Distribution of GLP-1 Binding Sites in the Rat Brain: Evidence that Exendin-4 is a Ligand of Brain GLP-1 Binding Sites

The distribution and biochemical properties of glucagon-like peptide (GLP)-1(7–36)amide (GLP-1) binding sites in the rat brain were investigated. By receptor autoradiography of tissue sections, the highest densities of [¹²⁵I]GLP-1 binding sites were identified in the lateral septum, the subfornical organ (SFO), the thalamus, the hypothalamus, the interpenduncular nucleus, the posterodorsal tegmental nucleus, the area postrema (AP), the inferior olive and the nucleus of the solitary tract (NTS). Binding studies with [¹²⁵I][Tyr39]exendin-4, a GLP-1 receptor agonist, showed an identical distribution pattern of binding sites. Binding specificity and affinity was investigated using sections of the brainstem containing the NTS. Binding of [¹²⁵I]GLP-1 to the NTS was inhibited concentration-dependently by unlabelled GLP-1 and [Tyr39]exendin-4 with K ₁ values of 3.5 and 9.4 nM respectively. Cross-linking of hypothalamic membranes with [¹²⁵I]GLP-1 or [¹²⁵I][Tyr39]exendin-4 identified a single ligand-binding protein complex with a molecular mass of 63 000 Da. The fact that no GLP-1 binding sites were detected in the cortex but that they were detected in the phylogenetically oldest parts of the brain emphasizes that GLP-1 may be involved in the regulation of vital functions. In conclusion, the biochemical data support the idea that the central GLP-1 receptor resembles the peripheral GLP-1 receptor. Furthermore, the presence of GLP-1 binding sites in the circumventricular organs suggests that these may be receptors which act as the target for both peripheral blood-borne GLP-1 and GLP-1 in the nervous system.     

Localization of Insulin-Like Growth Factor-II Receptors in Rat Brain by in vitro Autoradiography and Immunohistochemistry

Insulin-like growth factor-ll (IGF-II) and its receptor, which is homologous with the mannose-6-phosphate (M6P) receptor, are found in high levels in adult rat and human brain, though their role remains unclear. In order to point to possible regional functions, we have mapped and quantified IGF-II/M6P receptors in sagittal sections of adult rat brain by in vitro autoradiography/computerized densitometry and immunohistochemistry. While in vitro autoradiography allowed mapping and quantitation, immunohistochemistry both confirmed mapping and allowed more detailed determination of cellular distribution of receptors. The two methods were generally in agreement with few areas of mismatching. By in vitro autoradiography, a discrete and characteristic distribution of IGF-II receptor binding was demonstrated, with specific binding representing 85% of total binding. Displacement and specificity competition curves in arcuate nucleus and choroid plexus were typical for authentic IGF-II receptors with half maximal displacement at 1 nM cold IGF-II. IGF-II receptor density, estimated by in vitro autoradiography, was very high in circumventricular organs, especially the median eminence, which had the highest binding in the brain. In the remainder of the brain there was concordance between the distribution of receptors identified by the two techniques, with greatest densities in the olfactory bulb and olfactory pathways, the hippocampus and discrete regions of the cerebral cortex, cerebellum, hypothalamus, thalamus and brainstem. There were however, some notable mismatches. Autoradiographic binding was high to very high in the median eminence, arcuate nucleus, suprachiasmatic nucleus and anterodorsal thalamic nucleus, whereas these areas were only poorly immunostained. Conversely, the septum showed moderate autoradiographic binding, but very prominent immunostaining of neurons in its dorsolat-eral aspect. Using the immunohistochemical technique IGF-II receptors were localized to specific neuronal groups such as the mitral cells of the olfactory bulb, Purkinje cells of the cerebellum and neurons in the red nucleus. Fibre pathways were not labelled by either technique. We conclude that IGF-II/M6P receptors are widespread throughout rat brain, specifically in neurons and blood vessels, with a similar, but distinct distribution to IGF-I and insulin receptors. Many of these regions have in common high rates of metabolic and synthetic activity, which may be mediated by IGF-II/M6P and their receptors.     

