Technique Of Receptor Autoradiography Research Articles

GH regulation of the Type 2 IGF receptor in regenerating skeletal muscle of rats.

GH enhances skeletal muscle growth, and IGF-II peptide is highly expressed during regeneration. We have therefore investigated the effect of GH administration on IGF-II binding and expression in regenerating rat skeletal muscle using the techniques of receptor autoradiography and in situ hybridisation. Notexin, a myotoxin, was injected into the right M. biceps femoris (day 0), causing affected fibres to undergo necrosis followed by rapid regeneration. Animals were administered either GH (200 micrograms/100 g body weight) or saline vehicle daily. Contralateral muscles were used as regeneration controls. GH administration during regeneration resulted in significant increases in body weight, and damaged and undamaged muscle weights (P < 0.001). IGF-II expression, which was examined in regenerating fibres, survivor fibres and undamaged fibres, varied according to tissue type (P < 0.001). Specifically, IGF-II expression in regenerating fibres was elevated relative to control and survivor fibres after day 3 (P < 0.05), with a peak on day 9 (P < 0.001). GH did not affect IGF-II message levels. 125I-IGF-II binding in regenerating muscle was examined in the same fibre types as well as in connective tissue. 125I-IGF-II binding in regenerating fibres was higher (P < 0.001) than in other tissue types on day 5. GH administration increased 125I-IGF-II binding in all damaged muscle tissues on day 5 (P < 0.001, regenerating fibres; P < 0.01, others). We believe that this shows for the first time an effect of GH on the Type 2 IGF receptor in regenerating skeletal muscle.

Somatostatin receptors in human prostate and prostate cancer.

Benign as well as malignant human prostatic tissues were evaluated for their content of somatostatin (SRIH) receptors (SRIH-R). In vitro receptor autoradiography techniques on cryostat sections were performed using 125I-labeled [Tyr3]octreotide as well as 125I-labeled [Leu8,D-Trp22,Tyr25]SRIH-28 as radioligands. SRIH-R were identified in all normal and hyperplastic prostates in the smooth muscles of the stroma, whereas the glands did not express the receptors. Muscular nodules were strongly receptor positive as well. The receptors were of high affinity (Kd = 0.4 nmol/L) and high specificity for biologically active SRIH analogs; high affinity for SRIH-14, SRIH-28, and octreotide was detected, suggesting the presence of the SSTR2 receptor subtype. In situ hybridization studies confirmed the presence of SSTR2 messenger ribonucleic acid in these tissues. Primary prostate cancers did not have SRIH-R identified with 125I-labeled [Tyr3]octreotide. However, they were expressing SRIH-R identified with 125I-labeled [Leu8,D-Trp22,Tyr25]SRIH-28, with a high affinity for SRIH-14 and SRIH-28, but low affinity for octreotide. The receptors were located on tumoral cells. In situ hybridization studies revealed a preferential expression of SSTR1. Primary human prostate cancers, therefore, express a different SRIH-R subtype than benign prostate tissue. Several veins and the ganglion cells from the prostatic plexus in the surroundings of the tumors were expressing SRIH-R with high affinity for octreotide as well. These data suggest that the human prostate as well as prostate cancers may be targets for SRIH therapy; however, SRIH analogs with different selectivities for SRIH-R subtypes are required in each case.

An immunocytochemical and autoradiographic investigation of the serotoninergic innervation of trigeminal mesencephalic and motor nuclei in the rabbit

