Distribution and postnatal ontogeny of adenosine A2A receptors in rat brain: comparison with dopamine receptors

In adult rat brain, adenosine A_2A receptors and dopamine D₂ receptors are known to be located on the same cells where they interact in an antagonistic manner. In the present study we wanted to examine when this situation develops and compared the postnatal ontogeny of the binding of the adenosine A_2A receptor agonist [³H]CGS 21680, the binding of the dopamine D₁ receptor antagonist [³H]SCH 23390 and the dopamine D₂ receptor antagonist [³H]raclopride.

All three radioligands bound to the striatum at birth and this binding increased several-fold during the postnatal period. [³H]SCH 23390 binding developed first (mostly during the first week), followed by [³H]raclopride binding (first to third week) and [³H]CGS 21680 binding (only during second and third week). For all three radioligands the binding tended to decrease between 21 days and adulthood. This occurred earlier and was more pronounced in the globus pallidus than in the other examined structures. The increase in [³H]CGS 21680 binding from newborn to adult was mainly due to four-fold increase in the number of binding sites. The pharmacology of [³H]CGS 21680 binding to caudate–putamen was similar in newborn, one-week-old and adult animals, and was indicative of A_2A receptors. The binding was inhibited by guanylyl imidodiphosphate at all ages, indicating that A_2A receptors are G-protein-coupled already at birth. In contrast to the large increase in [³H]CGS 21680 binding, there was a decrease in the levels of A_2A messenger RNA during the postnatal period in the caudate–putamen. In cerebral cortex [³H]CGS 21680 bound to a different site than the A_2A receptor. From birth to adulthood cortical binding of [³H]CGS 21680 increased four-fold and that of the adenosine A₁ agonist [³H]cyclohexyladenosine 19-fold. During early postnatal development [³H]SCH 23390 binding was higher in deep than in superficial cortical layers, but this difference disappeared in adult animals. There was binding of both [³H]CGS 21680 and [³H]cyclohexyladenosine to the olfactory bulb, suggesting a role of the two adenosine receptors in processing of olfactory information. [³H]CGS 21680 binding was present in the external plexiform layer and glomerular layer, and increased during development, but the density of binding sites was about one tenth of that seen in caudate–putamen. [³H]cyclohexyladenosine showed a very different labelling pattern, resembling that observed with [³H]SCH 23390.

Postnatal changes in adenosine receptors may explain age-dependent differences in stimulatory caffeine effects and endogenous protection against seizures. Since A_2A receptors show a co-distribution with D₂ receptors throughout development, caffeine may partly exert such actions by regulating the activity of D₂ receptor-containing striatopallidal neurons     

Calcitonin gene-related peptide-mediated increase in K(+)-induced [(3)H]-dopamine release from rat caudal striatal slices

The calcitonin-gene receptor peptide (CGRP) receptor is present in high levels in the caudal striatum of the rat. Previous behavioural experiments have highlighted a possible correlation between CGRP-mediated effects and the dopaminergic system. In this study, we examined the effect of CGRP on K⁺-evoked [³H]-dopamine release in a slice preparation of the rat caudal striatum. The unstimulated release of [³H]-dopamine was not affected by CGRP. However, CGRP increased the release of [³H]-dopamine evoked by K⁺ (30 mM) in a concentration-dependent manner. The stimulatory effect of CGRP was blocked by the CGRP1 antagonist hCGRP(8–37) (without effect on its own). The stimulatory effect of 1 μM CGRP was blocked by dizocilpine (MK-801), suggesting that excitatory transmission is involved in mediating the facilitated release. This study suggests that the peptide CGRP, modulates dopamine release in the rat caudal striatum probably indirectly via glutamatergic transmission.     