Quantitative Autoradiographic Demonstration of Changes in Binding to NMDA-sensitive [3H]Glutamate and [3H]MK801, but not [3H]AMPA Receptors in Chick Forebrain 30 min After Passive Avoidance Training

Day-old domestic chicks ( Gallus domesticus ) were trained on a one-trial passive avoidance task in which the aversive stimulus was an unpleasant tasting substance, methyl anthranilate. Thirty minutes later, localization of N -methyl- d -aspartic acid (NMDA)-sensitive [³H]glutamate receptor binding sites, [³H]MK801 and [³H]AMPA binding sites in 17 regions of the forebrain of methylanthranilate-trained and control (water-trained) chicks was determined using quantitative receptor autoradiography. Significant differences in binding to both MK801- and NMDA-sensitive glutamate receptors, but not α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, were found in three regions of the forebrain of trained compared to control chicks; two of these regions have been implicated from previous lesion, biochemical and morphological studies as playing a key role in the process of memory formation and storage following passive avoidance training. For NMDA-sensitive [³H]glutamate receptors, significant elevations in binding were observed in two regions, the left intermediate and medial hyperstriatum ventrale (IMHV) (39%) and the lobus parolfactorius (LPO) (34%), at 30 min post-training, but a decrease (44%) occurred in binding to the lateral neostriatum. Significant increases in binding to MK801 receptors were observed in the left IMHV (19%) and right IMHV (13%), and left LPO (22%) at 30 min post-training, though there was a decrease in the right LPO (15%). These findings, coupled with those described in a previous paper from our group (Burchuladze and Rose, Eur. J. Neurosci. , 4 , 533–538, 1992), demonstrate that a glutamate receptor subtype is involved in learning and memory formation in the chick.     

Quantitative autoradiography of binding sites for [H]AMPA, a structural analogue of glutamic acid

Binding sites for the potent glutamate agonist [³H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were localized in rat brain frozen sections by quantitative autoradiography. Highest levels of binding were seen in stratum radiatum and stratum oriens of the CA1 hippocampal subfield and in the dorsal subiculum. Substantially less but still high amounts of [³H]AMPA binding occurred in other hippocampal subfields and in rostral forebrain structures. The heterogeneous nature of [³H]AMPA binding is discussed in relation to [³H]glutamate binding visualized by similar methods. From these data it is suggested that [³H]AMPA may label a particular subclass of the glutamate receptor population which exhibits a high affinity for quisqualic acid.     

A Comparative Study of α2- and β-Adrenoceptor Distribution in Pigeon and Chick Brain

The pharmacological properties and anatomical distribution of α₂, β₁ and β₂-adrenoceptors in pigeon and chick brains were studied by both homogenate binding and tissue section autoradiography. [³H]Bromoxidine (α₂-adrenoceptor-), [³H]CGP 12177 (β-adrenoceptor) and [¹²⁵1]cyanopindolol (β-adrenoceptor) were used as radioligands. In both species, [³H]bromoxidine binding to avian brain tissue showed a pharmacological profile similar to that previously reported for α₂-adrenoceptors in mammals. Regarding the anatomical distribution, the areas with the highest densities of α₂-adrenoceptors in the pigeon brain included the hyperstriatum, nuclei septalis, tectum opticum and some brainstem nuclei. Most β-adrenoceptors found in tissue membranes and sections from chick and pigeon brain were of the β₂ subtype, in contrast to what has been reported in the mammalian brain, where the β₁ subtype is predominant. A striking difference was found between the two species regarding the densities of these receptors: while pigeon brain was extremely rich in [¹²⁵1]cyanopindolol binding throughout the brain (mainly cerebellum) in the pigeon, the levels of labelling in the chick brain were much lower; the exception was the cerebellum, which displayed a higher density than other parts of the brain in both species. Overall, our results support the proposed anatomical equivalences between a number of structures in the avian and mammalian encephalon.     

