Autoradiographic localization of cholecystokinin receptors in rodent brain

Cholecystokinin (CCK) receptor binding sites have been localized by autoradiography in the guinea pig and rat central nervous system. [125I]CCK-triacontatriapeptide labeled the sites in brain slices with an observed association constant equal to 0.041 min-1 and a dissociation constant equal to 0.008 min-1. CCK-triacontatriapeptide (CCK-33) and the C-terminal octapeptide of CCK-33 (CCK-8) potently inhibited [125I]CCK-33 binding with Ki's of 2 nM, whereas desulfated CCK-8 (CCK8-ds) and the C-terminal tetrapeptide of CCK-33 (CCK-4) were much weaker. Receptors were concentrated in the olfactory bulb, in the superficial laminae of the primary olfactory cortex, in the deep laminae of the cerebral cortex, and in the pretectal area. Substantial numbers of sites were also found in the basal ganglia, in the amygdala, and in the hippocampal formation. [125I]CCK-33 binding sites appear to be located on fibers of the optic tract and probably on olfactory tract fibers as well. These results are discussed in terms of physiological functions associated with CCK, presynaptic receptors, and axonal flow of CCK receptors.     

Autoradiographic localization of muscarinic receptors in rabbit carotid body

Muscarinic receptors in the rabbit carotid body were studied utilizing the specific muscarinic antagonist. [3H]quinuclidinyl benzilate. Biochemical and autoradiographic assays of ligand binding showed that receptor numbers and distributions remained unchanged following degeneration of axons and terminals of the carotid sinus nerve. The data suggest that muscarinic receptors are associated with carotid body parenchymal cells and are absent on afferent nerve terminals.     

Autoradiographic localization of angiotensin receptors in the sheep brain

Binding of [125I]-(Sar1,Ile8)angiotensin II (AII) to frozen sections of sheep brain was determined by in vitro autoradiography. Greatest AII-binding occurred in the organum vasculosum of the lamina terminalis, subfornical organ, median preoptic and periventricular nuclei situated in the anterior third ventricle wall. Other binding sites included the hypothalamic supraoptic and paraventricular nuclei and the medullary nucleus tractus solitarius. These regions may be central receptor sites for AII involvement in fluid and electrolyte balance and blood pressure regulation.     

Autoradiographic localization of [3H] gamma-aminobutyric acid in neuronal elements of the rat gastric antrum and intestine

High-affinity uptake and localization of radiolabelled gamma-aminobutyric acid (GABA) has been examined using light microscopic autoradiography in laminar preparations and transverse paraffin sections of the rat stomach, and small and large intestine. In the presence of beta-alanine (10(-3) M), a substrate specific inhibitor of high-affinity GABA transport into glia, tritiated GABA was accumulated by a high-affinity uptake system into myenteric ganglia and a subpopulation of mucosal cells. In the small and large intestine high-affinity uptake of [3H]GABA was evident in myenteric ganglion cells, extra-ganglionic sites and in the deep muscular nerve plexus of the circular muscle layer. Such labelling could be prevented in tissue treated with the specific neuronal high-affinity uptake blocker, L-2,4-diaminobutyric acid dihydrochloride (L-DABA; 10(-3) M), and therefore represented the selective distribution of [3H]GABA uptake sites to intrinsic neuronal elements of the rat gastrointestinal tract.     

Autoradiographic localization of thyrotropin releasing hormone receptors in human brain

We used quantitative autoradiography to localize thyrotropin releasing hormone (TRH) receptors in human brain. Highest concentrations of TRH receptors were localized within the cortical, basal, and lateral nuclei of the amygdala and the molecular layer of the hippocampus. Low levels were found in the cortex, diencephalon, and basal ganglia. The radioligand bound with similar affinity and pharmacology to pituitary gland as to brain. These data suggest that authentic TRH receptors in the hippocampus and amygdala may mediate the putative effects of TRH on the human brain.     

Compartmental localization of gamma-aminobutyric acid type B receptors in the cholinergic circuitry of the rabbit retina