Localization of the dopamine uptake complex using [3H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine ([3H]BTCP) in rat brain

[³H]N-[1-(2-Benzo(b)thiophenyl)cyclohexyl]piperidine ([³H]BTCP) is a novel phencyclidine derivative with considerable selectivity for dopamine uptake sites. [³H]BTCP was used to label dopamine uptake sites in vitro, in rat brain, and the regions containing these sites were visualized with an autoradiographic technique. The binding was found to be highest in the striatum, where > 90% of binding was specific. Furthermore, 6-hydroxydopamine lesions of the nigrostriatal pathway obliterated striatal [³H]BTCP binding ipsilaterally, whereas ibotenic acid injection into the caudate-putamen failed to significantly reduce [³H]BTCP binding in that structure. These results indicate that [³H]BTCP labels dopamine uptake sites in mammalian brain and that it can be employed for autoradiographic studies of this transport complex.     

Localization of NADPH-diaphorase activity in the salt gland of the saltwater-acclimated Pekin duck

The pharmacological properties and the anatomical localisation of dopamine D₃ receptor were assessed in the rat cerebellar cortex using radioligand binding techniques associated with light microscope autoradiography and 7-[³H]hydroxy-N,N-di-n-propyl-2-aminotetralin (7-[³H]OH-DPAT) as a ligand. 7-[³H]OH-DPAT was specifically bound to sections of rat cerebellar cortex with a dissociation constant (K_d) of 0.5 nM and a maximum density of binding sites (B_max) of 97 ± 4 fmol/mg tissue. The rank order of potency of competitors of 7-[³H]OH-DPAT binding and the observation that guanosine triphosphate did not affect radioligand binding suggest the labelling of a dopamine D₃ receptor. 7-[³H]OH-DPAT binding sites are located mainly in the molecular layer and in lesser amounts in the Purkinje neuron layer, primarily within the cell body of Purkinje neurons. No specific accumulation of silver grains was observed in the granule neuron layer or in the white matter of the cerebellar cortex. The localisation of a putative dopamine D₃ receptor within Purkinje neurons suggests that this site may have functional relevance in the cerebellar cortex.     

Distribution of dopaminergic receptors in the primate cerebral cortex: Quantitative autoradiographic analysis using [H]raclopride, [H]spiperone and [H]SCH23390

A widespread distribution of dopamine D₁ receptors in the neocortex is well recognized. However, the presence of dopamine D₂ receptors in this structure has only recently been established [Martres et al. (1985) Eur. J. Pharmac.118, 211–219; Lidow et al. (1989) Proc. natn. Acad. Sci. U.S.A.86, 6412–6416]. In the present paper, a highly specific antagonist, [³H]raclopride, was used for autoradiographic determination of the distribution of D₂ receptors in 12 cytoarchitectonic areas of the frontal, parietal, and occipital lobes of the rhesus monkey. A low density of D₂-specific [³H]raclopride binding (1.5–4.0 fmol/mg tissue) was detected in all layers of all cortical areas studied. Throughout the entire cortex, the highest density of binding was consistently found in layer V. This is a unique distribution not observed so far for any other neurotransmitter receptor subtype in monkey cerebral cortex, including D₁ receptor. In addition, a comparison was made of the distribution of [³H]raclopride and [³H]spiperone, which has been commonly used in previous attempts to label cortical D₂ receptors. We found marked differences in the distribution of these two radioligands. In the prefrontal cortex, the pattern of [³H]spiperone binding in the presence of ketanserin resembled the combined distribution of 5-HT_ic serotoninergic and ₂-adrenergic sites as well as D₂ receptors. Thus, [³H]raclopride provides a better estimation of the D₂ receptor distribution than does [³H]spiperone. The distribution of D₂-specific binding of [³H]raclopride was also compared with the D₁-specific binding of [³H]SCH23390 in the presence of mianserin to block labeling to 5-HT₂ and 5-HT_IC sites. The density of D₁-specific [³H]SCH23390 binding was 10–20 times higher than that of D₂-speciflc [³H]raclopride binding throughout the cortex. The densities of both [³H]raclopride and [³H]SCH23390 binding sites display a rostral-caudal gradient with the highest concentrations in prefrontal and the lowest concentrations in the occipital cortex. However, the binding sites of these two ligands had different laminar distributions in all areas examined. In contrast to preferential [³H]raclopride binding in layer V, a bilaminar pattern of [³H]SCH23390 labeling was observed in most cytoarchitectonic areas, with the highest concentrations in supragranular layers I, II and IIIa and infragranular layers V and VI. Whereas [³H]raclopride binding was similar in all cytoarchitectonic areas, [³H]SCH23390 exhibited some region-specific variations in the primary visual and motor cortex.

