³H]zolpidem binding was also observed in hippocampal membranes from Fischer 344 rats. Moreover, in both rat strains, GABA induced a greater enhancement of [³H]zolpidem (5 nM) binding to type I (ω₁) sites (GABA shift) in aged than in adult hippocampal membranes. Quantitative autoradiographic analysis of [³H]zolpidem binding to coronal brain sections from adult and aged Fischer 344 rats demonstrated that the aging-associated increases in the density of type I (ω₁) binding sites were restricted to the hippocampus. Moreover, increases in binding density were larger in the dentate gyrus and in the CA2 field than in the CA1 and CA3 fields.     

Carbachol-induced accumulation of inositol phosphates and its modulation by excitatory amino acids in cortical slices of young and aged rats with down-regulation of muscarinic M-1 receptors

The effects of a subacute intoxication with diisopropyl fluorophosphate (DPF) on total muscarinic acetylcholine receptor sites (mAChRs) and M-1 AChRs were evaluated in the cerebral cortex of young (2–4 months) and aged (22–24 months) Fischer 344 rats. Since M-1 AChRs are coupled to the metabolism of phosphoinositides, carbachol-induced accumulation of inositol phosphates (IP) and its inhibition by glutamate and NMDA was also measured in the cortical slices. DFP treatment caused about 75% inhibition of cholinesterase and 35% down-regulation of mAChRs (measured as [³H]quinuclidinyl benzylate binding) in both young and aged rats. The down-regulation of M-1-ACHRs (measured as [³H]pirenzepine binding) was more pronounced in aged (30%) than in young (17%) DFP-treated rats. There was a significant increase in carbachol-induced IP accumulation in aged, with respect to young, untreated rats. DFP treatment caused a considerable decrease in such IP accumulation in aged but not in young rats. Glutamate and NMDA antagonized carbachol-induced IP accumulation in untreated young and aged rats (and the effects of NMDA were reversed by carboxy-piperazinyl-propyl phosphonic acid). In DFP-treated rats such antagonism was somewhat less pronounced. The data appear of interest in relation to the use of anticholinesterase compounds in the therapy of senile dementia of Alzheimer's type. They suggest that beside their primary action (increasing brain ACh levels) such compounds also act on post-receptor mechanisms and on the interactions between cholinergic and glutamatergic neurotransmitter systems.     

Quantitative autoradiography of the development of mu opiate binding sites in rat brain

The regional developmental appearance of mu binding sites in rat brain was examined by quantitative autoradiography of 3H-dihydromorphine binding in rats 2, 14, 21, and 28 days old. Labeling with 3H-dihydromorphine was heterogeneous in adult rat brains, as previously reported by other laboratories. Levels of 3H-dihydromorphine binding ranged from approximately 250 nCi/g tissue in the interpeduncular nucleus and 100 nCi/g tissue in the habenula to 40 nCi/g tissue in the hypothalamus and periaqueductal gray. Some areas, particularly white matter regions, had no detectable specific binding. The density of 3H-dihydromorphine binding increased in all regions between 2 and 28 days of age. The increases in 3H-dihydromorphine binding in various regions of rat brain developed at different rates. Maximal densities were seen by 14 days of age in most regions examined, including the caudate, hippocampus, amygdala, and hypothalamus. Binding in the medial thalamus and quadrigeminal plate, however, did not reach maximal levels until 21 days. Although quantitative autoradiography offers major advantages in the examination of the regional distribution of opiate binding sites, variability both between sections from the same brain and between sections from different brains demonstrate some of the difficulties associated with this type of experimental approach.     

Dietary lithium during development: Changes in amino acid levels,ionic content,and [3H]spiperone binding in the brain of rats

We found that chronic lithium diet affects the sensitivity of neuroleptic receptors and the content of amino acids in the brain, and that the changes in adult animals differ from those in young rats. Pregnant rats were kept on lithium diet (pellets with 0.21% Li2CO3 and 0.21% NaCl) during the gestation period and the offspring were kept on lithium for six weeks after delivery. Control rats were kept on normal diet under identical conditions. In corpus striatum and cerebral cortex of lithium-treated young rats a reduction in apparent dissociation constant and no change in (3H)spiperone total binding sites were found, suggesting a sensitization of the neuroleptic receptor; this result was unlike that obtained with adult lithium-treated rats, where the total number of binding sites was decreased. The lithium content of brain was very high (2.32 meq/kg of wet weight), whereas in the serum only 0.75 meq/l was recorded. K+ and Na+ levels increased by 20% and 9% respectively in the brain and remained at normal levels in the serum. Analysis of free amino acids in the cerebral cortex, midbrain, and cerebellum showed increases in GABA and glycine levels in all three regions, a significant increase in taurine in midbrain, and an increase in lysine in cerebral cortex and cerebellum. The results indicate that the effect of chronic dietary lithium given during pregnancy on the neuroleptic receptor in young rats is different from that in adult animals. It produces an increase in the number of the neuroleptic receptor sites instead of the decline in the number of binding sites found in adult rats. It remains to be established whether this effect is related more to the age of the animal tested or to the stage of development of the CNS at which the lithium was administered.     

