The use of 3H standards in 125I autoradiography

doi:10.1016/0165-0270(90)90139-7

Journal of Neuroscience Methods

Volume 32, Issue 3, June 1990, Pages 185-192

https://doi.org/10.1016/0165-0270(90)90139-7 Get rights and content

Abstract

To avoid repeated production of ¹²⁵I tissue mash standards and to decrease inter-experiment variability, a set of commercially available, high activity plastic ³H standards was calibrated for use with ¹²⁵I autoradiographs. Standards with a range of 2.0–7390 μCi ³H/g plastic were used since they produced autoradiographs of suitable optical density (darkness) at exposure times of 4 to more than 72 h. Tissue mash standards at 12 concentrations of ¹²⁵I were produced by homogenizing rat brains and adding ¹²⁵I. These were frozen and sectioned at thicknesses ranging from 6 to 120 μm and mounted on microscope slides. Each set of tissue standards, along with slides bearing the plastic ³H standards, was apposed to film for various times. The calibration of the ³H standards was done separately for each time point and section thickness. Autoradiographs were analyzed using a computer assisted image analysis system. The standard curve generated for each set of ¹²⁵I-labelled tissue standards was correlated with the ³H standards. It was found that the relationship between the ³H plastic standards and the ¹²⁵I tissue mash standards was not simple and depended on exposure time, thickness of the section, as well as the concentration of radioactivity in a given standard. However, this accurate calibration of the ³H standards yields long-lived standards for ¹²⁵I autoradiography useful over a wide range of radionuclide concentrations, tissue thicknesses and exposure times.