AMPA,kainate, and NMDA receptor densities in the hippocampus of untreated male rats and females in estrus and diestrus

Steroid hormones systematically affect numerous neuronal targets, thus influencing, in a permanent or a transitory manner, the way the brain reacts to external and internal stimuli. The hippocampus is an important brain region for learning and memory and the glutamatergic intrahippocampal pathway plays a major role in performing such functions. We applied quantitative in vitro receptor autoradiography to examine how the in vivo hormone milieu affects the densities of AMPA, kainate, and NMDA receptors in the hippocampus of adult male rats and females in estrus and diestrus. All three examined receptor types presented significant gender-specific differences in their densities. The hippocampus of male rats contains significantly more AMPA, kainate, and NMDA receptors than that of female rats. Female rats in diestrus have significantly higher AMPA receptor densities than female rats in estrus. AMPA changes occurred to the same extent in CA1-3 and in the dentate gyrus. Significant differences in the densities of NMDA receptors were observed in the CA1-3 regions, whereas kainate receptor differences were restricted to the CA1 region. These results further support that steroid hormones, through their modulation of AMPA and NMDA receptors, may be involved in the control of synaptic efficacy and, therefore, influence learning and memory.     

Effect of Temperature and Calcium on the Binding Properties of the AMPA Receptor in Frozen Rat Brain Sections

The elucidation of the mechanisms regulating the properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptors is important for understanding glutamatergic transmission. Here we report that qualitative as well as quantitative analysis of tritiated ligand binding to the AMPA receptor on thin frozen rat brain tissue sections reveals the existence of several mechanisms regulating the binding properties of AMPA receptors. Preincubation of tissue sections at 35 degrees C results in a decreased amount of [3H]AMPA binding as compared to that measured following preincubation at 0 degrees C. The decrease in binding appears to be mainly localized to cell bodies as evaluated by autoradiography, and could be due to proteolysis. Preincubation with calcium at 35 degrees C produces increased levels of [3H]AMPA binding. The effect of calcium is mimicked by manganese and to a lesser extent by magnesium; it is concentration-dependent with a 50% effective concentration for calcium of approximately 150 microM, time-dependent and temperature-dependent. The calcium-induced increase in [3H]AMPA binding is different among various brain structures, being larger in area CA1 of the hippocampus and in the superficial layers of the cerebral cortex. The effect of calcium is partly reduced by preincubation with the calpain inhibitor leupeptin and potentiated by preincubation with purified calpain II. The calcium-induced increase in [3H]AMPA binding is associated with a decrease in the binding of an antagonist of AMPA receptors, [3H]6-nitro-7-cyanoquinoxaline-2,3-dione. The results indicate that the binding properties of the AMPA receptor are rapidly regulated by calcium-dependent processes, and possibly by calcium-dependent proteases. They suggest that modulation of the binding properties involves changes in the configuration of the receptor, producing opposite changes in the affinities of the receptor for agonists and antagonists. Finally, these results strengthen the hypothesis that changes in the properties of AMPA receptors might underlie various forms of synaptic plasticity.     

Distribution of mu, delta, and kappa opioid receptor binding sites in the brain of the one-day-old domestic chick (Gallus domesticus): an in vitro quantitative autoradiographic study

Three highly specific opioid ligands--[D-Ala2,Gly-ol]-enkephalin (DAGO) for mu (mu) receptor sites, [D-Pen2,D-Pen5]-enkephalin (DPDPE) for delta (delta) sites, and U-69593 for kappa (kappa) sites--were used to determine the regional distribution of the three major subtypes of opioid receptor binding sites in the brains of 1-day-old domestic chicks by the technique of quantitative receptor autoradiography. Whilst there was a degree of heterogeneity in the binding levels of each of the ligands, some notable similarities existed in the binding of the mu and kappa ligands in several forebrain regions, and in the optic tectum of the midbrain where mu and delta binding was very high. In the forebrain there was a high level of binding of mu and kappa ligands in the hyperstriatum, and for the mu ligand there was a very distinct lamination of binding sites in hyperstriatum accessorium, intercalatum supremum, dorsale and ventrale. Levels of binding of the mu and kappa ligands were also high in nucleus basalis, and (for mu only) in the neostriatum. The distribution of binding of the delta specific ligand in the forebrain showed marked differences to that of mu and kappa, being particularly low in the hyperstriatum and neostriatum. Very high levels of labelling of delta binding sites were, however, found in the nucleus rotundus. Binding of the three ligands was generally low or absent in the cerebellum and medulla, apart from a distinct labelling of the granule cell layer by the mu-ligand. A kinetic analysis was made of the binding of the three ligands to whole forebrain sections using scintillation counting methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization and Characterization of Insulin-Like Growth Factor-I Receptors in Rat Brain and Pituitary Gland Using in vitro Autoradiography and Computerized Densitometry* A Distinct Distribution from Insulin Receptors

