Regional coupling of blood flow and methionine uptake in an experimental tumor assessed with autoradiography

Regional distribution of L-[methyl-¹⁴C] methionine (¹⁴C-MET) and 4-[¹⁸F] fluoro-antipyrine was compared using experimental rat tumors (AH109A) and a computerized autoradiogram image processor. Tissue distributions of the two tracers were found to be inhomogeneous in the tumor with nearly identical image patterns. Analysis of tissue radioactivities revealed that 82% of ¹⁴C-MET was derived from the acid insoluble fraction at 60 min after injection. The present study showed that ¹⁴C-MET uptake closely relates to tissue blood flow and may depend on its blood to tissue transport. Rapid incorporation of MET in the acid insoluble fraction implies that it is rapidly metabolized after transport into tumor tissue.     

Regional Distributions of Hippocampal Na+, K+-ATPase,Cytochrome Oxidase,and Total Protein in Temporal Lobe Epilepsy

Summary Na⁺, K⁺-ATPase (the sodium pump) is a ubiquitous enzyme that consumes ATP to maintain an adequate neuronal transmembrane electrical potential necessary for brain function and to dissipate ionic transients. Reductions in sodium pump function augment the sensitivity of neurons to glutamate, increasing excitability and neuronal damage in vitro. Temporal lobe epilepsy (TLE) is one disease characterized by hyperexcitability and marked hippocampal neuronal losses that could depend in part, on impaired sodium pump capacity secondary to changes in sodium pump levels and/or insufficient ATP supply. To assess whether abnormalities in the sodium pump occur in this disease, we used [³H]ouabain to determine the density of Na⁺, K⁺-ATPase for each anatomic region of hippocampus by in vitro autoradiography. Tissues were surgically obtained from epileptic patients with hippocampal sclerosis and compared with specimens from patients with seizures originating from temporal lobe tumors and autopsy controls. Changes in cellular population arising from neuronal losses or gliosis were assessed by protein densities derived from quantitative computerized densitometry of Coomassie-stained tissue sections. We estimated regional differences in capacity for ATP generation by determining cytochrome c oxidase (CO) activity. Principal neurons of hippocampus exhibit high levels of sodium pump enzyme. Both epilepsy groups exhibited slight but significant increases in sodium pump densityhnit mass of protein in the dentate molecular layer, CA2, and subiculum as compared with autopsy controls. Greater hilar sodium pump density was also observed in sclerotic hippocampi. In contrast, CO activity was reduced in both epilepsy types throughout hippocampus. Results suggest that although sodium pump protein in surviving neurons appears to be upregulated in epilepsy, sodium pump capacity may be limited by the reduced levels of CO activity. Functional reduction in sodium pump capacity may be an important factor in hyperexcitability and neuronal death.     

Repair of DNA damage in light sensitive human skin diseases

Summary Repair of UV-light induced DNA damage and changes in the semiconservative DNA synthesis were studied by in vitro autoradiography in the skin of patients with lightdermatoses (polymorphous light eruption, porphyria cutanea tarda, erythropoietic protoporphyria) and xeroderma pigmentosum as well as in that of healthy controls. In polymorphous light eruption the semiconservative DNA replication rate was more intensive in the area of the skin lesions and in the repeated phototest site, the excision repair synthesis appeared to be unaltered. In cutaneous porphyrias a decreased rate of the repair incorporation could be detected. Xeroderma pigmentosum was characterized by a strongly reduced repair synthesis.     

Autoradiographic localization of muscimol and baclofen binding sites in rodent cingulate cortex