The results of a previous experiment suggest that the cell bodies of many jaw closing muscle spindle afférents in the trigeminal mesencephalic nucleus of the rabbit are phasically inhibited during fictive mastication. The aim of this study was to investigate one possible neurotransmitter system that could be involved in this modulation, serotonin, by use of receptor autoradiography techniques and immunofluorescence combined with retrograde labelling of masseteric spindle afférents and motoneurons. A second objective was to compare the serotonin innervation of neurons in the trigeminal mesencephalic nucleus with that of masseteric motoneurons. Serotoninergic fibres were seen surrouncling labelled masseteric spindle afférents, as well as unlabelled neurons, in the trigeminal mesencephalic nucleus. These fibres were close to the cell bodies and sometimes to the axon hillocks of the neurons. Although it has been reported that many neurons of the trigeminal nucleus are multipolar in some species, none of the labelled spindle afferent in this study had more than one process. Throughout the motor trigeminal nucleus, serotonin fibres were found in close proximity with cell bodies and with the proximal portions of axons and dendrites of labelled and unlabelled motoneurons. Serotonin fibres were also seen adjacent to cell bodies and processes of efferent neurons in cell group k. Autoradiography with several tritiated ligands was used to reveal the presence of receptors for serotonin as well as its uptake sites. Only serotonin 2 receptors were found to be abundant in the trigeminal mesencephalic nucleus. The motor nucleus and cell group k contained serotonin 2 and serotonin 3 receptors, as well as serotonin uptake sites. Serotonin 1A receptors appear to be absent from both nuclei. The finclings suggest that release of serotonin from fibres in close proximity to trigeminal primary afferent somata could modify the transmission of action potentials from muscle spindle receptors during mastication through an action on serotonin, receptors. In the motor nucleus and cell group k, serotonin may alter neuronal properties through actions on at least two receptor subtypes (serotonin 2 and serotonin 3).

Regional effects of pertussis toxin in vivo and in vitro on GABA B receptor binding in rat brain

Agonist binding to GABA B receptors modulates the activity of the guanine nucleotide binding proteins, Go and Gi. These G proteins are ADP-ribosylated by pertussis toxin and this prevents them from coupling to the GABA B receptor resulting in a reduction in high-affinity GABA B binding. GTP, which binds to a different site on the G protein α subunit, also reduces the affinity of the receptor for the G protein, and this can be used as a “marker” for G protein-GABA B receptor linkage. We have examined GABA B binding site distribution in rat brain after unilateral intrahippocampal pertussis toxin injection in vivo, and after incubating brain slices in pertussis toxin in vitro, using the technique of receptor autoradiography. The effect of pertussis toxin was compared with that of GTPγS on GABA B binding. Intrahippocampal pertussis toxin administration reduced GABA B but not GABA A receptor binding and the effects appeared to be limited by pertussis toxin diffusion. More widespread reductions in GABA B binding were seen after incubation of brain slices in vitro but the extent varied in different brain regions. No reduction was detected in the corpus striatum. GABA B binding was also reduced in membranes prepared from cerebral cortex, hippocampus and cerebellum but there was no sigificant reduction in the corpus striatum after pertussis toxin treatment. GTPγS reduced GABA B binding to a similar extent in all areas studied irrespective of their sensitivity to pertussis toxin suggesting that while GABA B binding sites are linked to G proteins throughout the rat brain, those in the corpus striatum may be predominantly pertussis toxin insensitive.

Calcitonin gene-related peptide (CGRP) in the regenerating rat sciatic nerve

Calcitonin-gene related peptide (CGRP) is a neuromodulatory peptide present in motoneurons and a subpopulation of sensory neurons of the adult peripheral nervous system. Here we have investigated the changes in axonal transport of CGRP and CGRP receptor expression in the injured and regenerating rat sciatic nerve using CGRP-immunocytochemistry, radioimmunoassay and quantitative in situ receptor autoradiography techniques. Axotomy led to a gradual and prolonged, 2.5- to 3.5-fold increase in specific CGRP binding to the distal part of the crushed sciatic nerve, beginning 4-6 days after axotomy. An even stronger, up to 30-fold increase was observed after 30-42 day denervation in the distal part of the transected sciatic nerve, where neurite reinnervation was prevented by retroversion and ligation of the proximal nerve stump. Reconnection of the proximal and distal nerve stumps 21 days after transection did not lead to a major reduction in specific CGRP binding but prevented a further increase that occurred between 21 and 42 days after transection without reconnection. In contrast, the anterograde axonal transport of CGRP decreased after axotomy to 40-50% of the control values 6-8 days after nerve crush but recovered towards normal levels during successful regeneration. Interestingly, the retrograde axonal transport of CGRP appeared to amount to only 10-20% of the anterograde transport, suggesting that the peptide may be released by the regenerating neurites into the endoneurium of the injured peripheral nerve. In view of the persistent upregulation in endoneural CGRP binding after axotomy these data indicate that axonal CGRP could play a regulatory role in mediating axonal-endoneural cell interaction during peripheral nerve regeneration.