Although many effects of gamma-aminobutyric acid (GABA) on retinal function have been attributed to GABA(A) and GABA(C) receptors, specific retinal functions have also been shown to be mediated by GABA(B) receptors, including facilitation of light-evoked acetylcholine release from the rabbit retina (Neal and Cunningham [1995] J. Physiol. 482:363-372). To explain the results of a rich set of experiments, Neal and Cunningham proposed a model for this facilitation. In this model, GABA(B) receptor-mediated inhibition of glycinergic cells would reduce their own inhibition of cholinergic cells. In turn, muscarinic input from the latter to the glycinergic cells would complete a negative-feedback circuitry. In this study, we have used immunohistochemical techniques to test elements of this model. We report that glycinergic amacrine cells are GABA(B) receptor negative. In contrast, our data reveal the localization of GABA(B) receptors on cholinergic/GABAergic starburst amacrine cells. High-resolution localization of GABA(B) receptors on starburst amacrine cells shows that they are discretely localized to a limited population of its varicosities, the majority of likely synaptic-release sites being devoid of detectable levels of GABA(B) receptors. Finally, we identify a glycinergic cell that is a potential muscarinic receptor-bearing target of GABA(B)-modulated acetylcholine release. This target is the DAPI-3 cell. We propose, based on these data, a modification of the Neal and Cunningham model in which GABA(B) receptors are on starburst, not glycinergic amacrine cells.     

High affinity GABA receptors — Autoradiographic localization

The distribution of the high affinity gamma-aminobutyric acid (GABA) receptor labeled by [3H]muscimol, has been studied in the rat brain by light microscopic autoradiography. Receptors in slide-mounted tissue sections were labeled in vitro with [3H]muscimol. Most of the gray matter areas presented grain densities significantly higher than background or white matter areas. Wide variations in receptor densities were found between different brain areas and nuclei. Areas with very high grain densities are the granule cell layer of the cerebellum, external plexiform layer of the olfactory bulb and nuclei of the thalamus, such as the ventral nucleus, lateral nucleus and dorsal geniculate body. The molecular layer of the hippocampus and the external (I-IV) layers of the cortex are also rich in GABA receptors. The basal ganglia have moderate concentrations of receptors, while the pons, medulla and brainstem have only low concentrations of autoradiographic grains. These distributions are discussed in correlation with the known distribution of GABAergic terminals and the presence of inhibitory GABAergic mechanisms.     

The ontogeny of glutamate receptors in rat barrel field cortex

The ontogeny of N-methyl-d-aspartate (NMDA) and non-NMDA excitatory amino acid receptors in rat barrel field cortex were characterized using receptor autoradiography. NMDA receptors showed a different pattern of development than that of non-NMDA receptors recognizing quisqualate (QUIS sites). During the first 14 days, high densities of QUIS sites were localized in barrel centers forming a sensory map of the rat whisker pad. After that time, the density of QUIS sites in barrel centers decreased so that the pattern was no longer apparent by postnatal day 21. In contrast to QUIS sites, NMDA sites did not exhibit a somatotopic pattern until postnatal day 21, when the lower density of sites in barrel septa formed an outline of barrel centers. At all ages examined, the density of NMDA sites did not differ significantly between barrel centers and surrounding cortex. Of the non-NMDA receptors examined in the postnatal day 10 old rat, both metabotropic sites and the NNKQ sites, which are [³H]glutamate binding sites that are not displaceable by NMDA, kainate or QUIS, showed a pattern of higher densities in barrel centers than surrounding tissue, whereas AMPA sites exhibited a complementary pattern. [³H]Glutamate binding to metabotropic sites was not significantly displaced by QUIS, whereas both NNKQ sites and metabotropic sites were potently blocked by the metabotropic agonist trans-ACPD. These results suggest that the NNKQ sites are low affinity QUIS metabotropic receptors, which, due to their high density in the immature barrel field, are in a position to influence barrel formation.     

Autoradiographic localization of alpha5 subunit-containing GABAA receptors in rat brain

Multiple subtypes of GABAA receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of alpha5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [3H]L-655,708 and [3H]Ro15-1788, an alpha5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [3H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where alpha5 receptors accounted for 20-35% of total [3H]Ro15-1788 binding sites. Low levels of [3H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [3H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.     

Autoradiographic localization of adenosine A1 receptors in brainstem of fetal sheep

Quantitative autoradiographic techniques were used to localize adenosine A1 receptors at the light microscopic level with the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine [( 3H]DPCPX) in the brainstem of fetal sheep. Since adenosine has been proposed as a neuromodulator, which effects the depression of fetal breathing movements during hypoxia, attention was directed to respiratory neuronal areas. The highest density of A1 receptors in respiratory related groups was found in an area of the rostral ventrolateral medulla, which is ventral to the facial nucleus, caudal to the superior olive and lateral to the rostral inferior olive. Intermediate densities were seen in the medial and lateral parabrachial nuclei. Adenosine A1 receptor density was low in the areas of the nucleus of the solitary tract and the nucleus ambiguous. These data suggest that moderate hypoxia in the fetus may depress respiration by withdrawing a tonic stimulus at the level of the ventral lateral medullary chemoreceptors.     