The different regional and laminar distributions of D₁ and D₂ dopaminergic receptors indicates that they may subserve different aspects of dopamine function in the cerebral cortex.     

Autoradiographic localization of [H]-cyclic GMP binding sites in the rat brain

Both the atriopeptides and nitric oxide act in the nervous system by activating guanylyl cyclases to stimulate the production of cyclic GMP. Thus a key to understanding the roles of these messengers is to understand the functions of cyclic GMP in the nervous system. Three potential targets for cyclic GMP have been identified, phosphodiesterases, protein kinases and ion channels. In this study we describe a method using autoradiography to localize specific [³H]-cGMP binding sites in the brain. The specific binding of [³H]-cGMP to rat brain sections was saturable (B_max = 1.5 pmol/mg protein) and of high affinity (K_D = 164 nM). The pharmacological characteristics were consistent with binding to the cGMP-dependent protein kinase. Highest densities of binding were seen in the medial habenula, basal ganglia, locus ceruleus and nucleus of the solitary tract. The CA1 pyramidal cells of the hippocampus, the neocortex, thalamus and cerebellum were also labelled. This method should prove useful in studies of potential targets for cyclic GMP in the brain.     

Oocytes from Xenopus laevis contain an intrinsic σ2-like binding site

In preparation for expression studies for rat brain σ-binding sites, Xenopus oocytes were tested for the presence of [³H]di-o-tolylguanidine (DTG)-binding sites. Native oocytes were found to contain two intrinsic [³H]DTG-binding sites, a high-affinity site (K_d = 32 ± 6 nM, B_max of 45.7 ± 19 pmol/mg protein) and a low-affinity binding site (K_d = 1.3 ± 0.7 μM, B_max of 3.2 ± 0.7 nmol/mg protein). In a series of radioligand-binding-displacement studies, the high-affinity binding sites were found to have a binding profile which has a similar K_d to that of the mammalian σ₂-binding site (32 vs. 38 nM). Comparison of the IC₅₀ values for inhibition of [³H]DTG binding in rat liver and oocytes for DTG, haloperidol (HAL), (−)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ((+)-3-PPP), (+(-pentazocine and Zn²⁺, showed similarity in rank (r² = 0.913) but a 7-fold lower potency in oocytes. These results suggest that the high-affinity [³H]DTG-binding site in oocytes represents a σ₂-like binding site.     

35S]tert.-butylbicyclophosphorothionate and avermectin bind to different sites associated with the gamma- aminobutyric acid-benzodiazepine receptor complex

Low concentrations of avermectin B₁a (AVM) stimulated the specific high affinity binding of [³⁵S]tert.-butylbicyclophosphorothionate ([³⁵S]TBPT) to membranes from rat cerebral cortex in the absence or presence of chloride or bromide ions. In contrast, TBPT either weakly stimulates or does not significantly influence the specific high affinity binding of [³H]AVM to the same membranes in the absence or presence of chloride ions, respectively. These results indicate that [³H]AVM and [³⁵S]TBPT bind to different but closely associated binding sites.     

GABA enhances acetylcholine release from hippocampal nerve endings through a mechanism blocked by a GABA uptake inhibitor

The effect of γ-aminobutyric acid (GABA) on the release of [³H]acetylcholine ([³H]ACh) was investigated using superfused rat hippocampal synaptosomes. GABA enhanced the basal efflux of [³H]ACh. The effect of GABA was bicuculline-insensitive. Muscimol, (±)-baclofen or (−)-baclofen did not increase [³H]ACh release. The effect of GABA was counteracted by SK&F 89976 A (N-(4,4-diphenyl-3-butenyl)-nipecotic acid), a GABA uptake inhibitor. One possible interpretation of the results is that a GABA transport system is present on cholinergic terminals, suggesting that GABA and ACh may coexist in some rat hippocampus nerve endings. Another possibility is that the effect of GABA is mediated by a novel subtype of GABA receptor sensitive to SK&F 89976 A.     

