Age-related changes in the turnover rates of D1-dopamine receptors in the retina and in distinct areas of the rat brain

The steady-state density and the turnover rates of D₁-dopamine receptors were investigated in the striatum, nucleus accumbens, substantia nigra, and retina of adult (3-month-old) and aged (23-month-old) rats. The turnover rates were measured by monitoring the repopulation kinetics of D₁-dopamine receptors labeled with [³H]-SCH 23390 after the irreversible inactivation induced by a single dose of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 10 mg/kg, s.c.). In all the neural tissues examined, the repopulation of D₁ dopamine receptors could be adequately described by a theoretical model that assumes a constant rate of receptor production (i.e. zero order) and a rate of degradation that is dependent on the receptor density at any time (i.e. first order). The results obtained indicate that the reduction in the density of D₁-dopamine receptors in the striatum, nucleus accumbens and substantia nigra of aged rats is the result of a larger decrease in the receptor production rate (−44 to −60%) than in the receptor degradation rate (−21 to −46%). By contrast, the production rate of D₁-dopamine receptors in the retina of aged rats remains unchanged, whilst the degradation rate is reduced by 25%. This results in an age-related increase in the density of D₁-dopamine receptors in the rat retina.     

Developmental and age-related changes in D1-dopamine receptors and dopamine content in the rat striatum

The relationship between the postnatal development of dopaminergic (DAergic) nerve endings and the maturation of D1 DA receptors in the rat striatum was analyzed by measuring the content of DA and dihydroxyphenylacetic acid (DOPAC), two biochemical markers of DAergic nerve terminal proliferation, and the ontogenetic changes in [3H]SCH 23390 binding sites. DA-stimulated adenylate cyclase (AC) activity was also measured in order to characterize the coupling of [3H]SCH 23390 binding sites to the responses mediated by the activation of D1 DA receptors. Striatal levels of DA and DOPAC, as well as the density and affinity of [3H]SCH 23390 binding sites and DA-stimulated AC activity were also measured in senescent rats. The striatal content of DA increased slowly after birth, reaching adult levels by postnatal day 60 and remaining constant through adulthood and senescence (up to 20 months of age). The density of [3H]SCH 23390 binding sites increased 14-fold from birth to postnatal day 35, when a peak value was reached, whereas a significant decrease was observed in the striatum of aged rats. In contrast, the affinity of D1 DA receptors for [3H]SCH 23390 remained unchanged from birth through senescence. The stimulation of cyclic AMP formation induced by 100 microM DA increased 4-fold from birth to postnatal day 14, when the maximal responsiveness to DA was observed and then returned to adult levels. No significant alterations were observed in the Km values during development, whereas the stimulatory effect of 100 microM DA on AC activity was significantly decreased in senescent rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes of vasopressin content of microdissected areas of the rat brain

Vasopressin contents of posterior pituitaries and intra- and extrahypothalamic nuclei of brains from 3-month and 24-month-old Long-Evans rats and from Brattleboro rats heterozygous (HEDI) and homozygous (HODI) for diabetes insipidus were measured by radioimmunoassay.The pituitary content of vasopressin was highest in young rats and was significantly lower in HEDI tissue. The peptide was not detected in HODI pituitaries.In the brain regions studied both aged and HEDI rats showed reduced vasopressin contents as compared to young animals. In light of evidence of memory deficiencies in both HEDI and HODI rats, the possibility arises that memory decrements associated with senescence may be related to altered vasopressin synthesis and/or secretion occurring with aging of these neuronal systems.     

The role of D1-dopamine receptors in working memory-guided movements mediated by frontal cortical areas

Like the striatum, the frontal motor cortices receive dopaminergic fibers from midbrain dopamine cells and contain high levels of dopamine receptors. Among frontal cortical areas, the dorsolateral PFC (PFd1) and the dorsal premotor cortex (PMd) have strong neural connections and play a major role for working memory-guided directional movements. To reveal the role of dopamine in this cognitive motor function, dopamine antagonists (SCH23390 for D1 receptors and sulpiride for D2 receptors) were applied locally or iontophoretically to the PFd1 and PMd in monkeys that performed delayed-response tasks with memory-guided directional movements. Applications of SCH23390, but not sulpiride, to these areas had significant effects at both the behavioral and neuronal levels. In the PFd1 and at the behavioral level, local injections of SCH23390 induced specific errors for memory-guided saccades, whereas it had no effects on visually guided saccades. In the PMd, local injections of SCH23390 induced directional errors and increased reaction time and movement time in memory-guided reaching movements. At the neuron level, iontophoretic applications of SCH23390 attenuated directional tuning of neurons of the PFd1 and PMd, which showed directional activities during the delay-and/or response-period(s). These findings suggest that the activation of D1-dopamine receptors in these frontal cortical areas plays a facilitating role in a series of neuronal processes of working memory-guided directional movements; the working memory process for guiding motor act in the PFd1 and preparation/control of directional manual movements in the PMd. In addition, our findings may provide insight into symptoms of schizophrenia and Parkinson's disease; the dysfunction of D1-dopamine receptors in the PMd1 and PMd may contribute to some symptoms, such as bradyphrenia and bradykinesia, in these disorders.     