Autoradiographic localization of thyrotropin-releasing hormone receptors in amyotrophic lateral sclerosis spinal cord

We used quantitative autoradiography to determine the density of thyrotropin-releasing hormone (TRH) receptors in discrete regions of spinal cord from four patients with amyotrophic lateral sclerosis (ALS). The density and distribution of [3H]-3-methyl-histidine-TRH binding to TRH receptors differed from reported values in normal individuals, with fewer TRH receptors in lamina II and lamina IX. The diminished concentration of TRH receptors in lamina IX may reflect the loss of motor neurons in ALS.     

Immunocytochemical localization of a Manduca sexta gamma-aminobutyric acid transporter

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in insect central and peripheral nervous systems. Although much work has focused on the downstream targets of GABA, signal termination at insect GABAergic synapses has received very little attention. One of the major mechanisms of terminating synaptic transmission involves transport of the neurotransmitter molecules into presynaptic neurons or surrounding glia. Here we report the immunolocalization of a GABA transporter in the tobacco hornworm, Manduca sexta (MasGAT), using an affinity-purified antibody developed to the C-terminus. This is the first demonstration of an insect neurotransmitter transporter immunolocalization study. Results showed strong staining in the neuropil regions of embryonic, larval, and pharate adult central nervous system. Expression pattern in the pharate adult brain mostly mimicked that observed for GABA, with staining in parts of the optic and antennal lobes, mushroom body, lateral protocerebrum, and central complex. Certain longitudinal and lateral connectives of ganglia were observed to have immunostained fibers representing axons. These data support the view that GABA is involved in visual and olfactory processing in the insect brain.     

Autoradiographic localization of thyrotropin-releasing hormone receptors in the rat central nervous system

We employed quantitative autoradiography to examine the distribution of thyrotropin-releasing hormone (TRH) receptors in the rat CNS. The binding of [3H]3-methyl-histidine-TRH [( 3H]MeTRH) to TRH receptors in frozen rat brain sections was saturable, of a high affinity (Kd = 5 nM), and specific for TRH analogs. Autoradiograms of [3H]MeTRH binding showed highest concentrations of TRH receptors in the rhinencephalon, including accessory olfactory bulb, nuclei of the amygdala, and the ventral dentate gyrus and subiculum of the hippocampus. Moderate TRH receptor concentrations were found within the thalamus and hypothalamus, in most regions of the rhombencephalon, such as the cranial nerve nuclei, and in the substantia gelatinosa of the spinal cord. Neocortex and basal ganglia contained low densities of TRH receptors. This distribution correlates well with the sensitivity of brain regions to the known effects of TRH, and suggests that TRH receptors may mediate the actions of TRH in the rat CNS.     

Autoradiographic localization of inhibitory and excitatory amino acid neurotransmitter receptors in human normal and olivopontocerebellar atrophy cerebellar cortex

We used standard techniques of receptor autoradiography to study the distribution of inhibitory and excitatory amino acid neurotransmitter receptors in human normal cerebellar cortex. Benzodiazepine (BDZ) receptor density was relatively high in both granule cell and molecular layers. GABAA receptor density was highest in granule cell layer with lower receptor density in molecular layer. There was a lower density of GABAB receptors than GABAA receptors in both molecular and granule cell layers with a relatively higher density of GABAB receptors in molecular layer than in granule cell layer. In granule cell layer, the density of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors was greatest whereas in molecular layer the quisqualate (QA) receptor subtype density was greatest. With [3H]N-(1-[2-thienyl]cyclohexyl)3-4-piperidine as a ligand, there was no specific binding to the phencyclidine receptor. Molecular layer was also characterized by relatively high density of a non-NMDA/non-QA displaceable glutamate binding site. We studied also the cerebellar cortex of 4 cases of olivopontocerebellar atrophy (OPCA), a syndrome in which Purkinje and granule cells degenerate. In these specimens, there was significant decrement of BDZ and GABAA receptors in both molecular and granule cell layers, with loss of GABAB receptors in molecular layer. NMDA receptors were depleted in granule cell layer while QA receptors and the non-NMDA/non-QA glutamate binding site were significantly depleted in molecular layer. Our normal human and OPCA data are largely consistent with animal data about the cellular localization of cerebellar cortical amino acid neurotransmitter receptors.     