Decreased density of beta-adrenergic and muscarinic cholinergic receptor sites in the vasa nervorum of aged rats

The pharmacological profile and the anatomical localization of beta-adrenergic and muscarinic cholinergic receptors of the vasa nervorum were studied in sections of sciatic nerve using radioreceptor binding and light microscope autoradiography techniques. Sprague—Dawley rats of 4 and 24 months of age were used. [³H]Dihydroalprenolol (DHA) and [³H]quinuclidinyl benzilate (QNB) were used to label beta-adrenergic and muscarinic cholinergic receptors, respectively. The ligands were bound to sections of rat sciatic nerve in a manner consistent with the labelling of beta-adrenergic or muscarinic cholinergic receptors in the 2 age groups investigated. The dissociation constant (K_d) values (about 1.37 nM for [³H]DHA and 0.75 nM for [³H]QNB) did not significantly change between 4- and 24-month-old rats. The maximum concentration of binding sites (B_max) for [³H]DHA was decreased by about 35% in 24 in comparison with 4-month-old rats. The B_max value autoradiogaphy revealed the development of specific silver grains in the medial layer of epineurial and perineurial arteries in sections of sciatic nerve exposed either to [³H]DHA or [³H]QNB. The number of silver grains developed in epineurial and perineurial arteries of rats of 24 months is significantly lower than in animals of 4 months. The above results suggest the occurrence of an age-dependent loss in the density of beta-adrenergic and muscarinic cholinergic receptors of vasa nervorum.     

Imaging of the super high affinity binding sites for [H]Ro15–4513 in rat hippocampus: comparison between in vitro and in vivo binding

The super high affinity binding sites for [³H]Ro15–4513, a partial inverse agonist of central benzodiazepine receptors, were analyzed in rat hippocampus both in vivo and in vitro. An ultra high sensitive method of autoradiography with an imaging plate system was employed for quantitative analysis of [³H]Ro15–4513 binding. In in vitro binding, the super high affinity binding sites in the hippocampus were observed when the [³H]Ro15–4513 concentration was below 0.5 nM. In vivo, the super high affinity binding sites were only found when the injected dose of Ro15–4513 was below 3.6 μg/kg and almost disappeared when the dose was increased to 10 μg/kg. These results both in vivo and in vitro indicate that there is a significant discrepancy between actual free ligand concentration in vivo and in vitro, and that concentrations in intact brain may be much lower than previously thought.     

Inhibition of inositol phospholipid breakdown by D2 dopamine receptors in dissociated bovine anterior pituitary cells

Inositol phospholipids have been labelled with [³H]inositol in a lactotroph-enriched preparation of dissociated bovine anterior pituitary cells. Stimulation of cells with thyrotropin-releasing hormone receptor agonists leads to accumulation of [³H]inositol phosphates, and this effect may be inhibited by dopamine (DA) agonists. The DA agonist effect may be prevented by D₂ DA receptor selective antagonists. Thus the D₂ receptors on these cells are linked to inhibition of inositol phospholipid metabolism, and this provides a functional assay for the receptor.     

Calcium entry activators: Distinct sites of dihydropyridine and aminopyridine action

The dihydropyridine compound BAY K 8644, a putative calcium entry activator, inhibits binding of the dihydropyridine calcium entry blocker, [³H]nitrendipine, to rat skeletal muscle membranes. In contrast, aminopyridine compounds also believed to stimulate calcium flux do not interact with [³H]nitrendipine binding sites in skeletal muscle or brain. These findings suggest that calcium entry activators, like calcium entry blockers, affect voltage-dependent calcium channels by diverse mechanisms.     