Age-related changes in kynurenic acid production in rat brain.

Two separate in vitro assays were used to examine the biosynthesis of the broad spectrum excitatory amino acid receptor antagonist kynurenic acid (KYNA) during the life span of the adult rat. Assessment of KYNA's anabolic enzyme kynurenine aminotransferase revealed steady increases between 3 and 24 months of age in all five brain regions examined. No changes were observed in the liver. The changes were particularly pronounced in the cortex and in the striatum where enzyme activity increased three-fold during the period studied. KYNA production from its bioprecursor L-kynurenine was also investigated in tissue slices and was found to be significantly enhanced in the cortex and hippocampus of old animals. The effect of depolarizing agents or sodium replacement was virtually identical in tissues from young and old rats. These data, which are in excellent agreement with reports on an age-dependent increase of KYNA concentration in brain tissue, suggest an enhanced KYNA tone in the aged brain. Together with the reported decline in cerebral excitatory amino acid receptor densities with age, increased production of KYNA may play a role in cognitive and memory dysfunction in old animals.     

Age-related alterations of somatostatin gene expression in different rat brain areas.

Numerous experimental and clinical studies have demonstrated that brain somatostatinergic neurotransmission plays an important role in the modulation of several brain functions, including learning and memory processes. Due to the gradual decline of cognitive performances occurring during aging, we evaluated whether an age-related modification of brain somatostatin gene activity occurred in discrete rat brain areas. Our study demonstrates that a significant reduction of pre-prosomatostatin mRNA levels occurred in aged animals (25 months) in the frontal cortex (-49%), in the parietal cortex (-80%) and in the striatum (-69%), despite the absence of changes in beta-actin gene expression. Conversely, no statistical differences were observed in the pre-prosomatostatin mRNA content of old animals in the hypothalamus. These results demonstrate that age-related alterations in somatostatin gene expression occur in the rat, and suggest that such alterations may be involved in the behavioral and cognitive impairments that occur during the aging process.     

Age-related changes of strychnine-insensitive glycine receptors in rat brain as studied by in vitro autoradiography

Age-related changes of strychnine-insensitive glycine receptors in the rat brain were studied through quantitative in vitro autoradiography with 3H-glycine. 3H-glycine binding sites were most concentrated in the hippocampus, cerebral cortex, and olfactory tubercle, and moderate densities of binding sites were located in the striatum, nucleus accumbens, amygdala, and certain thalamic nuclei. Low densities of 3H-glycine binding sites were observed in the lateral septal nucleus, midbrain nuclei such as the superior colliculus and central gray matter, and granule cell layer of the cerebellum. In aged animals, severe decline of 3H-glycine binding sites was observed in the telencephalic regions including the hippocampus and cerebral cortex. On the other hand, decrease of binding sites in the midbrain nuclei was of lesser degree, and there were no changes in the cerebellum. These results suggest that the decrease of glycine receptors in particular brain regions has some relation with changes of neuronal functions associated with aging process in these areas. The glutamatergic neuronal system, particularly the N-methyl-D-aspartate (NMDA) subtype, has been considered to play an important role in learning and memory. Taking into consideration that strychnine-insensitive glycine receptors are contained in the NMDA receptor complex, the present study implies that the decrease of glycine receptors may be involved in impairments of learning and memory occurring in aged brains.     

Binding characteristics and daily rhythms of melatonin receptors are distinct in the retina and the brain areas of the European sea bass retina (Dicentrarchus labrax)