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. GABA A receptor density was highest in granule cell layer with lower receptor density in molecular layer. There was a lower density of GABA B receptors than GABA A receptors in both molecular and granule cell layers with a relatively higher density of GABA B receptors in molecular layer than in granule cell layer. In granule cell layer, the density of theN-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 GABA A receptors in both molecular and granule cell layers, with loss of GABA B 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.

Neurotensin receptors in parkinson's disease and progressive supranuclear palsy: an autoradiographic study in basal ganglia

The technique of receptor autoradiography was used to study the distribution of neurotensin receptors in post mortem brain tissues from patients affected by Parkinson's disease, progressive supranuclear palsy and from age-matched controls. [ 125I]Neurotensin was used as ligand. Significant receptor decreases were found in the substantia nigra, both pars compacta and reticulata, and in the putamen in Parkinson's disease and progressive supranuclear palsy. In addition, significant decreases of neurotensin receptors were found in the ventral tegmental area, nucleus accumbens and dorsal part of caudate head in patients with Parkinson's disease but not in patients with progressive supranuclear palsy, indicating differential involvement of neurotensin receptors in these two neurological disorders. In addition, both in Parkinson's disease and progressive supranuclear palsy the decrement of striatal neurotensin binding sites was less than expected from the reported decrease of dopamine content in this nucleus, suggesting only partial localization of neurotensin receptors on mesostriatal dopaminergic projections.

VIP receptors and content after bowel transplantation

Advances in immunosuppressive therapy have renewed interest in small bowel transplantation. Little is known, however, about the functional capacity of transplanted intestine. To clarify the potential for normal function, we investigated whether elements of the enteric nervous system are preserved after denervation in our rat model of intestinal transplantation. We investigated whether VIP, a major peptide neurotransmitter of the enteric nervous system, and its receptors are preserved in the bowel after transplantation. In our model of transplantation, avascular fetal jejunum from term Fisher rats is transplanted to the subcutaneous tissues of host syngeneic rats. This “neogut” becomes vascularized and develops characteristics of native small bowel. VIP content was measured by RIA and the in situ distribution of VIP receptors was determined by the technique of receptor autoradiography. Neogut was studied 1 and 3 weeks after transplantation and compared with age-matched rat pup jejunum. Autoradiographs showed high silver grain density, representing VIP binding sites, in the mucosal layers of all tissues studied. VIP content in the transplanted bowel was comparable to that of native gut and showed a rise with developmental age similar to that of native gut. VIP levels (pmole/mg protein, x ± SEM) were neogut 1 week, 0.26 ± 0.14; jejunum 1 week, 0.25 ± 0.07; neogut 3 weeks, 0.60 ± 0.21; and jejunum 3 weeks, 0.69 ± 0.16. These results show that VIP receptors and content are preserved in this model of transplantation. This suggests that the enteric nervous system and receptors for peptide neurotransmitters remain intact after transplantation and may retain the potential for regulatory function.

Transient elevation of locus coeruleus α 2-adrenergic receptor binding during the early stages of amygdala kindling

Enhancement of noradrenergic neurotransmission retards, but does not prevent, the development of kindling. The firing activity of noradrenergic locus coeruleus (LC) neurons is partially regulated by axon collateral recurrent inhibition mediated via alpha 2-adrenergic receptors. We tested the hypothesis that LC autoinhibitory alpha 2-adrenergic receptors may change during the kindling process thereby altering LC excitability. Specific binding of the alpha 2-adrenergic receptor antagonist [3H]RX781094 (idazoxan) was measured in the LC of rats at 3 different stages of kindling development using in vitro neurotransmitter receptor autoradiography techniques. Specific [3H]RX781094 binding was elevated significantly in rats kindled to two Class 1 kindled motor seizures. No differences in binding were observed in animals kindled to Class 3 or Class 5 kindled motor seizures. Saturation of binding experiments indicated that the increase in binding following two Class 1 kindled motor seizures was due to an increase in the total number of alpha 2-receptors without a change in the affinity of the binding site for [3H]RX781094. The transient increase in number of LC alpha 2-adrenergic receptors is consistent with the idea that noradrenergic neurotransmission inhibits the early progress of kindling development, but then subsequently becomes ineffective in maintaining the inhibition during later stages of kindling development.