Alterations in atropine sites of the brain of rats as a function of age

Binding of (³H)-atropine to synaptosomal fractions prepared from cerebral and cerebellar cortices of young, adult and old male and female rats were studied. Picomoles of labelled atropine bound/mg protein was highest in the cerebral cortex of young rats and decreased with increasing age in both sexes, whereas in cerebellar cortex the peak binding was in adult rats. Acetylcholinesterase activity of the same fractions showed corresponding changes with age.     

A comparative study of the effect of methionine enkephalin upon the stereospecific binding of an opiate agonist and an antagonist in mice and rats.

The synthetic enkephalins especially methionine enkephalin are more potent in inhibiting the stereospecific binding of ³H-dihydromorphine than that of ³H-naloxone in mouse brain homogenates. Methionine enkephalin is a more potent inhibitor of ³H-dihydromorphine binding in whole mouse brain homogenates than in washed mouse brain membranes. No difference was observed with regard to the inhibitory effect of methionine enkephalin on the binding of ³H-dihydromorphine in whole rat brain homogenates or washed rat brain membranes. The use of different radiolabelled drugs (agonist versus antagonist), different species (mouse versus rat) and/or the variation in the preparation (brain homogenates versus washed membranes) may account for the difference between the IC₅₀ of methionine enkephalin versus ³H-dihydromorphine and versus ³H-naloxone stereospecific binding. The increased inhibitory effect of methionine enkephalin when the supernatant was added to the washed brain membranes supports the hypothesis that methionine enkephalin may be one part of the real endogenous morphine ligand.     

Seasonal variation in the apparent number of binding sites for 3H-opioid agonists and antagonists

Throughout the year, a radioreceptor assay was used to investigate the number of binding sites for several opioid ligands in C57BL/6J mouse brain homogenates. The mean number of sites observed for ³H-morphine and ³H-dihydromorphine was about one third less than the mean for ³H-D-ala²-met⁵-enkephalinamide, ³H-naloxone or ³H-naltrexone. Some seasonal variation was observed in all the opioids studied; the seasonal fluctuation observed in the apparent number of naloxone binding sites was the greatest. These seasonal alterations may play a role in the annual pattern of several physiological processes in which endorphin systems are directly or indirectly involved.     

High affinity renal [3H]flunitrazepam binding: Characterization,localization, and alteration in hypertension

The binding of [³H]flunitrazepam was studied in membranes prepared from the kidney and cerebral cortex of unilaterally nephrectomized rats made hypertensive by simultaneous deoxycorticosterone acetate (DOCA) and NaCl administration. A significant 35–43% increase in the number of [³H]flunitrazepam binding sites (Bmax) was found in the renal membranes prepared from the hypertensive rats; there was no change in the density of binding sites in the membranes obtained from the cerebral cortex. The K_d of [³H]flunitrazepam binding did not change either in the renal or in the cerebral membranes (～ 12 nM in the kidney and ～2.0 nM in the brain). Drug specificity studies with renal membranes showed that the inhibition of [³H]flunitrazepam binding by various benzodiazepines did not jibe with their pharmacologic potency as anxiolytic agents. An intrarenal distribution of specific [³H]flunitrazepam binding was found in the bovine kidney; specific binding was greatest in the outer cortex and virtually absent in the medulla, the minor calyx and the renal artery. The evidence that the renal benzodiazepine binding site is of high affinity, is specific, has a unique distribution, and is regulated during hypertension suggests that it may be associated with an important pathophysiologic structure.     