In order to identify likely sites of action of insulin-like growth factor-I (IGF-I) in rat brain and pituitary gland, we have used the technique of in vitro autoradiography and computerized densitometry to map, characterize and quantify its receptors in coronal and sagittal sections. A discrete and characteristic distribution of IGF-I receptor binding was demonstrated, with specific binding representing 85% of total binding. Displacement and specificity competition curves in the olfactory bulb were typical for authentic IGF-I receptors and computer analysis indicated a single class of binding site with a dissociation constant (K_d) of 13 nM for the choroid plexus and 5.1 nM for the olfactory cortex. IGF-I receptor density was very high in the choroid plexus in ail ventricles, but the binding in other circumventricular organs was variable, with high levels in the median eminence and the sub-fornical organ, and low levels in the organum vasculosum of the lamina terminalis. Highest binding was seen in the glomerular layer of the olfactory bulb and its associated regions the taenia tecta and anteromedial olfactory nucleus. The preoptic and septal regions showed moderate binding, while the hypothalamus, with the exception of the median eminence, showed low IGF-I binding. The pituitary gland showed very high binding density in both anterior and posterior lobes, similar to the median eminence. The thalamus had high IGF-I binding density, while it was low in basal ganglia. In the limbic system the hippocampal CA2, CAS, CA4 layers showed high binding, with little in CA1, while binding was high also in the adjacent amygdala. Binding was low in the mid and hindbrain, with the exception of the geniculate bodies, and the sensory nucleus of the trigeminal nerve. Binding was high in the primary olfactory and endopyriform cortex and in specific superficial layers. Cerebellar binding was also high in the molecular layer. Fibre layers showed no binding. Comparison with insulin receptors revealed common distribution in the choroid plexus, paraventricular nucleus, cerebellum, entorhinal cortex and amygdala, with receptor density three- to five-fold higher for IGF-I than for insulin. In contrast, in the hippocampus, insulin binding was high in the CA1 field, and low in CA2, CA3, CA4 while for IGF-I binding the converse was seen. The arcuate nucleus showed prominent insulin labelling and minimal IGF-I binding, while the median eminence showed low insulin and high IGF-I binding. The hypothalamus was more widely labelled with insulin, while in the thalamus the converse was true. Olfactory bulb laminae were labelled with differing intensity by insulin and IGF-I. In common with insulin receptor distribution was the high density of IGF-I receptors over areas of extensive dendritic arborizations which receive rich synaptic inputs, in the cerebellum, hippocampus and olfactory bulb. We conclude that IGF-I receptors are widespread throughout rat brain and pituitary gland, with concentration in regions concerned with olfaction, autonomie and sensory processing, as well as in regulation of growth hormone release, via feedback at the median eminence and pituitary gland. Many of these regions have in common high rates of metabolic and synthetic activity, which may be mediated by IGF-I and its receptors.     

Localization of ionotropic glutamate receptors in caudate-putamen and nucleus accumbens septi of rat brain: Comparison of NMDA,AMPA, and kainate receptors