Mapping dopamine receptors in the human brain.

We have investigated the anatomic localization of dopamine D1 and D2 receptors in the human brain using selective high affinity ligands for both types of dopamine receptors and the technique of receptor autoradiography. Dopamine D1 receptors were labeled in postmortem human brain tissue sections using the antagonist [3H]SCH 23390. Dopamine D2 receptors were labeled in consecutive tissue sections using the agonist [3H]205-502 and the antagonist [3H]spiroperidol. D1 and D2 dopamine receptors presented a heterogeneous distribution in the human brain. The highest concentrations of both D1 and D2 receptors were found in parts of the basal ganglia, particularly the nucleus caudatus and putamen. Lower concentrations were seen in other areas for example, the lateral globus pallidus was enriched in D2 receptors and the medial globus pallidus in D1 receptors. The substantia nigra contained intermediate densities of both D1 and D2, D1 receptors being present in higher concentrations. Dopamine D1 receptors were also localized in areas outside of the basal ganglia, particularly in the neocortex, amygdala and hippocampal formation. Dopamine D2 receptors were also present in areas outside of the basal ganglia, the most significant densities being found in the hippocampal formation. We observed a marked age-dependent decline in the density of D1 receptors during the first decades of life. In contrast, D2 receptor concentrations appeared to be unaltered with age. The distribution and densities of dopamine receptors were examined in 12 cases of Parkinson's disease and compared to a control adult population. No significant differences in density and distribution were seen for either D1 nor D2 receptors.

Evaluation of N-isopropyl-(125I) p-iodoamphetamine affinity sites in rat brain by autoradiography.

Affinity sites of N-isopropyl-[125I]p-iodoamphetamine (IMP) were studied in rat brain employing in vitro technique of receptor autoradiography. High degrees of IMP accumulation were observed in almost regions of white matter and some of gray matter. Phenethylamine, glutamic acid and serotonin suppressed IMP accumulation in all brain regions, in cerebral cortex and in hypothalamus, respectively. These results indicated that IMP was accumulated mainly due to its physicochemical property of lipophilicity and might have some interactions with neurotransmitter receptors.

Alterations in muscarinic cholinergic receptor densities induced by thiamine deficiency: autoradiographic detection of changes in high- and low-affinity agonist binding.

Animals fed a diet deficient in thiamine or treated with a drug preventing the utilization of thiamine (thiamine antagonist) exhibited alterations in ligand binding to muscarinic receptors in several brain regions. Using quantitative techniques of receptor autoradiography, an increase in muscarinic receptor binding was demonstrated in such regions as the corpus callosum, lamina VI of the parietal cortex, caudate-putamen, ventral nucleus of the thalamus, stratum lacunosum moleculare and stratum oriens of the hippocampus, and the hilus of the area dentata. As a result of thiamine deficiency, this increase in muscarinic receptor populations was primarily due to an increase in the binding of the low-affinity agonist site. In the same experiment, a decrease in muscarinic receptor binding was found in the ventromedial region of the hypothalamus. Thiamine deficiency thus causes an up-regulation of muscarinic receptor binding in several regions of rat brain while causing a down-regulation of these same receptors in other brain areas.

Distribution of muscarinic cholinergic high and low affinity agonist binding sites: A light microscopic autoradiographic study

The distribution of high vs. low affinity muscarinic agonist binding sites has been determined using quantitative techniques of receptor autoradiography. The low affinity agonist sites predominate in many regions of the forebrain including the cerebral cortex, striatum, hippocampus, amygdala and thalamus. The high affinity agonist sites predominate in the brainstem and represent exclusively the type of muscarinic cholinergic receptor normally present in the principal nucleus of the trigeminal nerve, facial nerve nucleus, hypoglossal nerve nucleus, and in the ventral horn of the spinal cord. The regional localization of these subpopulations provides valuable information for future studies which seek to determine the functional importance of subtypes of muscarinic agonist binding sites.