Buprenorphine: Characteristics of binding sites in the rat central nervous system

The binding of the mixed opiate agonist-antagonist ³H-buprenorphine to rat CNS membranes was stereospecific, saturable and had high affinity. Maximal specific binding of ³H-buprenorphine at 25°C was reached by 30 minutes and dissociation from the receptor was slow. ³H-Buprenorphine labelled a single class of high affinity binding sites (K_D = 0.86nM, B_max = 30.2pmole/g tissue). The B_max for ³H-buprenorphine was about two times that for the μ-opiate receptor drugs ³H-naloxone and ³H-dihydromorphine, and three times the B_max for the σ-opiate receptor ligand ³H-D-Ala², L-Met⁵-enkephalinamide. The regional distribution of ³H-buprenorphine binding was qualitatively similar to the distribution of ³H-naloxone and ³H-dihydromorphine binding. Changing the incubation temperature from 25°C to 37°C increased ³H-buprenorphine binding in all regions of the CNS yet decreased ³H-naloxone and ³H-dihydromorphine binding in most regions. These effects of increasing temperature were a result of changes in ³H-opiate affinity for the receptor with no significant changes in receptor number. Sodium chloride (154mM) enhanced both ³H-buprenorphine and ³H-naloxone binding, and decreased ³H-dihydromorphine binding. The potency of opiate alkaloids and peptides in displacing ³H-buprenorphine was relatively weak with IC₅₀ values ranging between 40nM and 600nM. Furthermore displacement curves were shallow, yielding curvilinear Scatchard plots. Buprenorphine was very potent in displacing ³H-naloxone (IC₅₀ = 0.52nM), ³H-dihydromorphine (IC₅₀ = 1.17nM) and ³H-D-Ala², L-Met⁵-enkephalinamide (IC₅₀ = 0.47nM). These findings suggest that buprenorphine binds to both μ- and δ-opiate receptors.     

NMDA receptor function is enhanced in the hippocampus of aged rats

The density and functional activity of theN-methyl-D-aspartate (NMDA)-sensitive glutamate receptor was examined in various brain areas of 3-, 18- and 24-month-old rats. The total numbers of binding sites for the NMDA receptor antagonists [³H]CGP 39653 and [³H]MK 801 binding sites were decreased in the hippocampus, cerebral cortex and striatum of 18- and 24-month-old rats, relative to 3-month-old animals. In the hippocampus of 18-month-old rats, the reduced number of NMDA receptors was associated with an increased sensitivity of [³H]MK 801 binding to the stimulatory action of glycine and glutamate. Thus, 10 M glycine and 10 M glutamate increased [³H]MK 801 binding in the hippocampus of 18-month-old rats by 75 and 160%, respectively; in 3-month-old animals, the same concentration of these amino acids increased binding by 37 and 95%, respectively. The sensitivity of [³H]MK 801 binding to glycine and glutamate was not increased in the cerebral cortex and striatum of aged rats. Moreover, an increased efficacy of glycine and glutamate in stimulating the binding of [³H]MK 801 in the hippocampus was no longer apparent in the 24-month-old rats. The increased sensitivity of [³H]MK 801 binding to glycine and glutamate in the hippocampus of 18-month-old rats may reflect an increase in NMDA receptor activity to compensate for the decrease in receptor number.     

Lateral differences in GABA binding sites in rat brain

An asymmetric distribution of GABA binding sites was found in the cerebral cortex, hippocampus, cerebellar hemispheres, striatum, and thalamus. Higher levels of [³H]GABA binding were observed in the left-side of most brain areas and in a greater percentage of adult rats, but the opposite asymmetry was found in the thalamus. A similar left-right difference in cerebral hemispheres was also found in five day-old rats, suggesting the genetic predetermination of asymmetry.     

Effects of the intracerebroventricular injection of antinociceptive doses of acetylcholine on the stereospecific binding of 3H-dihydromorphine

The antinociceptive effects of intraventricularly administered acetylcholine (ACh) and its congeners have been demonstrated by previous investigators. The opiate receptor binding concept was used in this study to investigate possible correlations between ACh antinociception and its effects on opiate stereospecific binding. ACh $\text{in vitro}$ decreased the stereospecific binding of ³H-dihydromorphine in mouse brain homogenates. Such decrease was also observed in the brain homogenates of mice which had been treated with ACh intracerebroventricularly (i.v.t.). The decrease in the stereospecific binding of ³H-dihydromorphine induced by (i.v.t.) acetylcholine was inhibited by naloxone, atropine, cyclazocine and pentazocine. The $\text{d}$-isomers of cyclazocine and pentazocine were more potent than the $\text{l}$-isomers in antagonizing the inhibitory effects of i.v.t. acetylcholine upon the stereospecific binding of ³H-dihydromorphine to mouse brain homogenates. The same stereospecificity of these two narcotic analgesics in blocking acetylcholine had been previously observed in the tail-flick test. It is suggested that the antinociceptive effects of acetylcholine are related to the inhibition of opiate stereospecific binding, the mechanism of which is yet to be understood.