Summary The laminar distribution of ³H-muscimol and ³H-baclofen binding was analyzed autoradiographically in areas 29c and 24b of rat cingulate cortex. Muscimol binding was heterogeneous in area 29c with a single peak in layer Ia of 320±26 grains per 2500 m². Binding in deeper layers was between 46% and 71% of that in layer Ia. There was a marked diurnal variation in muscimol binding in area 29c such that binding was elevated by 320% in layer Ia of brains perfused at 2200–0100 versus those perfused at 1100–1400. Muscimol binding in area 24b was uniform across all layers and was higher than that in area 29c except for in layer Ia. Baclofen binding was homogeneous in both areas, but was 120% greater in area 24b than in area 29c, and showed no diurnal variations. To localize muscimol binding sites at the cellular level, two types of lesion experiments were conducted in area 29c. First, ablation of neurons intrinsic to this cortex with the neurotoxin ibotenic acid reduced muscimol binding to homogeneity with a 70% reduction in layer Ia and a 29–42% reduction in deeper layers. Second, knife cuts, which were placed to isolate cingulate cortex from fiber pathways originating extrinsically, increased muscimol binding in all layers except layer Ia. Conversely, knife cuts which isolated superficial from deep layers yielded a marked drop in muscimol binding in all layers. In conclusion, muscimol binding sites are heterogeneously distributed in area 29c with peak binding in layer Ia at night. Since experimental observations suggest that muscimol binding is located on pyramidal cell apical tuft dendrites, it is possible that excitatory thalamic and intrinsic inhibitory input via GABA_A receptors on apical dendrites interact before arriving at the soma.     

[<Superscript>3</Superscript>H]LY334370, a novel radioligand for the 5-HT<Subscript>1F</Subscript> receptor. II. Autoradiographic localization in rat,guinea pig,monkey and human brain

LY334370 is a high affinity, selective agonist at the 5-HT_1F receptor. On this basis, the tritiated compound was examined for its utility in autoradiography to localize the 5-HT_1F receptor in rat and guinea pig brain regions. Specific 5-HT_1F receptor binding in rat brain was found in layers 4–5 of all cortical regions examined, as well as olfactory bulb and tubercle, nucleus accumbens, caudate putamen, parafascicular nucleus of the thalamus, medial mammillary nucleus, the CA3 region of the hippocampus, subiculum, and several amygdaloid nuclei. In guinea pig brain, the [³H]LY334370 binding sites were found at highest density in claustrum, but also in a layer of the cortex, caudate putamen, nucleus accumbens, thalamus, and medial mammillary nucleus. Some species differences in the distribution of the 5-HT_1F receptor were noted. Side by side comparison of rat brain autoradiography with [³H]LY334370 and [³H]sumatriptan showed labeling in the same brain regions. Preliminary binding studies in rhesus monkey and human brain sections showed [³H]LY334370 binding in cortical layers 4–5, subiculum (in the monkey), and the granule cell layer of the cerebellum. These findings suggest a discrete localization of the 5-HT_1F receptor in the rat, guinea pig, monkey and human brain, and confirms the utility of [³H]LY334370 as a potential tool to explore further the localization and possible functions of the 5-HT_1F receptor.     

Localization of radioactive125I-Labelled botulinus toxin at the neuromuscular junction of mouse diaphragm

Summary Autoradiography at the light microscopic level demonstrated that the¹²⁵I-labelled neurotoxin fromClostridium botulinum type A crystalline toxin binds specifically to the neuromuscular junction of the mice diaphragm.     

The tryptophan hydroxylase activation inhibitor, AGN-2979, decreases regional 5-HT synthesis in the rat brain measured with α-[C]methyl-l-tryptophan: An autoradiographic study

Many experimental conditions are stressful for animals. It is well known that stress induces tryptophan hydroxylase (TPH) activation, resulting in increased serotonin (5-HT) synthesis. In our experimental procedure to measure 5-HT synthesis using alpha-[(14)C]methyl-L-tryptophan (alpha-MTrp) autoradiographic method, the hind limbs of animals are restrained using a loose-fitted plaster cast such that the forelimbs of the animal remain free. The objective of the present investigation was to evaluate the changes, if any, in 5-HT synthesis, after injecting these restrained rats with the TPH activation inhibitor AGN-2979. The effect on regional 5-HT synthesis was studied using the alpha-MTrp autoradiographic method. The hypothesis was that the TPH activation inhibitor would reduce 5-HT synthesis, if TPH activation was induced by this restraint. The rats received injection of AGN-2979 (10 mg/kg, i.p.) or distilled water vehicle (1 mL/kg, i.p.) 1 h prior to tracer administration. The free- and total tryptophan concentrations were not significantly different between the treatment and control groups. The results demonstrate that 5-HT synthesis in AGN-2979 treated rats is significantly decreased (-12 to -35%) in both the raphe nuclei and their terminal areas when compared to the control rats. These findings suggest that restrained conditions, such as those used in our experimental protocol, induce TPH activation resulting in an increased 5-HT synthesis throughout the brain. The reduction in 5-HT synthesis in the AGN-2979 group is not related to a change in the plasma tryptophan. Because there was no activation in the pineal body, the structure having a different isoform of TPH, we can propose that it is only the brain TPH that becomes activated with this specific restraint.     