Melatonin is synthesized, with a circadian rhythm, in the pineal organ of vertebrates, high levels being produced during the scotophase and low levels during the photophase. The retina also produces melatonin, although in the case of the European sea bass, its secretion pattern appears to be inverted. In the study described here, radioreceptor assay techniques were used to characterize the melatonin binding sites, their regional distribution and their daily variations. Brain and retina membrane preparations were used in all the binding assays and 2-[125I]iodomelatonin ([125I]Mel) as radioligand at 25 degrees C. The specific binding of [125I]Mel was seen to be saturable, reversible, specific and of high affinity. In all the tissues assayed, the power of the ligands to inhibit [125I]Mel binding decreased in the following order: melatonin>4-P-PDOT>luzindole> or =N-acetylserotonin, which points to the presence of Mel1-like receptors. The inhibition curves of 4-P-PDOT suggested the presence of two different binding sites in the brain areas, but only one type of site of low affinity in the neural retina. No daily variations in [125I]Mel binding capacity (Bmax) or affinity (Kd) were detected in the brain areas, while a clear rhythm in Kd melatonin receptor affinity and Bmax binding capacity was observed in the retina. Kd and Bmax retinal rhythms were out of phase with the lowest Kd and the highest Bmax occurring at scotophase. This result suggests that retinal melatonin is a paracrine factor able to control receptor desensitization during photophase when ocular melatonin is higher in this species.     

Differential effect of aging on3H-SCH 23390 binding sites in the retina and in distinct areas of the rat brain

Summary The effects of age on the binding parameters of the selective D-1 dopamine (DA) receptor antagonist³H-SCH 23390 were studied in membrane preparations from rat striatum, substantia nigra, olfactory tubercle, prefrontal cortex and retina. When compared with 3-month-old animals, there was a significant decrease in the density of³H-SCH 23390 binding sites in the striatum (–25%), substantia nigra (–24%), and olfactory tubercle (–23%), but not in the prefrontal cortex of senescent (23-month-old) rats. The affinity of³H-SCH 23390 for D-1 DA receptors did not change with age in any of the brain areas analyzed. In contrast, the density of³H-SCH 23390 binding sites was higher (+53%) in the retina of aged rats that in 3-month-old controls. Confirming previous studies, we observed that light deprivation induced a significant increment in the density of³H-SCH 23390 binding sites in the retina of adult rats (+31%) but not in the retina of aged animals. The ability of light exposure to activate DAergic neurons in the rat retina was not altered by normal aging. In fact, a similar increase in the concentration of DOPAC was observed in the retina of light-adapted adult and senescent rats when compared to their respective dark-adapted controls (+94% and +95%, respectively).The results indicate that aging has a differential effect on D-1 DA receptors in the retina and different areas of the rat brain.Finally, the age-related increment in the density of retinal D-1 DA receptors does not appear to depend on presynaptic mechanisms, since DA metabolism is increased by light to the same extent in young and aged rats.     

Age-related changes in the rat hippocampus.

The human brain is uniquely powerful in its cognitive abilities, yet the hippocampal and neocortical circuits that mediate these complex functions are highly vulnerable during aging. In this study, we analyzed age-related changes in the rat hippocampus by studying newborn (1 month), middle-aged (12 months), and older (24 months) male and female Sprague-Dawley rats. We evaluated neuronal dystrophy, neuron scattering, and granulovacuolar degeneration in the hippocampal area using light microscopy, according to age and gender. We detected significant neuronal dystrophy in the CA1, CA2, and CA3 areas in male rats, and in the CA1, CA3, and CA4 areas in female rats. Degenerative changes, indicated by neuron scattering, were observed in the CA1, CA2, and CA3 areas of male and the CA2 and CA4 areas of female rats. Changes in all areas of the hippocampus were observed with increasing age; these changes included neuronal dystrophy and neuron scattering and did not differ significantly between male and female rats.     

Age-related changes of the nitric oxide system in the rat brain

This work examines the age-related changes of the NO pathway in the central nervous system (CNS), analyzing nitric oxide synthase (NOS) isoform expression, the level of nitrotyrosine-modified proteins, and the NOS activity in the cerebral cortex, decorticated brain (basal ganglia, thalamus, hypothalamus, tegtum and tegmentum) and cerebellum of young, adult and aged rats. Our data demonstrate that the different NOS isoforms are not uniformly expressed across the CNS. In this sense, the nNOS and eNOS isoenzymes are expressed mainly in the cerebellum and decorticated brain, respectively, while the iNOS isoenzyme shows the highest level in cerebellum. Concerning age, in the cerebral cortex nNOS significantly increased its expression only in adult animals; meanwhile, in the cerebellum the eNOS expression decreased whereas iNOS increased in adult and aged rats. No age-related changes in any isoform were found in decorticated brain. NOS activity, determined by nitrate plus nitrite quantification, registered the highest levels in the cerebellum, where the significant increase detected with aging was probably related to iNOS activity. The number of nitrotyrosine-modified immunoreactive bands differed among regions; thus, the highest number was detected in the decorticated brain while the cerebellum showed the least number of bands. Finally, bulk protein nitration increased in cerebral cortex only in adult animal. No changes were found in the decorticated brain, and the decrease detected in the cerebellum of aged animals was not significant. According to these results, the NO pathway is differently modified with age in the three CNS regions analyzed.     