Reduced density of dopamine D2-like receptors on peripheral blood lymphocytes in Alzheimer's disease

Clinical and pathological evidence points to an involvement of dopamine in Alzheimer's disease (AD). The present study was designed to assay dopamine D1-like and D2-like receptors on peripheral blood lymphocytes (PBL) in 20 patients with AD and in 25 healthy controls by radioligand binding assay techniques with [³H][R]-(+)-(−)chloro-2,3,4,5 tetrahydro-5-phenyl-1H-3-benzazepin-al-hemimaleate (SCH 23390) and [³H]7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7OH-DPAT) as radioligands. The density of dopamine D1-like receptors and the affinity of [³H]SCH 23390 and [³H]7OH-DPAT binding to PBL were similar in both groups investigated. AD patients revealed a lower density of dopamine D2-like receptors on PBL than controls (P=0.0016). The pharmacological profile of [³H]SCH 23390 and [³H]7OH-DPAT binding to PBL was consistent with the labeling of dopamine D5 and D3 receptor subtypes, respectively. The reduced density of dopamine D2-like receptors on PBL is consistent with the observation of changes in the expression of D2-like receptors in dopaminergic brain areas in AD. Our findings support the hypothesis of an involvement of dopamine in AD, even in those patients with no evidence of Parkinsonism, behavioral abnormalities or psychosis.     

Quantitative autoradiography of [H]prazosin binding sites in rat forebrain

We have used the LKB Ultrofilm method of autoradiography to localize and quantify in rat forebrain the binding sites for [³H]prazosin, a highly-selective antagonist for the ₁ adrenoreceptor subtype. Frozen 32 μm thick brain sections were labeled in vitro with 1 nM [³H]prazosin and applied against LKB Ultrofilm for 60 days to generate autoradiograms. Non-specific binding was defined as the labeling in the presence of 10 μM phentolamine. The highest levels of prazosin binding were found in layer V of the motor portion of the frontoparietal cortex and in all nuclei of the thalamus. Moderate levels of ₁ receptors were observed in the remaining layers of the cerebral cortex and in most regions of the limbic system. Low levels of prazosin binding occurred in the caudate-putamen and the accumbens nucleus. Our results indicate that ₁ adrenoreceptors are distributed heterogenously throughout the rat forebrain.     

Quantitative autoradiography of nicotinic [H]acetylcholine binding sites in rat brain

Quantitative autoradiography was used to localize nicotinic [³H]acetylcholine (ACh) binding sites in rat brain. High concentrations of nicotinic [³H]ACh binding sites were observed in the anterior and medial nuclei of the thalamus, the medial habenula and the superficial layer of the superior colliculus. Moderate levels of binding sites were observed in a variety of brain regions such as the frontoparietal cortex and the hippocampus. Low levels of nicotinic ACh sites occurred throughout the hypothalamus and the primary olfactory cortex.     

Autoradiographic localization of binding sites for vasotocin in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Specific binding sites for vasotocin (VT) were detected in brain and pituitary of a teleost fish, the sea bass, after in vitro incubation of tissue sections with [³H]arginine-vasopressin (AVP) and light microscopic autoradiography. Conditions for the binding assay were optimized and as a result the binding was saturable and specific. In the brain [³H]AVP binding was found to occur in the pars lateralis and the pars ventralis of the ventral telencephalon, in the pars centralis of the dorsal telencephalon, in the hypothalamic region (especially in the nucleus preopticus, in the tuberal hypothalamus and around the posterior recess), in the tectum opticum and in the noncellular layer of the corpus cerebelli. In the pituitary a high density of [³H]AVP binding was observed in the areas of the pars distalis (PD) occupied by ACTH-, TSH- and GH-cells and also in the pars intermedia (PI). The present study presents the first anatomical evidence for the presence of VT specific binding sites in teleost brain and pituitary.     

Light-microscopic distribution and parasagittal organisation of muscarinic receptors in rabbit cerebellar cortex