Changes in binding of selective radioligands at NMDA ([³H]MK-801), AMPA ([³H]CNQX), and kainate ([³H]kainic acid) glutamate (GLU) ionotropic receptors in rat caudate-putamen (CPu) and nucleus accumbens (NAc) were examined by quantitative autoradiography following: 1) unilateral surgical ablation of frontal cerebral cortex to remove descending corticostriatal GLU projections, 2) unilateral injection of kainic acid (KA) into CPu or NAc to degenerate local intrinsic neurons, or 3) unilateral injections of 6-hydroxydopamine (6-OH-DA) into substantia nigra to degenerate ascending nigrostriatal dopamine (DA) projections. Cortical ablation significantly decreased NMDA receptor binding in ipsilateral medial CPu (20%), and NAc (16%), similar to previously reported losses of DA D₄ receptors. KA lesions produced large losses of NMDA receptor labeling in CPu and NAc (both by 52%), AMPA (41% and 45%, respectively), and kainate receptors (40% and 45%, respectively) that were similar to the loss of D₂ receptors in CPu and NAc after KA injections. Nigral 6-OH-DA lesions yielded smaller but significant losses in NMDA (17%), AMPA (12%), and kainate (11%) receptor binding in CPu. The results indicate that most NMDA, AMPA, and kainate receptors in rat CPu and NAc occur on intrinsic postsynaptic neurons. Also, some NMDA, but not AMPA or kainate, receptors are also found on corticostriatal projections in association with D₄ receptors; these may, respectively, represent excitatory presynaptic NMDA autoreceptors and inhibitory D₄ heteroceptors that regulate GLU release from corticostriatal axons in medial CPu and NAc. Conversely, the loss of all three GLU receptor subtypes after lesioning DA neurons supports their role as excitatory heteroceptors promoting DA release from nigrostriatal neurons. Synapse 30:227–235, 1998. © 1998 Wiley-Liss, Inc.     

Quantitative autoradiography of serotonin receptors in the rat brain

The distribution of serotonin receptors in the rat brain was studied using a new, quantitative autoradiographic technique utilizing tritium-sensitive LKB sheet film, and densitometric analysis of the film images. OD values above 13 were found in the dentate gyrus, subiculum, substantia nigra and ependymal tissue throughout the brain. ODs between 7 and 11 were found in the globus pallidus, anterior hippocampus and CA1 field, dorsal raphe nucleus and interpeduncular nucleus. The lowest densities were observed in the thalamus, cerebellum, and over white matter, between 0.1 and 0.4 OD units. Other brain nuclei have intermediate values. Serotonin₁ receptor distribution is found to correlate with 5-HT levels, corticosteroid and estrogen receptors in some but not all brain regions.     

Regional ontogeny of a unique glutamate recognition site in rat brain: An autoradiographic study

The developmental pattern of L-[3H]glutamate binding to rat brain in the presence of saturating concentrations of unlabeled N-methyl-D-aspartate (NMDA), kainate (KA) and quisqualate (QQA) was examined in an autoradiographic assay. The unique glutamate binding site defined by this assay displayed four distinct, regionally specific patterns of development. (1) In reticular nucleus of thalamus there was an initial very high level of binding at postnatal day 1 (PND1) followed by a progressive 80% decline in binding during maturation. (2) In entorhinal cortex, a progressive 500-1100% increase in binding was seen during development. (3) In ventral posterior medial nucleus of thalamus, there was an initial transient 200-300% increase in binding, peaking at PND10, followed by a steady decline in binding. (4) In frontal cortex, binding remained relatively stable throughout development. At all stages of development, the distribution of these recognition sites was different from NMDA, KA or QQA receptors. The function of this glutamate binding site remains to be determined, but the distinct regional and temporal patterns of binding suggest that it may be important in normal development of the central nervous system.     

Melatonin Receptor Sites in the Syrian Hamster Brain and Pituitary. Localization and Characterization Using [125|]lodomelatonin*

A high-affinity, discretely localized melatonin receptor has been characterized and mapped within the brain and pituitary of the Syrian hamster using the high specific activity ligand [¹²⁵|]iodomelatonin and a combination of in vitro autoradiography and membrane homogenate receptor assays. Specific binding of radioligand was found in regions of the epithalamus and hypothalamus in the brain and the pars tuberalis of the pituitary. Excitatory amino-acid lesions destroyed [¹²⁵|]iodomelatonin binding within the brain, demonstrating that binding sites are located on neurons. Analysis of [¹²⁵|]iodomelatonin binding to membrane homogenates of the pars tuberalis revealed a linear relationship between specific ligand binding and the amount of tissue. The time-course of specific binding at 37°C reached equilibrium after 30 min and remained stable thereafter. The addition of increasing concentrations of [¹²⁵|]iodomelatonin alone and in the presence of 1 μM melatonin showed that specific binding reached equilibrium at 80 to 100 pM. Analysis of the saturation isotherm using a one-site binding model was consistent with a single receptor site with a K_d of 29.3 (±5.9 SEM) pM and B_max of 2.54 (±0.19 SEM) fmol/mg protein.     