Age-related changes in bindings of second messengers in the rat brain

Age-related alterations in bindings of major second messengers in the brain were studied in 3-week- and 6-, 12-, 18- and 24-month-old Fisher 344 rats using receptor autoradiography. [³H]Phorbol 12,13-dibutyrate (PDBu) and [3H]forskolin were used to label protein kinase C (PKC) and adenylate cyclase, respectively. In immature rats (3-week-old), [³H]PDBu binding showed a significant decrease only in the cerebellum as compared to adult rats (6-month-old), whereas [³H]forskolin binding exhibited a significant reduction in the neocortex, nucleus accumbens, thalamus and substantia nigra. In aged rats, [³H]PDBu binding showed no significant change in all brain areas. In contrast, [³H]forskolin binding showed a conspicuous reduction in various brain areas in 18-month-old rats as compared to adult animals. The age-related reduction was especially observed in the cerebral cortex, hippocampal CA3 pyramidal cell layer, dentate gyrus, thalamus and molecular layer of cerebellum of 24-month-old rats. The results indicate that adenylate cyclase system in the rat brain is more susceptible to aging processes than phosphoinositide cycle system. Furthermore, our data demonstrate that the change in the adenylate cyclase system is more pronounced than that in the phosphoinositide cycle system in immature rat brain. These findings suggest that the adenylate cyclase system is primarily affected in aging processes and this may lead to age-related neurological deficits.     

Blockade of both D1- and D2-dopamine receptors inhibits amphetamine-induced ascorbate release in the neostriatum

In vivo recordings with electrochemically modified microvoltammetric electrodes revealed that several neuroleptic drugs, including haloperidol, clozapine, and thioridazine, blocked the rise in extracellular ascorbate produced by amphetamine in the neostriatum of urethane-anesthetized rats. This effect was also observed in animals that received a combined injection of Sch-23390 and sulpiride, but not when either of these drugs were administered alone or in combination with the 5-HT₂ blocker, ritanserin. These results indicate that a combined blockade of D₁- and D₂-dopamine receptors blocks amphetamine-induced ascorbate release.     

Dopaminergic neurotransmission in somatodendritic and terminal areas of the rat brain: susceptibility to modulation by D 1 and D2 receptors and to axotomy

Summary We have investigated the influence of D1 and D2 dopamine receptor active drugs on dopamine (DA) release in substantia nigra (SN), striatum and limbic forebrain in intact and in hemisected rats in vivo. DA release was indirectly assessed as 3-methoxytyramine (3-MT) accumulation following monoamine oxidase inhibition by pargyline. Hemisection per se had no effect on the 3-MT accumulation in the SN. Neither, had SCH 23390, SK & F28393, or cisflupentixol any effect in the SN in intact animals or in the lesioned side in hemisected animals. SCH 23390 slightly increased the 3-MT accumulation both in the striatum and limbic forebrain, indicating a stimulatory action on DA release, but SK & F38393 had no effect in these brain regions. A difference between the striatum and the limbic forebrain was that the effects of SCH 23390, and cis-FPX were almost abolished following hemisection in the limbic fore-brain, but only partially reduced in the striatum. In summary, our data give further support for the concept that neither D 1 nor D 2 dopamine receptors have any pronounced influence on the DA release in the SN. The data also indicate operational differences in the feedback regulation of limbic versus striatal dopaminergic transmission.     

Binding of (+)PHNO and other D2-dopamine agonists to D1-dopamine receptors labelled by [3H]SCH 23390

Summary The affinity of (+)PHNO for the D₁-dopamine receptor subtype in striatal tissue was determined in a radioligand binding assay using the D₁-selective compound, [³H]SCH 23390. The IC₅₀ for (+)PHNO was 22M, a value considerably higher than obtained for reference agents (lisuride, apomorphine, pergolide, [–]EOE and bromocriptine). The low potency of (+)PHNO at the D₁-receptor subtype contrasts with the compound's high potency at the D₂-receptor subtype, as reported previously. (+)PHNO can, therefore, be classified as a very selective D₂-receptor agonist.     