Recent studies on the effects of intrafloccular injections of muscarinic agonists and antagonists on compensatory eye movements in rabbit, indicate that muscarinic receptors may play a modulatory role in the rabbit cerebellar circuitry. It was previously demonstrated by Neustadt et al. (1988), that muscarinic receptors in rabbit cerebellar cortex are distributed into alternating longitudinal zones of very high and very low receptor density. In the present study, the zonal and cellular distribution of muscarinic receptors in the rabbit cerebellar cortex is investigated in detail using in vitro ligand autoradiography with the non-selective high-affinity antagonist [³H]quinuclidinyl benzilate (QNB), and the M2-specific antagonist [³H]AF-DX384, and immunocytochemistry with a monoclonal antibody specific for the cloned m2 muscarinic receptor protein. [³H]QNB and [³H]AF-DX384 binding sites and m2-immunoreactivity had similar overall distributions: dense labeling occurred in the dendritec arbors of a subset of Purkinje cells that are organized into parasagittal bands. A high level of muscarinic receptor labeling was also observed in a thin substratum of the molecular layer immediately above the Purkinje cell layer of the vestibulo-cerebellar lobules, i.e. the nodulus, the ventral uvula and the flocculus. Labeling in this stratum was associated with densely packed fibres, which were putatively identified as parallel fibres. Also Golgi cells, which were localized in part in the molecular layer, and a subset of mossy fibre rosettes, primarily concentrated in lobule VI, were immunoreactive for the m2 receptor. The parasagittal bands of labeled Purkinje cell dendrites were most prominent in the anterior lobe (lobules I–V), in crus 1 and 2, in the flocculus, the ventral paraflocculus and the rostral folium of the nodulus. In other lobules, only infrequent Purkinje cells contained muscarinic receptors. The parasagittal organisation of muscarinic receptors differed from that of zebrin I, a Purkinje cell-specific protein which is often used as a marker of parasagittal parcelation of the cerebellar cortex. In the anterior lobe, however, there was a partial correspondence between muscarinic receptor and zebrin I bands. In the flocculus the distribution of muscarinic-receptor-positive Purkinje cells was related to the distinct white matter compartments as revealed with acetylcholinesterase (AChE) histochemistry. Muscarinic receptor-containing Purkinje cells were located primarily in the floccular zone 1, which is implicated in the control of eye movements about a horizontal axis. In order to relate the distribution of muscarinic receptor labeling to that of cholinergic nerve terminals, [³H]QNB binding sites and sodium-dependent [³H]hemicholinium-3 binding were compared. Sodium-dependent [³H]hemicholinium-3 binding sites mainly occurred in the granule cell layer of the vestibulo-cerebellum, which corresponds well with the distribution of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT). However, sodium-dependent [³H]hemicholinium binding complemented, rather than co-localized with, muscarinic receptors which were primarily distributed in the molecular layer of the lobules of the vestibulo-cerebellar lobules. Their functional significance is puzzling, since their distribution does not correspond to that of markers of cholinergic innervation.     

Age-correlated loss of dopamine uptake sites labeled with [H]GBR-12935 in human putamen

The effects of age (19–100 years) upon dopamine uptake sites labeled with [³H]GBR-12935 in human postmortem putamen from 20 individuals were studied. There was a 70% decrease in binding density (B_max) over the adult age range. No significant changes in binding affinity (K_d) were detected, the mean K_d being 1.0 ± 0.2 NM (mean ± S.E.M.). Nor were there any changes in binding related to the postmortem delay. Based on the findings that [³H]GBR-12935 labels the uptake site for dopamine, it is suggested that the age-related loss of [³H]GBR-12935 binding in human putamen reflects a degeneration of dopamine neurites.     

Excitatory and inhibitory amino acid neurotransmitter binding sites in the cerebellar cortex of the pigeon (Columba livia).

We used receptor autoradiography to determine the distribution of excitatory and inhibitory amino acid neurotransmitter binding sites in the cerebellar cortex of the pigeon (Columba livia). -Amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate and metabotropic binding sites had highest levels in the molecular layer. N-methyl- -aspartate binding sites, assayed with both [³H]glutamate under selective conditions and with [³H]glycine binding to the associated strychnine-insensitive glycine site, had highest levels in the granule cell layer. There was little specific binding of the non-competitive N-methyl- -aspartate antagonist, [³H]MK-801. The level of gamma-aminobutyric acid (GABA)-A binding sites was higher than GABA-B binding sites in both molecular and granule cell layers with the highest level of GABA-A sites in the granule cell layer. The highest level of GABA-B binding sites was in the molecular layer. [³H]Flunitrazepam binding levels were approximately the same in both molecular and granule cell layers. With the exception of kainate binding sites, the distribution of binding sites was identical to that seen in the cerebellar cortex of mammals. Our results support the concept that the chemoarchitecture of the cerebellar cortex has been conserved in the course of vertebrate evolution.