Selective effects of aniracetam across receptor types and forms of synaptic facilitation in hippocampus.

Aniracetam reversibly increased synaptic responses mediated by the AMPA but not the NMDA subclass of glutamate receptors in hippocampus and was considerably more potent than structurally similar nootropics. The drug had greater effects on field excitatory postsynaptic potentials (EPSPs) in the dentate gyrus and CA1 region than it did in the CA3 region, suggesting that it differentiates between variants of the AMPA receptor. Ligand binding to glutamate receptors in synaptosomal membrane fractions was minimally changed by aniracetam. Finally, the percent facilitation produced by aniracetam in the CA1 region was not reduced by any of three treatments (4-aminopyridine, changes in extracellular calcium concentrations, paired-pulse stimulation) that affect release but, in accord with a previous report, was substantially decreased by long-term potentiation. These results support the conclusion that aniracetam selectively increases the conductance of a subgroup of synaptic AMPA receptors in hippocampus and suggest that receptor changes underlie the expression of long-term potentiation.     

Glutamate Receptors of a Quisqualate-Kainate Subtype are Involved in the Presynaptic Regulation of Dopamine Release in the Cat Caudate Nucleus in vivo

Experiments were conducted with halothane-anesthetized cats implanted with a push-pull cannula in the caudate nucleus in order to estimate the effects of glutamate (GLU) agonists on the release of 3H-dopamine continuously synthesized from 3H-tyrosine. In the presence of tetrodotoxin (TTX), glutamate (10-8 M, 10-4 M) and kainate (KAI) (10-5 M) stimulated the release of 3H-dopamine while quisqualate (10-5 M) and N-methyl-D-aspartate (NMDA) (10-5 M) were without effect. The stimulatory effect of kainate (10-5 M) on 3H-dopamine release did not seem to be mediated by glutamate released from corticostriatal fibers, as not only kainate, but also quisqualate (QUI) and N-methyl-D-aspartate enhanced the efflux of glutamate through a tetrodotoxin-resistant process. Riluzole (10-5 M), gamma-D-glutamyl-glycine (GDGG) (10-5 M) and glutamine-diethyl-ester (10-5 M) prevented the stimulatory effect of kainate (10-5 M) while 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) (10-5 M), kynurenate (10-5 M) and 2-amino-5-phosphonovalerate (APV) (10-5 M) were without effect. In the presence of concanavalin A (CONA) (10-7 M), a lectin which is known to prevent the quisqualate-evoked desensitization of glutamate receptors, quisqualate (10-5 M) stimulated the release of 3H-dopamine. In addition, in the absence of concanavalin A, quisqualate (10-5 M) blocked the stimulatory effects of kainate (10-5 M) or glutamate (10-4 M) on 3H-dopamine release. These results suggest the involvement of receptors of the quisqualate/kainate subtype in the direct glutamate-induced presynaptic facilitation of dopamine release. In contrast to what was observed in the presence of tetrodotoxin, in the absence of the neurotoxin, high concentrations of glutamate (10-4 M) and kainate (10-5 M) reduced rather than stimulated the release of 3H-dopamine. A weak inhibitory effect was also observed with quisqualate (10-5 M) while N-methyl-D-aspartate (10-5 M) was without effect. In the light of previous studies, these latter observations suggest that glutamate can also exert an indirect inhibitory presynaptic influence on the release of dopamine from nerve terminals of the nigrostriatal dopaminergic neurons by acting on receptors of the quisqualate/kainate subtype located on striatal GABAergic neurons.     