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Optogenetic activation of striatal cholinergic interneurons regulates L-dopa-induced dyskinesias
2016, Neurobiology of Disease
Citation Excerpt :
The ImageQuant program from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK) was used to obtain optical density values from autoradiographic images, which were converted to fmol/mg tissue using standard curves generated from 3H-microscale standards. These were calibrated for 125I autoradiography as described (Artymyshyn et al., 1990). Sample optical density readings were within the linear range of the standards.
L-dopa-induced dyskinesias (LIDs) are a serious complication of L-dopa therapy for Parkinson's disease. Emerging evidence indicates that the nicotinic cholinergic system plays a role in LIDs, although the pathways and mechanisms are poorly understood. Here we used optogenetics to investigate the role of striatal cholinergic interneurons in LIDs. Mice expressing cre-recombinase under the control of the choline acetyltransferase promoter (ChAT-Cre) were lesioned by unilateral injection of 6-hydroxydopamine. AAV5-ChR2-eYFP or AAV5-control-eYFP was injected into the dorsolateral striatum, and optical fibers implanted. After stable virus expression, mice were treated with L-dopa. They were then subjected to various stimulation protocols for 2 h and LIDs rated. Continuous stimulation with a short duration optical pulse (1–5 ms) enhanced LIDs. This effect was blocked by the general muscarinic acetylcholine receptor (mAChR) antagonist atropine indicating it was mAChR-mediated. By contrast, continuous stimulation with a longer duration optical pulse (20 ms to 1 s) reduced LIDs to a similar extent as nicotine treatment (~ 50%). The general nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine blocked the decline in LIDs with longer optical pulses showing it was nAChR-mediated. None of the stimulation regimens altered LIDs in control-eYFP mice. Lesion-induced motor impairment was not affected by optical stimulation indicating that cholinergic transmission selectively regulates LIDs. Longer pulse stimulation increased the number of c-Fos expressing ChAT neurons, suggesting that changes in this immediate early gene may be involved. These results demonstrate that striatal cholinergic interneurons play a critical role in LIDs and support the idea that nicotine treatment reduces LIDs via nAChR desensitization.
α4β2 nicotinic receptors play a role in the nAChR-mediated decline in l-dopa-induced dyskinesias in parkinsonian rats
2013, Neuropharmacology
l-dopa-induced dyskinesias are a serious long-term side effect of dopamine replacement therapy for Parkinson's disease for which there are few treatment options. Our previous studies showed that nicotine decreased l-dopa-induced abnormal involuntary movements (AIMs). Subsequent work with knockout mice demonstrated that α6β2* nicotinic receptors (nAChRs) play a key role. The present experiments were done to determine if α4β2* nAChRs are also involved in l-dopa-induced dyskinesias. To approach this, we took advantage of the finding that α6β2* nAChRs are predominantly present on striatal dopaminergic nerve terminals, while a significant population of α4β2* nAChRs are located on other neurons. Thus, a severe dopaminergic lesion would cause a major loss in α6β2*, but not α4β2* nAChRs. Experiments were therefore done in which rats were unilaterally lesioned with 6-hydroxydopamine, at a dose that led to severe nigrostriatal damage. The dopamine transporter, a dopamine nerve terminal marker, was decreased by >99%. This lesion also decreased striatal α6β2* nAChRs by 97%, while α4β2* nAChRs were reduced by only 12% compared to control. A series of β2* nAChR compounds, including TC-2696, TI-10165, TC-8831, TC-10600 and sazetidine reduced l-dopa-induced AIMs in these rats by 23–32%. TC-2696, TI-10165, TC-8831 were also tested for parkinsonism, with no effect on this behavior. Tolerance did not develop with up to 3 months of treatment. Since α4α5β2 nAChRs are also predominantly on striatal dopamine terminals, these data suggest that drugs targeting α4β2 nAChRs may reduce l-dopa-induced dyskinesias in late stage Parkinson's disease.
Nicotine-mediated improvement in l-dopa-induced dyskinesias in MPTP-lesioned monkeys is dependent on dopamine nerve terminal function
2013, Neurobiology of Disease
Citation Excerpt :
The ImageQuant program from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK) was used to obtain optical density values from autoradiographic images, which were converted to fmol/mg tissue using standard curves generated from 3H-microscale standards. These were calibrated for 125I autoradiography as described (Artymyshyn et al., 1990). Sample optical density readings were within the linear range of the standards.
l-Dopa-induced dyskinesias (LIDs) are abnormal involuntary movements that develop with long term l-dopa therapy for Parkinson's disease. Studies show that nicotine administration reduced LIDs in several parkinsonian animal models. The present work was done to understand the factors that regulate the nicotine-mediated reduction in LIDs in MPTP-lesioned nonhuman primates. To approach this, we used two groups of monkeys, one with mild-moderate and the other with more severe parkinsonism rendered dyskinetic using l-dopa. In mild-moderately parkinsonian monkeys, nicotine pretreatment (300 μg/ml via drinking water) prevented the development of LIDs by ~ 75%. This improvement was maintained when the nicotine dose was lowered to 50 μg/ml but was lost with nicotine removal. Nicotine re-exposure again decreased LIDs. By contrast, nicotine treatment did not reduce LIDs in monkeys with more severe parkinsonism. We next determined how nicotine's ability to reduce LIDs correlated with lesion-induced changes in the striatal dopamine transporter and ³H-dopamine release in these two groups of monkeys. The striatal dopamine transporter was reduced to 54% and 28% of control in mild-moderately and more severely parkinsonian monkeys, respectively. However, basal, K⁺, α4β2* and α6β2* nAChR-evoked ³H-dopamine release were near control levels in striatum of mild-moderately parkinsonian monkeys. By contrast, these same release measures were reduced to a significantly greater extent in striatum of more severely parkinsonian monkeys. Thus, nicotine best improves LIDs in lesioned monkeys in which striatal dopamine transmission is still relatively intact. These data suggest that nicotine treatment would most effectively reduce LIDs in patients with mild to moderate Parkinson's disease.