Development of the spinal-medullary projection from the mouse barrel field

Neurons in layer V of the murine posteromedial barrel subfield (PMBSF) project to structures at or caudal to the spinal-medullary junction. During postnatal development a reduction occurs in the density of the neurons which form this projection. In principle, three processes might be expected to contribute to this reduction: cell death, tissue growth, and axon pruning. Three different paradigms in which cells of origin of the projection are labeled retrogradely with True Blue, injected into the spinal-medullary junction, taken together with an estimate of the relative growth of layer V, provide separate estimates of the magnitude and rate of reduction consequent to these different processes during the first 3 postnatal weeks. The density of neurons in an index sector of layer V of the PMBSF which contribute to the projection at varied ages is estimated by injections made at a range of ages from postnatal day 1 (P1) to P16, with a survival of 4 days in each instance. Overall reduction in density is 80%. The component due primarily to axon pruning is estimated to be 50% by injections delivered at graded ages from P1 to P16 with survival to P20 in each instance. The component of the reduction attributable to increase in volume is estimated at 30% by a series of injections delivered at P1 with graded survival times from P5 through P20. A reduction due to cell death is not identified. The reduction in density due to tissue growth is essentially linear through the interval P5-P11. At all ages, neuronal somata of origin of the spinal-medullary projection are located within layer V. Subsequent to P15 they are confined to sublayer Vb; at earlier ages somata in Va and Vc also contribute axons to the projection. Although volume increase due to growth of the neuropil reduces the density of the population contributing to the projection equally in all three sublayers, final elimination of all contributions from Va and Vc depends upon axon pruning.     

Ontogeny of non-NMDA glutamate receptors in rat barrel field cortex: I. metabotropic receptors

The ontogeny of metabotropic excitatory amino acid receptors (mGluRs) in rat barrel field cortex was characterized by using receptor autoradiography and immunocytochemistry to test the hypothesis that changes in mGluR expression coincide with the emergence of somatotopic patterns in this region. On postnatal days 1 (P1) and 3, [³H]glutamate binding to mGluRs was not distributed in a somatotopic pattern. By P5, mGluRs exhibited a whisker-related pattern, with higher densities of mGluRs in barrel centers than in surrounding cortex. Between P5 and P14 and at P60, the overall binding density remained higher in barrels than in surrounding cortex. At P60, a somatotopic pattern of binding was not apparent. The majority of mGluR sites in the barrel field were blocked by the metabotropic agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid but were not significantly displaced by quisqualate. Immunocytochemical studies of phosphoinositide-linked mGluRs, mGluR5 and mGluR1α, showed that the developmental expression of mGluR5 mirrored that of the pattern of autoradiographically labeled mGluRs. The immature barrel field (ages P5–P14) was enriched in mGluR5, with greater concentrations of mGluR5 immunoreactivity in barrels than in surrounding cortex. Within barrel centers, mGluR5 was localized within the neuropil, on the surfaces of cell bodies and dendrites in layer IV. A somatotopic pattern of mGluR5 immunoreactivity persisted into adulthood, although the pattern was less pronounced after P14. In contrast, mGluR1α was never localized in a somatotopic pattern in barrel field cortex. We conclude from the developmental localization of mGluRs that the spatiotemporal regulated expression of these receptors may influence barrel maturation and plasticity. J. Comp. Neurol. 386:16–28, 1997. © 1997 Wiley-Liss, Inc.     

Subcellular localization and endocytosis of homomeric gamma2 subunit splice variants of gamma-aminobutyric acid type A receptors.

The expression of alpha and beta gamma-aminobutyric acid type A receptor subunits produces GABA-gated channels which require the incorporation of either the gamma2 or gamma3 subunit for benzodiazepine modulation. Here we examine the role of the gamma2 subunit splice variants, gamma2S and gamma2L which differ by eight amino acids in the major intracellular domain, in mediating cell surface expression. Using immunocytochemistry we have demonstrated that when expressed alone, the gamma2S subunit can access the cell surface and internalize constitutively. In contrast, alpha1, beta2 and gamma2L are retained predominantly in the endoplasmic reticulum (ER) when expressed alone. Replacing the insert which differentiates gamma2L from gamma2S (LLRMFSFK) with eight alanines produces a phenotype identical to gamma2S. Both gamma2 subunits fail to produce high molecular weight oligomers observed for alpha1beta2 and alpha1beta2gamma2 heterooligomers and do not form functional ion channels. Surface expression of gamma2S is repressed upon the coexpression of alpha1 or beta2 subunits, resulting in ER-retained heterooligomers, suggesting that homomeric gamma2S is unlikely to occur in vivo. However, its independent maturation to surface competence and preferential assembly with alpha and beta subunits may ensure the production of functional benzodiazepine-sensitive receptors. Furthermore, the presence of the gamma2 subunit appears to confer an endocytotic capacity to these heterooligomeric receptors.