Influence of age, postmortem delay and freezing storage period on cannabinoid receptor density and functionality in human brain

It has been suggested that cannabimimetic drugs could be of interest in the treatment of several nervous disorders. Thus, it is important to analyse the distribution and properties of cannabinoid (CB) receptors directly in human brain. As postmortem human tissue is subjected to the effects of several biological variables, we have analyzed by autoradiography the influence of age, postmortem delay and freezing storage period (at -25 degrees C) on two parameters corresponding to cannabinoid CB1 receptors in human frontal cortex: receptor density and degree of activation of G-proteins ([35S]GTPgammaS assays). A significant decrease in the amount of both receptor density and agonist-stimulated G-protein activity was observed with age, revealing a mean reduction of about 10% per decade. In contrast, no significant correlations were found with postmortem delay either for CB1 receptors density or functionality. Finally, both parameters (receptor density and [35S]GTPgammaS response) were significantly reduced with freezing storage period at -25 degrees C in frontal cortical layers. Non-linear analysis of these data yielded values between 12 and 24 months of storage for a 50% reduction. In conclusion, when studying CB1 receptor properties in human brain samples, a careful analysis (and matching) for variables such as age and freezing storage period has to be carried out.     

Metabotropic glutamate 1 receptor distribution and occupancy in the rat brain: a quantitative autoradiographic study using [3H]R214127

We used the selective metabotropic glutamate (mGlu) 1 receptor antagonist [3H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone ([3H]R214127) to investigate the distribution of mGlu1 receptor binding sites in rat brain. We found high mGlu1 receptor binding in the cerebellum, thalamus, dentate gyrus and medial central gray, moderate binding within the CA3 of the hippocampus and hypothalamus, and low mGlu1 receptor binding in the basal ganglia and cortex. The mGlu1 receptor is also present in variable degree in the dorsal lateral septal nucleus, amygdala, interpeduncular nucleus and median raphe nucleus. Additionally, we employed [3H]R214127 autoradiography as a means of investigating the occupancy of central mGlu1 receptors following in vivo administration of mGlu1 receptor antagonists that prevent binding of this radioligand. We found that the mGlu1 receptor antagonist (3aS,6aS)-6a-naphtalan-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on (BAY 36-7620), administered subcutaneously (s.c.) at 10 mg/kg, only occupied about 30% of cerebellar and thalamic mGlu1 receptors. The mGlu1/5 receptor antagonist 2-quinoxaline-carboxamide-N-adamantan-1-yl (NPS 2390) exhibited a relatively high potency in occupying mGlu1 receptors in rat cerebellum (ED50 = 0.75 mg/kg, s.c.) and thalamus (ED50 = 0.63 mg/kg, s.c). In the future, this method can be employed to gain more insight into the in vivo profile and central activity of potential therapeutic agents that act upon the mGlu1 receptor.     

Mild thyroid hormones deficiency modifies benzodiazepine and mu-opioid receptor binding in rats

The effects of a mild hypothyroidism condition on benzodiazepine (BDZ) and mu opioid receptor levels was investigated. Female Wistar rats were randomly divided into two groups: 1) hypothyroid rats (n=7), which received methimazole (60 mg/kg per day) in drinking water for four weeks, and 2) euthyroid rats (n=8), which drank only tap water. Animals were sacrificed and their brains were used for autoradiography experiments. When compared to the euthyroid group, the hypothyroid group presented reduced benzodiazepine receptor binding in medial amygdala (24%) and high mu-receptor levels in frontal (25%), sensorimotor (65%) and temporal (29%) cortices, basolateral amygdala (50%) and ventroposterior thalamic nucleus (49%). The present data suggest that alterations in BDZ and mu-receptor binding could be associated with the higher excitability observed in animals with triiodothyronine (T(3)) deficiency.