Regional differences in reappearance of D2-dopamine receptors in the rat caudate-putamen complex after irreversible inactivation

The reappearance of D2-receptors in the striatum of the rat was studied by autoradiography after in vivo labeling with [3H]N-n-propylnorapomorphine ([3H]NPA) at various time intervals after the inactivation of dopamine receptors by intraperitoneal administration of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Within two days after inactivation the labeling had decreased to 18% of controls. Thereafter, the label reappeared and after 8 days or more reached levels of 80% of that of untreated controls. Autography showed that 4 h after EEDQ treatment no preferential labeling of the striatum can be seen. Five days after EEDQ a slight difference in labeling density between the medial and lateral striatum was detected, whereas after 18 days a prominent lateromedial gradient in silver grain density was seen, resembling the gradient seen without EEDQ treatment. This silver grain gradient is not paralleled by the density of medium-sized neuronal cell bodies. This suggests a difference in synthesis rate of receptors either in other cells than the medium-sized neuron or, alternatively, in otherwise indistinguishable medium-sized neurons. Five days after EEDQ treatment, clusters of silver grains in the lateral striatum were seen. These clusters have a diameter of 150-400 microns and are separated from each other at 200-500 microns. Each cluster may represent newly synthesized receptors of a single neuron (e.g. cholinergic or somatostatinergic interneuron).     

Quantitative autoradiographic localization of the D1 and D2 subtypes of dopamine receptors in rat brain

The distribution of D1 and D2 receptors was studied in coronal sections of rat brain, using quantitative autoradiography. D1 receptors were labeled with 1.8 nM 3H-SKF-83566 (a brominated analog of 3H-SCH-23390), while D2 receptors were labeled with 1.0 nM 3H-spiroperidol (3H-SPD). The binding of both ligands to sections from brain and from a homogenate of caudate putamen (CPu mash) reached equilibrium within 80 min at 37 degrees C. CPu mash provided a virtually unlimited number of homogeneous sections that contained a high density of both D1 and D2 receptors. Sections of CPu mash were used in competition studies that confirmed that the specific binding of 3H-SKF-83566 was selective for D1 receptors, and that the binding of 3H-SPD was selective for D2 receptors. Scatchard analysis of equilibrium binding of the 2 ligands in the CPu in horizontal sections of rat brain revealed Kd values of 1.1 +/- 0.07 nM for 3H-SKF-83566 and 0.7 +/- 0.09 nM for 3H-SPD. Studies of the distribution of D1 and D2 receptors were carried out in coronal sections of brains from 5 rats. D1 receptors were found throughout the forebrain and were present in greater density than were D2 receptors in all regions examined except the olfactory nerve layer. In the CPu, nucleus accumbens, and olfactory tubercle, the densities of D1 and D2 receptors were, respectively, approximately 2,500 and 600-800 fmol/mg protein. In the substantia nigra, the density of D1 receptors was approximately 2,500 fmol/mg protein in both the compacta and the reticulata, but the density of D2 receptors was 230 fmol/mg protein in the compacta and 70 fmol/mg protein in the reticulata. The ventral tegmental area contained only 90 fmol/mg protein of D1 receptors, and D2 receptors were undetectable. The entopeduncular nucleus, zona incerta, and region of the ventral internal capsule had densities of D1 receptors of 550-950 fmol/mg protein and D2 receptor densities of less than 100 fmol/mg protein. Densities of D1 and D2 receptors were, respectively, 2,700 and 900 fmol/mg protein in the choroid plexus. Knowledge of the differences in the relative distributions of D1 and D2 receptors in various brain regions may increase our understanding of the functions of brain dopaminergic systems and may aid in the development of new therapeutic approaches for neuropsychiatric disorders.     

Topographic nonoverlapping distribution of D1 and D2 dopamine receptors in the amygdaloid nuclear complex of the rat brain.

The distribution of D1 and D2 dopamine (DA) receptors in the nuclei and subnuclear zones of the rat amygdaloid complex was mapped using quantitative light microscopic autoradiography. [125I]iodosulpiride and [125I]SCH 23982 (in the presence of 50 nM ketanserin) were used to label D2 and D1 DA receptors, respectively. The DA receptor subtypes exhibited a topographic, nonoverlapping distribution which generally conformed to the cytoarchitectonic boundaries of the component nuclei and subnuclear zones of the amygdaloid complex. The highest density of [125I]iodosulpiride binding sites was observed in the main intercalated cell group and the central amygdaloid nucleus where a medial to lateral gradient of binding sites was localized to its subnuclear zones. [125I]SCH 23982 binding sites were localized in the main intercalated cell group and the basolateral amygdaloid nucleus with a uniform low density in the central nucleus. The functional topography of mesoamygdaloid DA neurons may therefore be mediated, in part, at the level of DA receptor subtypes. The pattern of distribution of [125I]iodosulpiride binding sites in subdivisions of the central amygdaloid nucleus and bed nucleus of the stria terminalis suggests that the functions of the "extended amygdala," a major system of the functional organization of the basal forebrain, may be regulated by DA afferents at multiple key sites of D2 receptor action.