Role for α6 nicotinic receptors in l-dopa-induced dyskinesias in parkinsonian mice
2012, Neuropharmacology
Citation Excerpt :
For quantitation of the autoradiograms, optical density measurements were assessed using the ImageQuant system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). These values were converted to fmol/mg tissue using standard curves generated from 3H standards as previously described (Artymyshyn et al., 1990). The optical density readings of the samples fell within the linear range of the standards.
l-Dopa-induced dyskinesias are a serious side effect that develops in most Parkinson's disease patients on dopamine replacement therapy. Few treatment options are available to manage dyskinesias; however, recent studies show that nicotine reduces these abnormal involuntary movements (AIMs) in parkinsonian animals by acting at nicotinic acetylcholine receptors (nAChRs). Identification of the nAChR subtypes that mediate this reduction in AIMs is important as it will help in the development of nAChR subtype selective drugs for their treatment. Here we investigate the role of α6β2* nAChRs, a subtype selectively present in the nigrostriatal pathway, using α6 nAChR subunit null mutant (α6(−/−)) mice. Wildtype and α6(−/−) mice were lesioned by unilateral injection of 6-hydroxydopamine (3 μg/μl) into the medial forebrain bundle. They were then given l-dopa (3 mg/kg) plus benserazide (15 mg/kg) 2–3 wk later. l-dopa-induced AIMs developed to a similar extent in α6(−/−) and wildtype mice. However, AIMs in α6(−/−) mice declined to ∼50% of that in wildtype mice with continued l-dopa treatment. Nicotine treatment also decreased AIMs by ∼50% in wildtype mice, although not in α6(−/−) mice. There were no effects on parkinsonism under any experimental condition. To conclude, the similar declines in l-dopa-induced AIMs in nicotine-treated wildtype mice and in α6(−/−) mice treated with and without nicotine indicate an essential role for α6β2* nAChRs in the maintenance of l-dopa-induced AIMs. These findings suggest that α6β2* nAChR drugs have potential for reducing l-dopa-induced dyskinesias in Parkinson's disease.
Nicotinic receptor agonists decrease L-dopa-induced dyskinesias most effectively in partially lesioned parkinsonian rats
2011, Neuropharmacology
Citation Excerpt :
The radioactivity in nCi/mg was then converted to fmol/mg tissue. The 3H standards were calibrated for 125I autoradiography using the corrections described, including exposure time, section thickness and concentration of radioactivity (Artymyshyn et al., 1990). Care was taken to ensure that the optical density readings of the samples were within the linear range of the film.
L-dopa therapy for Parkinson’s disease leads to dyskinesias or abnormal involuntary movement (AIMs) for which there are few treatment options. Our previous data showed that nicotine administration reduced L-dopa-induced AIMs in parkinsonian monkeys and rats. To further understand how nicotine mediates its antidyskinetic action, we investigated the effect of nicotinic receptor (nAChR) agonists in unilateral 6-OHDA-lesioned rats with varying striatal damage. We first tested the drugs in L-dopa-treated rats with a near-complete striatal dopamine lesion (>99%), the standard rodent dyskinesia model. Varenicline, an agonist that interacts with multiple nAChRs, did not significantly reduce L-dopa-induced AIMs, while 5-iodo-A-85380 (A-85380), which acts selectively at α4β2* and α6β2* subtypes, reduced AIMs by 20%. By contrast, both varenicline and A-85380 reduced L-dopa-induced AIMs by 40-50% in rats with a partial striatal dopamine lesion. Neither drug worsened the antiparkinsonian action of L-dopa. The results show that selective nicotinic agonists reduce dyskinesias, and that they are optimally effective in animals with partial striatal dopamine damage. These findings suggest that presynaptic dopamine terminal α4β2* and α6β2* nAChRs are critical for nicotine’s antidyskinetic action. The current data have important implications for the use of nicotinic receptor-directed drugs for L-dopa-induced dyskinesias, a debilitating motor complication of dopamine replacement therapy for Parkinson’s disease.
Effect of Chronic Administration of Duloxetine on Serotonin and Norepinephrine Transporter Binding Sites in Rat Brain
2007, Biological Psychiatry
Citation Excerpt :
Non-specific binding was determined by 100 μM l-isoproterenol (Sigma), and was 10–15% of total binding. Tritium labeled sections on slides were exposed to Kodak MR film (Kodak, Rochester) for six weeks along with tritium standards (American Radiolabeled Chemicals) calibrated against [3H] brain mash per Geary et al (1985); [125I] labeled sections were exposed for 18 hours to the same film and tritium standards calibrated to [125I] labeled brain mash per Artymyshyn et al (1990). Films were developed using Kodak GBX developer.
Chronic treatment of rats with certain selective serotonin or norepinephrine reuptake inhibitors produces significant decreases, respectively, in serotonin and norepinephrine transporter binding sites in brain. Duloxetine may be a dual serotonin/norepinephrine reuptake inhibitor, as it is only a slightly more potent inhibitor of serotonin than norepinephrine uptake in vitro. Consequently, we hypothesized that chronic duloxetine treatment, at doses producing serum levels within its therapeutic range, would affect both monoamine transporters dose-dependently, with a higher dose causing greater reductions of binding sites for both transporters.
Rats were treated with either 4 or 8 mg/kg/d of duloxetine, paroxetine, desipramine, or vehicle via subcutaneous osmotic minipumps for 21 days. Binding sites for serotonin and norepinephrine transporters were measured in amygdala and hippocampus using quantitative autoradiography.
Both doses of duloxetine and paroxetine produced equivalent and significant decreases in [³H] cyanoimipramine binding to serotonin transporters, but only desipramine treatment significantly reduced [³H] nisoxetine binding to norepinephrine transporters.
At doses producing rat serum concentrations in the range achieved in patients at recommended daily doses of the drug, duloxetine behaves in vivo more as a selective serotonin reuptake inhibitor than a dual reuptake inhibitor in its capacity to selectively reduce serotonin transporter density.

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Research paperThe use of 3H standards in 125I autoradiography

Abstract

J. Neurosci. Methods

J. Neurosci. Methods

Brain Res.

J. Biol. Chem.

Research paper
The use of ³H standards in ¹²⁵I autoradiography