Distribution of Galanin mRNA Containing Cells and Galanin Receptor Binding Sites in Human and Rat Hypothalamus

The distribution of cells containing galanin mRNA and that of galanin receptor binding sites were investigated using in situ hybridization histochemistry and receptor autoradiography in male rat hypothalamus and in postmortem hypothalamic tissues from control human brains. Oligonucleotide probes labelled with 32P were used for hybridization experiments. The specificity of the hybridization signal was ascertained using several probes, competition assays and Northern blot analysis. High levels of hybridization were found in the paraventricular, supraoptic and arcuate nuclei of rat and human hypothalamus. Human intermediate nuclei and scattered cells of the posterior perifornical nucleus also contained galanin mRNA. Galanin mRNA was also found in the dorsomedial nucleus of the rat. The distribution of galanin receptor sites was investigated by receptor autoradiography using 125I-labelled porcine galanin. The specificity of the binding was assessed by competition with different neuropeptides. While galanin blocked the binding at nanomolar concentrations, the other neuropeptides examined were ineffective at 10-7 M concentrations. The highest densities of galanin binding sites were seen in the preoptic area, ventromedial and lateral nuclei, of rat and human hypothalamus. In contrast, very low densities of binding sites were observed in the paraventricular, supraoptic and arcuate nuclei. Our results show that the distribution of neurons expressing galanin is complementary to that of galanin receptors in the rat and human hypothalamus. This suggests that receptors for galanin are not located on the cell bodies of galaninergic neurons, but are probably presynaptic on or postsynaptic to the processes of these cells.     

Neurotransmitter receptor sites in human hippocampus: a quantitative autoradiographic study

The distribution of serotonin₁, cholinergic-muscarinic, α-adrenergic and opiate-type receptors was studied in the human hippocampus by quantitative autoradiography. Each receptor type exhibited a unique distribution. Serotonin₁ receptors were found to predominate in the subiculum, whereas α₁-adrenergic receptor are absent in that area and present at high levels in the dentate and the CA fields. High densities of muscarinic receptors appear in the subiculum and dentate gyrus. Opiate receptors are restricted to a medial aspect of the subiculum, with lower levels in the dentate and CA fields. The high anatomical resolution and quantitative character of the data may make it useful in the investigation of hippocampal pathology in humans.     

Distribution of [H]AMPA binding sites in rat brain as determined by quantitative autoradiography

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) is a potent neuroexcitatory compound which acts at the quisqualate class of excitatory amino acid receptors. In this study we describe the pharmacological characteristics and anatomical distribution of [³H]AMPA binding sites in rat brain using quantitative autoradiography. These binding sites exhibit the appropriate pharmacological characteristics and are found in high concentrations in the hippocampus, cerebral cortex (especially layers I–III), induseum griseum, and dorsal lateral septum. Intermediate concentrations are found in the corpus striatum and deeper layers of cerebral cortex. Lower concentrations are found in the diencephalon, midbrain and brainstem. These results demonstrate that [³H]AMPA binding sites are found throughout the CNS and suggest brain regions which may use quisqualate receptors as glutamate neurotransmitter receptors.     

Non-NMDA glutamate receptor binding in canine brain after global cerebral ischemia and reperfusion

We employed a canine model to test the effects of global cerebral ischemia and reperfusion on binding to α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA), kainate (KA), and metabotropic glutamate receptors. Ischemia was induced by 10 min of cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. Frozen sections were prepared from parietal and temporal cortex, hippocampus, and striatum, and in vitro autoradiography was performed with one of three radioligands: [³H]AMPA, [³H]KA, or [³H]glutamate (using conditions allowing specific labeling of the metabotropic binding site). In striatum, metabotropic binding was unchanged, whereas AMPA and KA binding decreased by 20–30% at 30 min postischemia, remaining depressed through 24h. In cortex, AMPA and metabotropic binding were decreased at several timepoints after ischemia and recirculation, particularly in parietal cortex, whereas KA binding was unaffected in this tissue. Binding to hippocampal regions was largely unchanged, except for a decrease in KA binding at 2 and 4 h postischemia. These findings contrast with results from parallel studies showing increased striatal binding to NMDA receptors following ischemia. Decreased binding to non-NMDA glutamate receptors in striatum and parietal cortex may serve to protect against damage mediated through these receptors.